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The Notch signaling pathway plays a crucial role in embryonic development as well as in maintaining tissue homeostasis throughout the lifespan of an adult organism. The involvement of this signaling pathway in the development of certain neurodegenerative diseases and other nervous system disorders is known, yet a comprehensive therapy related to the Notch signaling pathway has not been developed. This underscores the importance of further investigating the molecular mechanisms of the Notch signaling pathway. This review explores the molecular mechanisms by which the Notch signaling pathway participates in neurogenesis and neural differentiation. Finally, we discuss the role of Notch in neurodegenerative pathologies and possible therapeutic approaches.
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Diabetes mellitus (DM) encompasses a range of metabolic disorders characterized by chronic hyperglycemia, including type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus (GDM). The pathophysiology of DM is intricate, exhibiting variations across its different types, involving factors such as autoimmunity, insulin resistance, and β-cell dysfunction. Prolonged hyperglycemia in DM leads to microvascular and macrovascular complications, driven by various pathophysiological processes such as inflammation, coagulation, oxidative stress, and endothelial dysfunction. Exosomes, a specific type of extracellular vesicles (EVs), are recognized contributors to the aforementioned pathological processes and are thus implicated in the pathogenesis of diabetes and its associated complications. This work begins with summarizing the key characteristics, biogenesis, composition, and essential physiological and pathological functions of exosomes. It then focuses on collating and analyzing the most recent data on the pathogenic roles of exosomes in T1DM, T2DM, GDM, and diabetes-associated complications, such as diabetic retinopathy, nephropathy, neuropathy, and cardiomyopathy.
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Ferroptosis is a form of iron-dependent cell death that differs from apoptosis. Key characteristics of cells undergoing ferroptosis include iron overload, lipid peroxidation, glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) expression inhibition, and cystine/glutamate antiporter system Xc- damage. Ferroptosis is common in nervous system diseases. The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) is a classic intracellular signal transduction pathway that responds to extracellular signals and promotes proliferation, growth, metabolism, and angiogenesis. The imbalance of the PI3K/AKT signaling pathway might lead to a series of diseases. Although there have been many studies on the role of this signaling pathway in central nervous system (CNS) diseases, the impact of this axis on mediating ferroptosis in CNS diseases has not been summarized. In this review, we will elucidate the role of PI3K/AKT in mediating ferroptosis in several main CNS diseases, including ischemic stroke (IS), intracranial cerebral hemorrhage (ICH), Alzheimer's disease (AD), Parkinson's disease (PD), and glioblastoma (GBM), to provide valuable insights for both clinical endeavors and scientific investigations in the future.
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Oxidative stress occurs when the accumulation of oxidants exceeds the body's antioxidant defenses. Reactive oxygen species (ROS) are the primary oxidants and their generation is influenced by various factors including disease states, surgical procedures, and anesthetics. Studies have shown that various perioperative complications are attributed to ROS, especially in immature organs and systems. The pediatric oxidative stress profile is distinct from that seen in adults. We conducted a search for publicly available studies on perioperative oxidative stress, including both reviews and original research articles, animal studies, and clinical trials. In this review, we provide an overview of the factors that influence oxidative stress, the impact of perioperative oxidative stress on the body, and the biomarkers and measurement methods for oxidative stress. We also summarize current knowledge of oxidative stress in children during the perioperative period. Emphasis was placed on various factors during the perioperative period that affect oxidative stress in children including specific surgical procedures, anesthetic agents and their use, fluid therapy, mechanical ventilation, and oxygen inhalation. This review also provides several insights into oxidative stress in children during the perioperative period. Current evidence indicates that the greater the surgical trauma, the stronger the oxidative stress response, and this is due to ischemia-reperfusion injury which is particularly evident in pediatric cardiac surgery. Regional anesthesia is helpful in reducing systemic oxidative stress levels compared to general anesthesia. Intravenous anesthetics have an inhibitory effect on the build-up of oxidative stress when compared to inhaled anesthetics. Further, compared with unrestrictive fluid therapy, restrictive fluid therapy, during the perioperative period, can enhance the body's antioxidant capacity. In addition, we found that mechanical ventilation also increases oxidative stress. Therefore, to minimize perioperative oxidative stress, children undergoing surgery should, whenever possible, be treated with minimally invasive techniques such as laparoscopy and arthroscopy. Children undergoing general anesthesia should be given intravenous anesthetics if possible. A restrictive fluid therapy strategy should be adopted, and mechanical ventilation should be avoided. Finally, during perioperative treatment, exogenous antioxidants could be included.
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Background: Excess consumption of fructose is a significant factor in the development of metabolic syndrome (MetS). It may also play a role in the progress of chronic kidney disease (CKD). Osteopontin (OPN) is a pleiotropic, multi-phosphorylated glycoprotein which plays important roles in diseases as well as in a wide range of biological activities. Based on these findings, the aim of this study was to evaluate OPN as a biomarker for the early detection of renal injury during an experimental model of fructose associated MetS in mice.
Methods: Male CD1 mice aged 8–10 weeks were used. Fructose mice were given 30% fructose solution in drinking water, while control mice were given normal drinking water for 56 days. At sacrifice, kidneys, blood and urine of mice were collected. Biochemical, histological (hematoxylin and eosin and Masson's trichrome), immunohistochemical and molecular analyses (western blot, real-time quantitative polymerase chain reaction (RT-qPCR)) were performed.
Results: Compared to controls, Fructose mice showed increased levels of glucose, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), as well as creatinine and blood urea nitrogen (BUN). In addition, significant histological kidney injury and fibrosis were observed in Fructose mice. These alterations were associated with increased levels of plasma and renal tissue OPN.
Conclusions: Thus, new biomarkers such as OPN can be clinically useful to help predict kidney damage in MetS.
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Background: Because of their anti-proliferative effects, 9-anilinoacridines are important as antitumor agents with DNA-intercalating properties. In this study, anticancer drugs with 9-anilinoacridines, such as amascrine and nitracrine, were developed. Pharmacophore modelling, molecular docking, Molecular Mechanics Generalized Born Surface Area (MM-GBSA), induced fit docking and a molecular dynamics (MD) study were performed to investigate the binding affinity of 9-anilinoacridines with heterocyclic substitutes as selective human epidermal growth factor receptor 2 (HER2) inhibitors for breast carcinoma.
Methods: The pharmacophore model was developed using the Schrödinger suite 2019-2 phase module. 3D structures of dataset compounds were generated using Maestro version 9.6 and optimised using the LigPrep design of the Schrödinger suite 2019-2. To predict the binding free energy of the ligands in the complex with Protein Data Bank (pdb), we performed post-docked energy minimisation using the Prime MM-GBSA module. Induced fit docking studies were performed to determine the ligand-modulated dynamic behaviour shown in the protein MD study. Using the Desmond module in Schrödinger 2019-2, the complex in the optimized potentials for liquid simulations 3 (OPLS3) force field's explicit solvent system was investigated. Using the pharmacophore hypothesis, a statistically substantial 3 Dimensional Quantitative Structure Activity Relationship (3D-QSAR) design was created.
Results: We obtained the top five hypotheses, and according to the scoring parameters, the best model was identified as the 3D-QSAR model A-Acceptor, D-Donar, R-Ring (model AADRRR)-21. From the contour map analysis, we found that the presence of a hydrogen-bond donor and electron-withdrawing and hydrophobic features are crucial for inhibiting the HER2 enzyme. The docking study showed that the ligands have significant G-score (Glide Score) values from –4.18 to –9.96 kcal/mol. A 15-ns MD simulation was also run to determine the molecular details involving the affinity of 3m in active site 3PP0.pdb. The binding free energy was determined using the Prime MM-GBSA module, and dG binding values were observed from –35.11 to –106.87 kcal/mol.
Conclusions: We designed and developed a pharmacophore hypothesis and 3D-QSAR model and then elucidated the structural features and spatial arrangement of atoms responsible for the HER2 inhibitory activity of 9-anilinoacridines. The predicted 3D-QSAR model significantly correlated with experimentally reported in vitro antitumor activity. The findings indicated that additional pharmacophore feature modifications might enhance the HER2 inhibitory activity of 9-anilinoacridines.
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Background: Glenohumeral osteoarthritis (GH OA) is a very common joint disease characterized by pain and functional limitation. No standardized protocols of treatment are available in clinical practice and literature data are confusing. This monocentric randomized open-label study aims to assess the efficacy and safety of a treatment with medium molecular weight Hyaluronic Acid (HA) compared to a treatment with corticosteroids on patients affected by glenohumeral osteoarthritis.
Methods: A total of 80 consecutive patients with GH OA (Kellegren Lawrence grade II or III) were randomized in two different groups: Hymovis® (24 mg/3 mL) (treatment group) and corticosteroids (control group), with a follow-up after 3 and 6 months. Modifications in terms of pain, range of motion and quality of life were observed during the treatment.
Results: After 6 months, the treatment group presented a mean active abduction (ABD) of 67.9° ± 3.6 (baseline 56.4° ± 4.5, a gain of 11.5°), the mean internal rotation (INT) was 14.4° ± 2.0 (baseline 13.6° ± 2.1. a gain of 0.8°) and the mean external rotation (EXT) was 24.3° ± 2.5 (baseline 23.5 ° ± 2.9, a gain of 0.8°) for patients belonging to the treatment group. The mean Constant-Murley Score (CMS) in the treatment group was 76.5 ± 4.3 (baseline 59.5 ± 5.4, a gain of 17.0 from the starting point). The mean Visual Analogue Scale (VAS) was 4.6 ± 0.7 (baseline 8.0 ± 1.1, a reduction of 3.4), and the EuroQol-5 Dimension (EQ-5D) was 70.6 ± 4.3 (baseline 53.7 ± 5.1, a gain of 16.9). For patients in the control group, after 6 months, the mean active ABD was 60.4° ± 4.8 (baseline 58.9° ± 4.8, a gain of 1.5°), the mean INT was 15.3° ± 1.9 (baseline 14.9° ± 2.0, a gain of 0.4°) and the mean EXT was 22.8° ± 2.1 (baseline 23.5° ± 2.9, a gain of 0.6°). The mean CMS was 66.5 ± 4.8 points, (baseline 61.2 ± 4.7, a gain of 5.3). The mean VAS score was 7.2 ± 1.1 (baseline 7.9 ± 1.1, a reduction of 0.7), and the EQ-5D was 62.1 ± 3.8 (baseline 54.9 ± 4.7, a gain of 7.2). At 6 months follow-up, patients treated with HA showed a significant reduction in the level of pain and a significant improvement in their daily activities in comparison with the control group.
Conclusion: The present study demonstrates that patients affected by mild-moderate GH OA treated with two intra-articular injections of HA (Hymovis® 24 mg/3 mL) achieved better results for a longer duration, in terms of reduction in shoulder pain and improvement in their daily activities, compared to patients treated with two intra-articular injections of corticosteroids.
Clinical Trial Registration: No. NCT06043544. URL:
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Background: Multiple myeloma (MM) is a malignant clonal plasma cell disorder in the bone marrow and is the second-most common hematologic malignancy in adults. It proliferates by inducing neovascularization of the bone marrow stroma. Both nuclear factor kappa B (NF-κB) and osteopontin (OPN) pathways are considered crucial in the development of various tumors, including MM. Bortezomib is a proteasome inhibitor well established as a first-line treatment in MM. This study aimed to demonstrate in vivo anti-angiogenic effects of bortezomib in MM patients who responded to therapy by analyzing whether the expression of NF-κB was associated with OPN and microvessel density (MVD) as well as overall survival (OS).
Methods: Thirty patients who were newly diagnosed MM were enrolled in a study to investigate possible associations between neovascularization, expressed as microvessel density, NF-κB, and osteopontin expression in myeloma cells from bone marrow biopsy (BMB) pretreatment and posttreatment samples. This study used paraffin-embedded BMB before and after therapy with bortezomib. Immunohistochemical staining was performed to analyze the samples.
Results: After bortezomib treatment, BMB samples showed significantly fewer plasma cell infiltrates (PCI) (p < 0.001), lower percentages of NF-κB (p < 0.001), and OPN (p = 0.023) in plasma cells, and reduced MVD (p = 0.009) compared to pretreatment. A significant positive correlation was observed between NF-κB and OPN expression in plasma cells before and after bortezomib treatment in BMB samples (rs = 0.57, p = 0.002; and rs = 0.50, p = 0.007; respectively). Additionally, significant positive correlations were observed between NF-κB and OPN with MVD in pretreatment BMB samples (rs = 0.500, p = 0.018; and rs = 0.502, p = 0.017; respectively). Patients with higher MVD posttreatment had significantly shorter overall survival (OS) rates (p = 0.025).
Conclusions: Bortezomib treatment results in a significant decrease of MVD (angiogenesis) and PCI in BMBs of MM patients who responded to treatment. Furthermore, lower posttreatment MVD is associated with a longer OS. It seems that NF-κB and OPN inhibition could be the new therapeutic targets for MM patients, whilst MVD assessment before and after treatment with bortezomib (and other proteasome inhibitors) should be incorporated into routine diagnostic procedures.
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Background: Physical activity after a joint replacement surgery is one of the most important factors for a satisfactory quality of life for the patients. This study aimed to assess postoperative pain, rate of return to sports, intensity of athletic involvement, postoperative functional outcome, and level of satisfaction after unicompartmental knee replacement (UKR) with a mobile-bearing prosthesis at a minimum follow-up of two years after surgery.
Methods: Forty-two patients who had previously undergone a mobile-bearing UKR surgery were enrolled in the study. The mean age at surgery was 67.8 ± 2.5 years. The average follow-up was 3.2 ± 1.5 years. Sports activity was reported for each patient. Visual Analog Scale (VAS), Tegner activity level, Knee Osteoarthritis Outcome Score (KOOS), and International Knee Documentation Committee Subjective score were determined. Data were analyzed to detect statistically significant differences between preoperatory status and follow-up.
Results: The mean KOOS demonstrated a statistically significant difference from preoperative (48.7 ± 5.4) to postoperative (86.5 ± 4.7; p < 0.0001) status. VAS scores significantly decreased from a median of 6.5 (range 2 to 9) to 1.0 (range 0 to 3) at follow-up (p < 0.0001). Tegner's activity level increased from a median of 3.5 (range 1 to 6) preoperatively to a median of 4.5 (range 1 to 8) postoperatively (p < 0.0001). Eighty-three percent of patients (35 of 42) returned to sports activities successfully. In the subgroup of patients engaged in a sport before surgery, the percentage increased to 92%.
Conclusions: Evidence suggests that mobile-bearing UKR enables a significant percentage of patients suffering from unicompartmental knee osteoarthritis to resume sports activity while also improving pain scores and overall functional and subjective outcomes. Most patients who successfully returned to physical activity engaged in low-impact sports.
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Background: Gastric cancer (GC) is a common malignancy of the digestive tract and about 95% of them are gastric adenocarcinomas (STAD). However, Helicobacter pylori (HP) infection does not only serve as a major risk factor for GC but is also linked to the differential expression of miRNAs. Moreover, miR-146a-5p has a crucial role in numerous human malignancies. Therefore, this study aimed to explore the molecular mechanisms underlying the role of miR-146a-5p in HP-associated STAD.
Methods: MiRNAs genes linked to HP-induced GC were retrieved from the GeneCards and g: Profiler databases. MiR-146a-5p expression in HP-induced STAD was determined using quantitative real-time PCR. The role of miR-146a-5p in HP-induced STAD was assessed using Cell Counting Kit-8, colony formation assay, wound healing analysis, transwell analysis, flow cytometry, and Western blot. Furthermore, the mechanism of miR-146a-5p in STAD was evaluated using a dual-luciferase reporter assay.
Results: MiR-146a-5p expression was significantly increased in HP-infected STAD cells. Moreover, HP infection promoted STAD cell growth by increasing the expression of miR-146a-5p. Mechanistically, miR-146a-5p targeted four-and-a-half LIM domain protein 1 (FHL1), and rescue assays further authenticated that HP facilitated STAD cell growth by increasing miR-146a-5p to reduce FHL1.
Conclusion: In summary, HP infection leads to elevated miR-146a-5p expression and promotes STAD by mediating the miR-146a-5p/FHL1 pathway. This study might provide a basis for considering miR-146a-5p as a diagnostic marker for STAD and may help in designing preventive measures and treatment strategies.
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Background: Retinoblastoma (RB) is a malignant tumor of the retina, and occurs usually in infants. However, the homeobox gene B5 (HOXB5)/signal transducer and activator of transcription 3 (STAT3) pathway is predominantly involved in many cancer types but has never been explored in RB. Therefore, we aimed to investigate the molecular mechanism underlying the HOXB5/STAT3 pathway in the progression of RB.
Methods: The expression level of HOXB5, both at RNA and protein levels, within RB cells and tissues was assessed using various approaches, including immunocytochemistry, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), and western blot analysis. Furthermore, three types of transfected cell models were developed, including a group of cells with knocked-down or deleted HOXB5, another group with overexpressed HOXB5, and the third group with HOXB5 deleted+STAT3 overexpressed. Finally, the viability, metastasis, and apoptosis of RB cells were determined using Cell Counting Kit-8 (CCK-8) assay, Transwell analysis, and flow cytometry.
Results: We observed an increase in the expression levels of HOXB5 and STAT3 in RB tissues and cells (p < 0.05). Furthermore, it was found that the overexpression of HOXB5 increased the viability (p < 0.01), migration (p < 0.05), and invasion capability (p < 0.05), and inhibited apoptosis in RB cells (p < 0.001). HOXB5 deletion reduced the viability and the number of migration and invasion of RB cells and increased the number of cell apoptosis (p < 0.001). Moreover, the silencing of HOXB5 decreased the expression level of STAT3 (p < 0.01), while the overexpression of STAT3 reversed the HOXB5 silence-induced RB cell inhibition effect (p < 0.001).
Conclusions: These findings suggest that HOXB5 enhances the proliferation and metastasis of RB cells by regulating the STAT3 signaling pathway.
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Background: Studies have shown that foam cell formation and cholesterol leakage are important causes of abdominal aortic aneurysm (AAA), long non-coding RNA (lncRNA) antisense non-coding RNA at the INK4 locus (ANRIL) is a biomarker for diagnosing AAA, and ATP-binding cassette transporter A1 (ABCA1) is involved in cholesterol transport. However, the mechanism of action of ABCA1 and ANRIL in AAA is unclear. This work mainly explored the effect of the RR, RK and KK genotypes of the R219K locus of the ABCA1 and ANRIL on the occurrence and development of AAA.
Methods: In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expression of ABCA1 and ANRIL in AAA and normal tissues. Eukaryotic expression vectors (RR, RK, KK) of the ABCA1 allele were introduced into vascular smooth muscle cells (VSMCs) or human monocytic leukemia cells (THP). Oil red O staining was used to detect the foam cell formation rate after THP-1 treated with oxidized low-density lipoprotein (oxLDL), and a kit was used to detect the intracellular lipid content. An enzyme-linked immunosorbent assay (ELISA) was used to detect apolipoprotein A-I (apoA-Ⅰ), high-density lipoprotein-C (HDL-C), IL-6 (interleukin-6) and TNF-α (tumor necrosis factor-α). Western blotting was used to detect MMP-2 (matrix metallo proteinase-2) and MMP-9. In addition, VSMCs were treated with angiotensin II (Ang II). The mitogen activity of VSMCs was detected by 5-ethynyl-2′-deoxyuridine (EdU) staining, and the cycle distribution and apoptosis of VSMCs were assessed by flow cytometry. Cell scratch and transwell were used to detect migration of VSMCs, and Western blotting was used to detect the expression of apoptosis-related proteins (Bcl-2 (B-cell lymphoma-2), Bax (Bcl-2 Associated X protein) and Caspase 3).
Results: In AAA, ABCA1 levels were downregulated, and ANRIL were upregulated (p < 0.001). Foam cell formation and a reduction in intracellular lipid content are induced by transfection with pc-ABCA1-KK, which promotes the outflow of cholesterol from THP-1 macrophages. pc-ABCA1-KK promoted expression of apoA-Ⅰ and HDL-C while inhibiting inflammation and matrix degradation (p < 0.01). Similarly, transfection of pc-ABCA1-KK promoted cell proliferation in VSMCs (p < 0.05). However, transfection with pc-ABCA1-RK and pc-ABCA1-RR did not yield any effects. The ABCA1-KK allele can affect cholesterol metabolism in THP-1 cells and alter the biological behavior of VSMCs by inhibiting ANRIL.
Conclusion: The ABCA1-KK allele affects the development of AAA by regulating ANRIL to modulate cholesterol metabolism in THP-1 cells and alter the biological behavior of VSMCs.
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Background: Hepatitis B virus (HBV) infection can lead to end-stage cirrhosis and hepatocellular carcinoma (HCC). Although approved antiviral drugs strongly inhibit viral replication, long-term treatment leads to drug-resistant variants and adverse side effects. Hence, the treatments of patients with HBV and virus carriers are likely to remain a major health problem for decades to come. The aim of this study was to investigate the effects of the distal upstream binding factor-far upstream element binding protein 1 (FUBP1) on the activities of the core promoter and enhancer II (EnhII)/basic core promoter (BCP) in Huh7 cells infected with HBV. In addition, we investigated the molecular mechanism of FUBP1 regulation of HBV transcription to find new targets for anti-HBV therapy.
Methods: The FUBP1 overexpression plasmid with HA label (pcDNA3.1-HA-FUBP1) was transfected and overexpressed in Huh7 cells. Western blot analysis was used to verify successful construction of the FUBP1 overexpression plasmid, as well as the overexpression of FUBP1. Co-transfection of pcDNA3.1-HA-FUBP1 and Plasmids containing the 1.24-fold HBV genomes (pUC19-HBV-Ce), together with real-time fluorescence quantitative PCR (RT-qPCR), was used to investigate the effects of FUBP1 overexpression on HBV pre-genomic RNA (pgRNA), spliced RNA (spRNA), and splicing efficiency. The results of RT-qPCR were further verified by Northern blot analysis. The poly(U)-binding splicing factor 60 kDa (PUF60) and FUBP1 are known to form a complex that exerts regulatory functions. Hence, the effects of PUF60, FUBP1, and their co-expression on HBV pgRNA level, pg promoter activity, and EnhII/BCP core promoter activity were investigated by RT-qPCR and sea kidney luciferase reporting assays. Alpha screen and RNA degradation experiments were also used to examine whether FUBP1 regulation of RNA transcription was related to post-transcriptional RNA extranuclear output and degradation. A series of HBV promoters and mutants were constructed, and the effects of FUBP1 overexpression on the activity of these HBV promoters and their mutants were evaluated. Gel migration assay was used to further investigate the targeting of the HBV EnhII/BCP region by FUBP1.
Results: Overexpression of FUBP1 significantly decreased the expression levels of pgRNA (p < 0.01) and spRNA (p < 0.001), and decreased the splicing efficiency (p < 0.001). These results were further verified by Northern blot. FUBP1 inhibited HBV transcription by down-regulating the activity of HBV EnhII/BCP (p < 0.001). Previous studies have reported that PUF60 often forms a complex with FUBP1. The present study found that FUBP1 independently targets the HBV EnhII/BCP region to inhibit the expression of HBV pre-genomic RNA (p < 0.001). Further studies showed that inhibition of HBV pgRNA by FUBP1 was not achieved by post-transcriptional regulation of extranuclear export and degradation of HBV RNA. FUBP1 regulates promoter activity by targeting promoters with the common sequence of del-1 (deletion of nt 1627—1652) and del-2 (deletion of nt 1640—1674) region nt1640–1652 (specific base sequence: GCCCAAGGTCTTG), thereby inhibiting HBV replication.
Conclusions: FUBP1 binds directly to the HBV nt1640-1652 region to down-regulate promoter activity and inhibit HBV transcription. FUBP1 plays a negative role in HBV transcription and is a potential target for anti-HBV therapy.
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Background: Numerous studies have demonstrated the function of long non-coding RNA (lncRNA) in the regulation of gene expression and its implication in the development of intracranial aneurysm (IA). However, the function and underlying mechanisms through which the lncRNA affects the occurrence and rupture of IA need to be further elucidated. The current study aimed to investigate the lncRNA and mRNA expression profiles in ruptured and unruptured IA patients and control individuals using RNA transcriptome sequencing (RNA-seq) and explore the underlying molecular mechanisms in the onset and rupture of IA mediated by lncRNA-mRNA axis.
Methods: RNA-seq was performed to evaluate the lncRNA and mRNA expression profiles in ruptured IA (IA group), unruptured IA (UIA group), and superficial temporal arteries (control group), followed by differential gene expression and functional enrichment analysis. The selected targets were then verified in an expanded cohort of 30 patients per group. Furthermore, the roles of target genes were evaluated in vascular smooth muscle cells (VSMCs) by transfecting them with an overexpression vector for lncRNA SLC12A5-AS1 (SLC12A5-AS1), either alone or in combination with small interfering RNA (si)-cell division cycle associated 3 (CDCA3), si-SLC12A5-AS1, or their corresponding negative controls. The cell proliferation, apoptosis, proteins expression, levels of pro-inflammatory factors, and the angiopoiesis of VSMCs were then determined.
Results: RNA-seq showed that SLC12A5-AS1 and CDCA3 gradually increased in a gradient pattern from control donors to UIA to IA patients. Furthermore, receiver operating characteristic (ROC) curve analysis confirmed SLC12A5-AS1 and CDCA3 as valuable biomarkers of UIA and IA patients. Moreover, pathway enrichment analysis of the overlapped differentially expressed (DE) mRNAs in UIA and IA patients revealed that they were mainly enriched in the nuclear factor-kappa B (NF-κB) pathway. Additionally, it was observed that SLC12A5-AS1 overexpression inhibited proliferation, angiopoiesis, and α-smooth muscle actin (α-SMA) and smooth muscle-22α (SM-22α) expression of VSMCs, whereas promoted apoptosis, levels of proinflammatory factors (interleukin-1β (IL-1β), interleukin 6 (IL-6), and tumour necrosis factor-alpha (TNF-α)), matrix metalloproteinase 9 (MMP9) and CDCA3 proteins expression, as well as phosphorylation of p65 and nuclear factor-kappa B inhibitor alpha (IKBα). Conversely, inhibition of CDCA3 had opposite effects on VSMCs, which can be overcome with SLC12A5-AS1 overexpression.
Conclusion: Our current findings indicated that the SLC12A5-AS1 is involved in IA occurrence and rupture by regulating the phenotypic transformation of VSMCs through modulating CDCA3.
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Background: Dezocine is a commonly used opioid receptor mixed agonist-antagonist in clinical practice. It has been proven to play a vital effect on the regulation of immune function in cancer patients. However, the latent mechanisms of Dezocine on gastric cancer cell viability and tumor growth in gastric cancer mice remain unclear.
Methods: Dezocine's impact on the viability of gastric cancer cells, apoptosis, and apoptosis-related proteins was evaluated through Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and western blot analysis, respectively. The effect of Dezocine on T-helper 1/T-helper 2 (Th1/Th2) cells and inflammatory cytokines was determined by flow cytometry and Enzyme-linked Immunosorbent Assay (ELISA). The role of Dezocine on tumor viability in gastric cancer mice in vivo was also determined.
Results: We discovered that Dezocine suppressed gastric cancer (GC) cell viability and accelerated apoptosis in a concentration-dependent pattern. In comparison with control, Dezocine reduced the tumor weight, volume, Th2 cell proportion, and interleukin-10 (IL-10) level. In contrast, it increased Th1 cell proportion, Th1/Th2 ratio, interferon-γ (IFN-γ), Tumor Necrosis Factor-alpha (TNF-α), and IL-6 levels in the mice with gastric cancer. Furthermore, Dezocine also inhibited the expression of Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6), while overexpression of TRAF6 reversed the suppression effect of Dezocine on gastric cancer (GC) cell viability and tumor growth.
Conclusions: Dezocine has an anti-GC effect by inhibiting the development of GC by regulating TRAF6, Th1/Th2 balance, and inflammatory cytokines.
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Background: Transfusion-related acute lung injury (TRALI) is a form of acute lung injury (ALI) that can cause complications such as respiratory distress, hypoxia, fever, and tachycardia in patients. A study using mice found that the focal adhesion kinase (FAK) inhibitor PF-562271 improved ALI. This study aimed to investigate whether PF-562271 could inhibit the progression of inflammation in a mouse model of TRALI.
Methods: Platelets were collected from experimental mice and stored for 5 days. The mice were injected intraperitoneally with lipopolysaccharide (LPS), and then the stored platelets were injected into the mice through the tail vein to establish the TRALI model. Lung tissue samples were collected from the mice after the experiment. Pathological changes in the lung tissue, lung wet/dry weight ratio, myeloperoxidase (MPO) activity, and the expression of Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-8 (IL-8), and FAK protein were analyzed to determine the effects of PF-562271 on TRALI in mice.
Results: Histological analysis revealed that the alveolar interstitium was filled with inflammatory cells and the alveolar septum was significantly widened in the TRALI model group. The lung wet/dry weight ratio confirmed that the pulmonary edema induced in the TRALI group was more severe than that in the LPS group (p < 0.001). MPO activity was higher in the TRALI group than in the LPS group (p < 0.001). The mRNA expression of TNF-α, IL-6, and IL-8, and protein expression of FAK in the lung tissue were up-regulated (p < 0.05, p < 0.01, p < 0.001). After 24 h of FAK inhibitor intervention, the pulmonary edema in TRALI mice was significantly reduced, the infiltration of inflammatory cells in the lung tissue was improved, the lung function was better, and the expression of inflammatory factors was downregulated (p < 0.05, p < 0.01, p < 0.001).
Conclusions: We successfully constructed a mouse TRALI model infused with aged platelets and found that the FAK inhibitor PF-562271 could alleviate the lung injury caused by TRALI and increase the survival rate of TRALI mice. Therefore, FAK inhibitors may have potential applications in the treatment of TRALI.
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Background: Hepatocellular carcinoma (HCC) is a kind of multi-vessel tumor with high morbidity and mortality. Anti-angiogenesis is a promising treatment for HCC. In recent years, transcatheter arterial chemoembolization (TACE) is a typical non-surgical anti-angiogenic therapy for HCC. This study aimed to explore the effect and mechanism of miR-125b-5p on TACE for treating HCC.
Methods: Twenty patients with HCC received surgery after TACE and were subjected to tumor tissues collection. Twenty patients with HCC underwent surgery only and tumor tissues and adjacent tissues were collected. Additionally, serum samples were obtained from patients before and after TACE, and from patients with remission or recurrence after TACE treatment. Knockdown or overexpression of miR-125b-5p and overexpression of vascular endothelial growth factor A (VEGFA) were carried out in human HCC cell line (HCCLM3). Real time quantitative PCR (qRT-PCR) was employed to measure the expression of miR-125b-5p and VEGFA. Cell proliferation, viability, and tube-forming ability of HCCLM3 cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and tube formation assay. Further, a dual luciferase reporter assay was carried out to determine the relationship between miR-125b-5p and VEGFA. Western blot was adopted to test the level of VEGFA in the tissues and MEK/ERK signaling pathway-related proteins in the cells were measured.
Results: MiR-125b-5p expression in the tumor tissues of patients with HCC receiving TACE and surgery was notably increased relative to those of patients receiving surgery only. The expression of miR-125b-5p mounted obviously in the serum after TACE (before TACE vs. after TACE), but showed a marked decline in the serum of patients with recurrence after TACE treatment (remission vs. recurrence). At cellular level, miR-125b-5p expression was noticeably reduced in HCC cell line, and its overexpression inhibited HCC cell proliferation and angiogenesis as well as the activation of the mitogen-activated protein kinase kinase (MEK1/2)-extracellular signal-regulated kinase (ERK) signaling pathway. Moreover, there was a targeting relationship between miR-125b-5p and VEGFA, and VEGFA expression was significantly decreased in the tumor tissues of patients after TACE treatment compared with the patients who received surgery alone. VEGFA could reverse the inhibitory effect of the overexpression of miR-125b-5p on HCC cells.
Conclusions: MiR-125b-5p targeted and inhibited VEGFA expression to affect angiogenesis, and it affected cell proliferation and viability by inhibiting the MEK1/2-ERK1/2 signaling pathway, thereby improving the efficacy of TACE in the treatment of HCC.
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Background: High-intensity interval training (HIIT) is an exercise modality that can induce reactive oxygen species (ROS) production in skeletal muscles, the extent of which depends on the intensity, duration, and volume of exercise, potentially leading to oxidative stress.
Objective: To analyze the effects of a single session of two HIIT protocols with different intensity levels on the markers of blood sugar, lactate, and hepatic damage in rats.
Methods: Overall, 24 male Wistar rats were included in the study and divided into three groups (n = 8/group): control group (CG); short-interval HIIT (H1): HIIT involving 14 swimming periods lasting 20 s each, with 10-s intervals between each period, totaling a duration of 7 min; long-interval HIIT (H2): HIIT involving 14 swimming periods lasting 35 s each, with 25-s intervals between each period, totaling a duration of 14 min.
Results: Regarding blood sugar levels, there were no significant differences in the H1 (113.00 ± 8.21 vs. 123.25 ± 10.78; p = 0.3303) and H2 (112.25 ± 12.55 vs. 114.25 ± 8.77; p = 0.9545) groups, respectively. Regarding lactate levels, there was a significant difference in both the groups compared with the CG (H1: 2.75 ± 0.55 vs. 10.06 ± 1.93; p = 0.0003; H2: 2.65 ± 1.07 vs. 9.99 ± 1.30; p = 0.0001). However, there was no difference in lactate levels between the H1 and H2 groups (10.06 ± 1.93 vs. 9.99 ± 1.30; p = 0.9965). Regarding hepatic damage, only H1 demonstrated a reduction in aspartate aminotransferase (AST) and alanine transaminase (ALT) levels compared with CG (30% and 29.65%, respectively; p < 0.05). Conversely, there were no significant differences in the AST levels between both the groups and CG (16.40 ± 1.91 [H2] vs. 13.98 ± 1.06 [CG]; –14.75%; p = 0.0918; 11.48 ± 1.59 [H1] vs. 13.98 ± 1.06 [H2]; p = 0.0792). Additionally, there was no significant difference in the ALT levels between the HIIT groups (8.54 ± 1.70 [H1] vs. 10.32 ± 0.78 [H2]; p = 0.1756).
Conclusions: A single session of HIIT training with different intensity levels does not induce changes in blood sugar and lactate markers. Furthermore, short-duration HIIT induced alterations in the hepatic markers (AST and ALT) compared with the control group. However, when comparing the HIIT groups, no significant alterations were observed following a single HIIT session.
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Background: Many malignancies are associated with C-Type Lectin Domain Family 3 Member B (CLEC3B), but the mechanism by which it affects esophageal squamous cell carcinoma (ESCC) cells is unclear. This study will investigate the changes of CLEC3 on invasion, migration, and cell cycle of ESCC. The effect of CLEC3 on the growth of Besophageal squamous cell carcinoma was also verified through transplanted tumors.
Methods: Differences in CLEC3B expression were explored by immunohistochemistry and RT-qPCR (real-time quantitative polymerase chain reaction). Effects of overexpression of CLEC3B on ESCC cell behavior were observed in vitro. ESCC cells were divided into ov-NC (negative control) and ov-CLEC3B groups. The effect of CLEC3B on the proliferative activity and invasive metastasis of ESCC cells was investigated. The transcriptome of human esophageal squamous carcinoma cells-30 (KYSE-30) was sequenced. The molecular mechanism of CLEC3B action on ESCC cells was verified by ELISA (enzyme linked immunosorbent assay), immunofluorescence staining, flow cytometry and western blot. The effect of CLEC3B overexpression on ESCC growth was measured in vitro. BALB/c nude mice were injected with adenovirus in subcutaneous tumor xenograft experiments. The size and weight of xenograft tumors were measured. Immunohistochemistry was performed to detect ki67. CLEC3B expression was detected by RT-qPCR and Western blot.
Results: CLEC3B expression was decreased in ESCC tissues and cells compared to normal tissues and cells. Esophageal cancer cells overexpressing CLEC3B showed reduced proliferative activity, metastasis and invasive ability. Overexpression of CLEC3B increased the level of inflammation in esophageal cancer cells, exacerbated the degree of DNA damage, resulting in cell cycle halt in the G1 phase. Possible pathways through which CLEC3B exerts these effects include the MAPK (Mitogen-activated protein kinases) pathway, NOD-like receptors pathway and NF-κB (nuclear factor kappa-B) pathway. Overexpression of CLEC3B inhibited the proliferation of subcutaneous xenograft tumors and increased the expression of CLEC3B.
Conclusions: CLEC3B suppresses esophageal squamous cell carcinoma cell growth, invasion, and metastasis by raising inflammation, increasing cellular DNA damage, and triggering cell cycle arrest.
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Background: Skin squamous cell carcinoma (SSCC) is a common malignancy of skin, and its occurrence is a multifactorial process. Glycolysis plays a vital role in its occurrence and development. This study investigated the effect of neurogenic locus notch homolog protein 1 (Notch1) on malignant behavior of SSCC cells through glycolysis mediated by hypoxia-inducible factor 1 alpha (HIF1A).
Methods: Notch1 expressions in normal human skin cells human keratinocytes (HaCaT) and three SSCC cell lines (SCL-1, Colo-16, and A431) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Colo-16 cells with Notch1 or HIF1A overexpression were constructed by liposome-mediated transfection technology. Colo-16 cells were divided into negative control of Notch1 overexpression (pc-NC1), Notch1 overexpression (pc-Notch1), Notch1 overexpression+negative control of HIF1A overexpression (pc-Notch1+pc-NC2), and Notch1 overexpression+HIF1A overexpression (pc-Notch1+pc-HIF1A) groups. Clone formation, scratch healing and Transwell tests were used to detect the effect of Notch1 overexpression on Colo-16 cell proliferation, migration and invasion ability, respectively. Glucose and lactic acid test kits were used to detect glucose and lactic acid levels. The protein expression levels of Notch1, HIF1A and key glycolytic proteases were detected by western blot.
Results: Notch1 expression in SSCC cells was lower than in the control group (p < 0.01, p < 0.001). Notch1 overexpression reduced cell proliferation, invasion and migration, and inhibited glucose consumption and lactic acid formation (p < 0.01, p < 0.001). Notch1 overexpression also inhibited expression of glucose transporter type 1 (GLUT1) and related key glycolytic proteases (p < 0.01, p < 0.001, p < 0.0001). Upregulation of Notch1 decreased HIF1A protein expression (p < 0.01, p <0.001). HIF1A overexpression partially reversed the inhibitory effect of Notch1 on cell malignant behavior and glycolysis.
Conclusions: Notch1 inhibits proliferation, migration and invasion of SSCC cells through HIF1A-mediated glycolysis and may be a target for SSCC diagnosis and treatment.
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Background: Although squamous cell carcinoma antigen (SCCA) is a tumor biomarker used in the diagnosis of cancer, it is also elevated in non-oncological diseases. However, the diagnostic efficiency of SCCA for different diseases has yet to be systematically investigated. The purpose of this study was to explore the diagnostic efficacy of serum SCCA in neoplastic and non-neoplastic diseases.
Methods: The clinical, pathological, and serological data for 15,844 patients admitted to Hainan Hospital of PLA General Hospital between November 2018 and May 2022 were collected and retrospectively analyzed.
Results: Patients diagnosed with cervical cancer, bladder cancer, esophageal cancer, lung cancer, psoriasis, uremia, chronic renal failure, and nephritis had a mean serum SCCA value which exceeded the upper limit of the reference range (2.0 ng/mL). Squamous cell carcinoma (SCC) had considerably higher serum SCCA levels than other pathological types. The severity of renal failure is positively associated with a gradual increase in serum SCCA levels. Multivariate logistic analysis showed that SCCA was an indicator of uremia [odds ratio (OR), 1.845; 95% confidence interval (CI): 1.040–2.100; p = 0.038]. Receiver operating characteristic (ROC) curves demonstrated that the sensitivity (Sen) of SCCA in the diagnosis of uremia and psoriasis was 89.80% and 86.73%, respectively, and the specificity (Spe) was 75.00% and 78.79%, respectively. The area under the ROC curve (AUC) (95% CI) was 0.920 (0.882–0.958) and 0.907 (0.841–0.974), respectively, which was higher than cervical SCC [Sen: 81.63%; Spe: 68.46%, AUC (95% CI): 0.751 (0.609–0.793)]. Calculations of positive and negative likelihood ratios also supported these findings.
Conclusions: While SCCA is a specific marker for SCC, its diagnostic accuracy is higher for noncancerous diseases such as uremia, psoriasis, and renal failure, indicating the need for clinicians to consider the correlation between SCCA and certain noncancerous diseases while evaluating tumor development and monitoring therapeutic efficacy.
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Background: Hypertrophic scar (HS) is a disorder characterized by hyperplasia of fibrous tissue during the process of wound healing. During this process, patients suffer from pain, itching and ulceration. There is currently no effective method to inhibit the formation of HS. It is reported that botulinum toxin A (BTX-A) can prevent the excessive proliferation of fibroblasts by regulating apoptosis. The aim of this study was to investigate the therapeutic effects of BTX-A on HS tissue in mice.
Methods: An animal model of HS was established and treated with low (1 U/kg), medium (2 U/kg) and high (4 U/kg) doses of BTX-A, respectively. The effects of different doses of BTX-A on the HS tissue and collagen fibers of mice were detected by hematoxylin-eosin (HE) and masson staining. The effects of different doses of BTX-A on the expression of proteins related to the mitogen-activated protein kinase (MAPK) pathway as well as alpha-smooth muscle actin (α-SMA) and Collagen-I were detected by western blot (WB). The effects of different doses of BTX-A on apoptosis were detected by TdT-mediated dUTP nick end labeling (TUNEL).
Results: There was no visible bulge at the site of the incision. The wound was healed after 2 weeks, and there was no significant scarring in the control group. In the model group, the HS tissue showed an obvious bulge with significant congestion and was hard in texture. After 2 weeks, the degree of bulge was reduced, and was slightly softer in texture, but still had a certain degree of congestion, as well as significant scarring, altered arrangement of tissue and abnormal accumulation of collagen fibers. As the dose of BTX-A increased, there was a significant reduction in congestion. The scar was softer in texture, the thickness was markedly thinner, the number and density of collagen fibers were reduced and the arrangement of tissue and accumulation of collagen fibers were improved. Compared with the control group, the expression of extracellular signal-regulated kinase (ERK)1/2, p-ERK1, Jun N-terminal kinase (JNK), p-JNK, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Cleaved-caspase 3, α-SMA and Collagen-I was increased in the model group (p < 0.05). With the increased dose of BTX-A, the expressions of p-ERK1, Bcl-2, α-SMA and Collagen-I were reduced and p-JNK, Bax and Cleaved-caspase 3 were increased (p < 0.05).
Conclusion: BTX-A inhibited the formation of HS in mice by inhibiting the activation of the JNK/ERK pathway, promoting the level of apoptosis in HS tissue, reducing the formation of myofibroblasts and deposition of collagen, down-regulating the expression of α-SMA and Collagen-I as well as improving the disordered arrangement of collagen fibers.
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Background: Sepsis, a complex and potentially life-threatening condition resulting from a dysregulated host response to infection, claims numerous lives and carries a high mortality rate. The development of multiple organ dysfunction syndrome (MODS), a cascade of events leading to the failure of vital organ systems, worsens the clinical prognosis and represents a devastating consequence of sepsis. Ginsenoside Rg3 (Rg3), a natural compound, has been demonstrated its potential in regulating mitochondrial dysfunction, offering a potential treatment avenue for sepsis-related complications. Our research aimed to explore the mechanism by which Rg3 treats sepsis-induced MODS.
Method: In vitro, we established a sepsis cell model by subjecting normal cells (H9C2 cardiac cells, L02 human liver cells, and HK2 renal tubular cells) to lipopolysaccharide (LPS) treatment, with untreated cells as the control group. We conducted investigations to assess the impact of Rg3 on mitochondrial metabolism, respiratory function, oxidative stress, and mitochondrial homeostasis in cardiac, hepatic, and renal cells. In vivo, we employed the cecal ligation and puncture (CLP) method to induce a sepsis model in rats and administered or withheld Rg3 treatment. We assessed the general well-being of the rats, quantified inflammatory factors, conducted histological examinations, and performed biochemical analyses of key markers within the liver, kidneys, heart, and lungs to evaluate the extent of multi-organ dysfunction in CLP rats.
Results: In vitro, the Rg3-treated group exhibited a significant amelioration in mitochondrial dysfunction, including mitochondrial metabolism, mitochondrial respiratory function of cardiomyocytes, oxidative stress in liver cells, and mitochondrial homeostasis of renal cells, compared to the control group (p < 0.05). In vivo, following Rg3 administration, a significant improvement in the overall Severity Scores was observed in CLP rats (p < 0.05). Rg3 was discovered to have notable organ protective properties. It repaired histopathologic abnormalities induced by CLP in the liver, kidney, heart, and lungs in addition to lowering inflammatory cytokine levels (p <0.05).
Conclusion: Rg3 improves mitochondrial metabolism, respiratory function, and mitochondria homeostasis, and suppresses oxidative stress. Furthermore, Rg3 exhibits significant organ-protective properties and capacity to reduce inflammatory cytokine levels, thereby suppressing sepsis-induced MODS.
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Background: Acute kidney injury (AKI) is the main complication of critical care and has a variety of causes. Silent information regulator 2-related enzyme 3 (SIRT3) plays an important role in kidney diseases, including AKI. The aim of this study was to investigate the effect of SIRT3 on AKI in mice and its possible mechanism.
Methods: Lipopolysaccharide (LPS) treatment was used to establish an animal model of AKI. Stable overexpression of SIRT3 in this model was achieved by injection of adeno-associated vector (Aav) expression system for overexpression of SIRT3 (Aav-SIRT3). Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were detected by biochemical analysis. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to evaluate SIRT3 and neurogenic locus notch homolog protein 1 (Notch1) expression levels. Enzyme-linked immunosorbent assay (ELISA) was used to quantify interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels, as well as oxidative stress indices. Western blot was used to evaluate the level of protein expression.
Results: The LPS treatment group showed increased SCr, BUN, IL-1β, TNF-α, IL-6, malondialdehyde (MDA), and Notch1 compared to the control group, while the superoxide dismutase (SOD) level and SIRT3 expression were decreased. However, overexpression of SIRT3 could reverse the above changes. Similar changes in the levels of IL-1β, TNF-α, IL-6, MDA, SOD, Notch1 and hairy and enhancer of split-1 (Hes-1) were observed after SIRT3 overexpression in LPS-induced HK-2 cells. These changes could be reversed by the addition of Notch1 activator.
Conclusions: SIRT3 can improve LPS-induced AKI by regulating Notch1 signaling, thus providing a new approach for the treatment of AKI.
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Background: Among the limited therapeutic strategies for late-stage or triple-negative breast cancer (TNBC), immunotherapy has become an available method to prolong lifespan. Therefore, we aimed to explore immunotherapy response markers in humanized mice models to identify sub-populations that respond positively to immunotherapy.
Method: A mixture of peripheral blood mononuclear cells (PBMCs) and human breast cancer cells MDA-MB-231 was injected into mice to develop a PBMC cell line-derived xenograft (CDX) mouse model. The mice were treated with pembrolizumab, a programmed cell death protein-1 (PD-1) specific antibody. The tumor growth was monitored and recorded, and tumor volume was calculated using caliper measurements. The immunophenotyping was carried out using flow cytometry. The screening of differentially expressed genes and functional enrichment analysis were done through RNAseq and bioinformatics.
Results: A gradual decrease in tumor size was observed on day 7, day 10, and day 14 within the PD-1 treated group compared to the control group, although no statistical significance was observed (p > 0.05). Compared to the control group, RNAseq analysis on day 14 of the PD-1 treated group revealed nine genes with significant changes, including one upregulated gene and eight downregulated genes. Using the GSE124821 dataset, it was found that Nucleoporin 210 kDa (NUP210) was downregulated in both studies (p < 0.05) and proved to be responsive to immunotherapy in the validation dataset (GSE169246). Additionally, pathway analysis revealed that NUP210 is crucial in an immune response process.
Conclusions: Our study demonstrated that pembrolizumab could control tumors driven by the MDA-MB-231 cell line. Additionally, we identified NUP210 as a potential responsive marker for immunotherapy of pembrolizumab in breast cancer. These findings may provide significant insights into the future of breast cancer treatment.
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Background: Osteosarcoma (OS) belongs to the most familiar malignant bone tumor. Ferroptosis-suppress-protein 1 (FSP1), Sirtuin 1 (SIRT1), Fibrillin-1 (FBN1) are important regulators of OS progression. FSP1 is abnormally expressed in a range of tumor tissues and is a cancer promoter, but its mechanism in OS is not clear. The purpose of this research is to explore the potential function and regulatory mechanism of the possibly existed FSP1/SIRT1/FBN1 axis in OS.
Methods: The relationship between FSP1, SIRT1, and FBN1 was verified by cell con-transfection assay. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression efficiency of FSP1, SIRT1, FBN1, and apoptotic factors. Malondialdehyde (MDA) levels in OS were detected by the thiobarbituric acid method. The viability and metastasis of OS cells were detected by cell counting kit-8 (CCK-8) and transwell assays.
Results: The results indicated that FSP1 expression was upregulated in OS cells. The silencing of FSP1 suppressed viability and metastasis of OS cells, and promoted the ferroptosis. Meanwhile, the results showed that FSP1 deletion increased the expression of SIRT1, and the overexpression of SIRT1 decreased the expression of FBN1. We also found that the Sh-FBN1 reversed the inhibitory action of SIRT1 on OS cells transfected with Sh-FSP1.
Conclusions: The expression pattern of FSP1 is upregulated in OS cells. The overexpression of FSP1 inhibits iron death in OS cells. In contrast, SIRT1's expression pattern is downregulated in OS, and overexpression of FSP1 suppresses SIRT1 expression. Additionally, the expression pattern of FBN1 is upregulated in OS. Overexpression of SIRT1 inhibits FBN1 expression, and this regulatory pattern can influence the metastasis and ferroptosis of OS.
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Background: Zinc oxide (ZnO) nanoparticles can induce cytotoxicity by generating reactive oxygen species (ROS), but they are promising in pharmaceutical applications. Metal doping to nanoparticles often mitigates cytotoxicity by altering nanoparticle behavior. This study aims to synthesize nanoparticles of undoped ZnO and ZnO doped with copper, focusing on their diverse biological activities.
Methods: The nanoparticles were characterized using various techniques, including energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), scanning electron microscopy (SEM), and fourier-transform infrared (FTIR) spectroscopy. Antibacterial activity was assessed through a modified Kirby Bauer method, while antifungal activity was evaluated using a test-tube dilution method. The anti-termite activity was assessed using established methods with minor modifications. Cytotoxicity on the 3T3 mouse fibroblast cell line was quantified using the methylthiazoletetrazolium (MTT) assay. The mutagenic and carcinogenic potential of the prepared nanoparticles was evaluated using the Ames test.
Results: Characterization results confirmed the successful synthesis of ZnO and copper-doped ZnO nanoparticles measuring 1.67 nm and 55.8 nm, respectively. Undoped ZnO displayed antibacterial and antifungal activity against B. subtilis and K. oryzae. Copper-doped ZnO efficiently inhibited the growth of S. marcescens, P. aeruginosa, E. Coli, A. flavus, and K. oryzae. Furthermore, the anti-termite activity of both nanoparticles increased significantly after 48 and 72 hours, resulting in a higher termite mortality rate. The Mutagenic potential of the nanoparticle samples was assessed in two bacterial strains, S. typhimurium TA-98 and TA-100, by observing color changes in the well plates. Purple wells with no color change indicated non-contamination. Zinc oxide was found toxic, with zero positive wells, while copper-doped ZnO showed neither mutagenicity nor toxicity, confirming its safety. Undoped ZnO and copper-doped ZnO nanoparticles exhibited mild cytotoxicity at 30 g/mL concentration, reducing cell viability by 10% and 13%, respectively. However, the cytotoxicity was limited, as evidenced by their 50% growth inhibition (IC50) values, which did not reach concentrations that significantly inhibited cell growth.
Conclusion: Both nanoparticles demonstrated antibacterial and antifungal activities, highlighting their potential in pharmaceutical applications, while their anti-termite efficacy suggests applications in pest management. Copper-doped ZnO nanoparticles showed no mutagenicity or cytotoxicity, confirming their safety. At higher concentrations, both nanoparticles displayed mild cytotoxicity. These findings emphasize the adaptability of copper-doped ZnO nanoparticles for various applications in biomedicine and pest control.
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Background: There is currently no standard follow-up treatment method for metastatic endometrial carcinoma (EC). Exploring the molecular mechanisms of EC is of great significance for the prevention and treatment of EC. This research aimed to study the impact of the receptor tyrosine kinase (AXL) on the surface of EC cells and the crucial mechanism of AXL in tumor immune escape.
Methods: Western blot and flow cytometry were used to detect programmed death ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) protein in EC cell line. The AXL level was determined using quantitative real-time polymerase chain reaction (qRT-PCR). AXL plasmid and AXL shRNA were transfected into EC cells. The killing outcome of activated peripheral blood mononuclear cells (PBMC) on EC cells was detected by the lactate dehydrogenase (LDH) method. Enzyme linked immunosorbent assay (ELISA) was used for Interferon γ (IFN-γ), Granzyme B (GZMB), Perforin, and cluster of differentiation 107a (CD107a) detection in the supernatant. Furthermore, Western blot was employed to detect PD-L1 in EC cell lines treated with the c-Myc inhibitor (JQ1) for 24 h. The survival rate of tumor cells killed by PBMC was observed using crystal violet staining.
Results: EC cell lines RL-952, KLE, and HEC-1B exhibited varying levels of PD-1 and PD-L1 content. RL-952 cells showed relatively high levels, while HEC-1B cells displayed low levels. AXL was found to confer resistance to lymphocyte killing and enhance PD-L1 levels in EC cells. At the cytotoxic molecules level, AXL promoted the deactivation of CD8+ T (Cytotoxic T lymphocytes) cells, whereas JQ1 was observed to reverse this effect. A preliminary mechanism study indicated that AXL enhanced PD-L1 levels via the c-Myc pathway in EC cells. Treatment of EC with JQ1 resulted in a decrease in cellular-myelocytomatosis viral oncogene (c-Myc) and PD-L1 levels, potentially reversing the tumor immune escape induced by AXL.
Conclusions: AXL can increase the levels of PD-L1 and reinforce the immune evasion of EC cells by activating c-Myc.
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Background: Myocardial damage is a severe complication of pneumonia in children. Early diagnosis and symptomatic intervention can improve the prognosis of affected children. This study aims to investigate the predictive value of serum growth differentiation factor-15 (GDF-15) and neutrophil cluster of differentiated lymphoid cells 64 (CD64) surface expressions in identifying myocardial damage in children with pneumonia.
Methods: A total of 177 children with pneumonia admitted to the Shandong Provincial Third Hospital from January 2019 to January 2021 were selected as the study subjects. The children were divided into two groups: the control group (CG) consisted of 91 children with simple pneumonia, and the study group (SG) consisted of 86 children with pneumonia complicated with myocardial damage. This study measured the levels of serum GDF-15 and cardiac troponin I (cTnI, a cardiac damage marker) using enzyme-linked immunosorbent assay and the expression of neutrophil surface CD64 by flow cytometry. The relationship among GDF-15, CD64 and cTnI was analyzed using the Pearson method. The diagnostic value of GDF-15 and CD64 expression on myocardial damage was analyzed by drawing the receiver operating characteristic (ROC) curve.
Results: Compared to the CG, the SG had significantly higher values for heat duration, peak heat, chest X-ray results, hospitalization time, course of disease, serum cTnI, GDF-15, CD64, erythrocyte sedimentation rate (ESR), lactate dehydrogenase (LDH), C-reactive protein (CRP), and red blood cell distribution width (RDW) (p < 0.05). Correlation analysis showed that serum GDF-15 and CD64 were positively correlated with cTnI in children with pneumonia and myocardial damage (p < 0.05). Taking the presence or absence of myocardial damage in all children as the dependent variable, and serum levels of GDF-15 and CD64 as independent variables, regression analysis revealed that heat duration >5 d, course of disease, serum GDF-15 and CD64 were independent influencing factors of myocardial damage in children with pneumonia (p < 0.05). Receiver operating characteristic (ROC) curve analysis showed that the course of disease, serum GDF-15 and CD64 had clinical value in predicting myocardial damage, and combined detection of serum GDF-15 and CD64 had a higher diagnostic value for myocardial damage (p < 0.05).
Conclusions: The serum levels of GDF-15 and CD64 in children with pneumonia are positively correlated with cTnI protein level, which effectively predicts the occurrence of myocardial damage. Their combination detection has clinical reference value for the diagnosis of pneumonia combined with myocardial damage in children.
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Background: Cervical cancer (CC) is a common malignant tumor in women, ranking fourth in terms of both incidence and mortality. It is often diagnosed in the intermediate or advanced stages and has an extremely low complete cure rate. Human umbilical cord mesenchymal stem cells (hUCMSCs) have been found to have anti-tumor potential. Therefore, we aimed to investigate the role of hUCMSCs in the occurrence and development of CC and the underlying mechanisms.
Methods: Initially, the in vitro effects of mesenchymal stem cells (MSCs) supernatant and the MSCs + HeLa cells co-culture on the viability, migration, invasion, and apoptosis of HeLa cells were determined. A CC mouse model was successfully established. Then, the model mouse was administered with MSCs to assess the role of MSCs on the growth of CC tumors. Furthermore, the expression levels of proteins associated with the Wnt/β-Catenin/c-Myc signaling pathway were evaluated in tumor tissues using Western blot analysis. Additionally, the levels of inflammatory cytokines were measured in peripheral blood samples using their respective enzyme-linked immunosorbent assay (ELISA) detection kits.
Results: Through in vitro experimental settings, it was found that MSCs supernatant significantly suppressed the viability and metastasis of HeLa cells while inducing the apoptosis of HeLa cells compared with HeLa cells cultured alone (p < 0.05). However, when HeLa cells were co-cultured with MSCs, there was no significant inhibitory or promoting effect on HeLa cells. Furthermore, in the in vivo experiments, compared to the control group, the injection of MSCs significantly suppressed the growth of CC cells in vivo, induced apoptosis in these cells, suppressed the expression of β-Catenin and c-Myc proteins, increased the expression of glycogen synthase kinase-3β (GSK-3β), upregulated the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ (IFN-γ), and downregulated the level of transforming growth factor-β (TGF-β) (p < 0.05).
Conclusion: hUCMSCs can significantly inhibit the occurrence and development of CC, concurrently suppress the Wnt/β-Catenin/c-Myc signaling pathway, and subsequently regulate the secretion of inflammatory cytokines.
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Background: Immune cell infiltration plays an important role in the occurrence, development, and prognosis of cancer. The purpose of this investigation was to explore the relationship between immunoscore and prognosis, and to build a clinical nomogram for predicting survival in hepatocellular carcinoma (HCC) patients.
Patients and Methods: A total of 368 subjects were gathered in this study from the Cancer Genome Atlas, and the immunoscore was determined using the ESTIMATE (Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data) model. To identify prognostic factors, a Cox proportional hazards regression model was used, and multivariate analysis was used to develop the nomogram. The predictive accuracy and discriminating ability of model were verified using concordance indices (C-indices), calibration, Kaplan-Meier analysis and receiver operating characteristic (ROC).
Results: We divided the patients into three subgroups based on their immunoscore: low, medium, and high. A higher immunoscore was associated with significantly better overall survival (OS) and disease-free survival (DFS) than one with a lower immunoscore (hazard ratios (HR) with 95% confidence intervals (CI): 0.50 (0.27–0.92) and 0.39 (0.19–0.82)). Prediction using C-indices for OS was 0.66 (95% CI: 0.61–0.72) and for DFS was 0.74 (95% CI: 0.68–0.80). Nomogram predictions and actual observations showed a significant degree of agreement by ROC and calibration curve for OS and DFS probabilities at 3 and 5 years.
Conclusions: HCC patients with high immunoscore have significantly improved OS and DFS. The immunoscore can be considered as a prognostic marker in HCC patients. Moreover, our nomograms may help establish a greater degree of accuracy in predicting prognosis.
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Background: Nasopharyngeal carcinoma (NPC) is a highly malignant head and neck tumor, with most patients having lymph node metastasis at the time of diagnosis. Studies have shown that forkhead box protein O-1 (FOXO-1) and nuclear factor kappa-B (NF-κB) are associated with resistance to radiotherapy and chemotherapy in NPC. Baicalein can play an anti-inflammatory role by negatively regulating the activation of NF-κB and FOXO. The aim of this study was to investigate the effect of baicalein on the FOXO-1/NF-κB pathway in NPC.
Methods: The human NPC cell line CNE-2 was treated with baicalein at low (20 μmol/L), medium (40 μmol/L) and high (80 μmol/L) concentrations. The effects of baicalein on cell proliferation, apoptosis and invasion were detected by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry and transwell assay. The expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Cleaved-caspase 3, FOXO-1, p-FOXO-1 and NF-κB in each group were evaluated by Western blot (WB) analysis. The animal model of NPC was established and treated with baicalein (50 mg/kg), the weight and volume of tumor were measured. Histopathological changes were detected by hematoxylin-eosin (HE) staining. The expressions of FOXO-1, p-FOXO-1 and NF-κB were detected by WB.
Results: Compared with the control group, the proliferation and invasive ability of CNE-2 decreased while the rate of cell apoptosis increased with the increase in the concentration of baicalein (p < 0.05). Compared with the control group, as the concentration of baicalein increased, the expression of TNF-α, IL-1β, IL-6, Bcl-2, p-FOXO-1 and NF-κB decreased while the expression of Bax and Cleaved-caspase 3 increased (p < 0.05). There was no significant change in the expression of FOXO-1 in each group. In the animal model of NPC, tumor in the control group grew rapidly and the tumor cells were closely arranged, whereas in the baicalein treated group, the tumor grew slowly and the cells were loosely arranged. There was no significant difference in the expression of FOXO-1 between the control and baicalein group (p > 0.05). However, the expressions of p-FOXO-1 and NF-κB were lower in the baicalein group compared to the control group (p < 0.05).
Conclusion: Baicalein exerts anti-tumor effects in NPC by blocking the activation of the FOXO-1/NF-κB pathway, inhibiting the proliferation and invasion of tumor cells, up-regulating the level of cell apoptosis, and down-regulating the expression of inflammatory factors.
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Background: Uterine Corpus Endometrial Carcinoma (UCEC) ranks second among most common gynecological tumors, and the overall treatment situation is inadequate. In-depth study of tumorigenesis mechanism and the discovery of effective therapeutic drugs are an urgent problem to be solved. Our study mainly focused on the construction of competing endogenous RNA (ceRNA) network and the exploration of potential treatment strategies of UCEC.
Methods: 35 normal and 543 endometrial cancer samples had RNA expression profile extracted from The Cancer Genome Atlas (TCGA) GDC data portal. DEGs of LncRNAs (DELncRNAs), DEGs of miRNAs (DEmiRNAs) and DEGs of mRNAs (DEmRNAs) were calculated by R software (version 4.2.1). The function of DEmRNAs was analyzed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Protein-protein interaction (PPI) network and long non-coding RNA (LncRNA)-microRNA (miRNA)-mRNA regulation network were constructed. Further evaluation included venn analysis for vital protein and prediction of potential drugs were based on Connectivity Map (CMap). Lastly, we verified the cyclin B1 (CCNB1) as a potential therapeutic target with wet experiment (western blotting, clonogenic assay and flow cytometry).
Results: Our results revealed that 1218 DELncRNAs, 187 DEmiRNAs and 1897 DEmRNAs were identified by comparing UCEC tissue with normal controls. A total of 106 DELncRNAs, 27 DEmiRNAs and 56 DEmRNAs were included to construct a ceRNA network. GO analysis revealed that upregulated mRNAs were involved in nuclear division process. CCNB1 was included in both hub genes of PPI network and ceRNA network, which might be a powerful potential therapeutic target. We predicted 10 potential compounds based on ceRNA network and 49 potential compounds based on DEmRNAs. Aminopurvalanol-a, purvalanol-a and AZD-8055 were candidate compounds most likely to have therapeutic effect. Based on the TCGA database, we successfully constructed a PPI network and ceRNA network. Downregulation of CCNB1 markedly suppressed the proliferation ability and promoted the apoptosis of the UCEC cell lines (p < 0.001).
Conclusions: We demonstrated that CCNB1, a gene involved in multiple bioinformatic models, is considered to be a potential therapeutic target for endometrial cancer. Aminopurvalanol-a, purvalanol-a and AZD-8055 are the three most potential therapeutic drugs for UCEC.
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Background: A thorough diagnosis of liposarcoma is essential to develop an optimal therapy. This study aimed to identify differentiation-related biomarkers in liposarcoma.
Methods: Expression profiling data were downloaded from the Gene Expression Omnibus (GEO) database. Modules correlated with dedifferentiated liposarcoma were identified using weighted gene co-expression network analysis (WGCNA). Differentially-expressed genes were identified utilizing the limma R package. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted with the clusterProfiler R package. Hub genes were identified by least absolute shrinkage and selection operator (LASSO) analysis. Survival analysis was performed using survival and survminer R packages.
Results: The brown module was the most positively correlated module with dedifferentiated liposarcoma, while the turquoise module exhibited the strongest negative correlation with dedifferentiated liposarcoma. Forty-nine upregulated common genes were found by intersecting the upregulated differentially-expressed genes with the co-expressed genes in the brown module, and 177 downregulated common genes were found by intersecting the downregulated differentially-expressed genes with the co-expressed genes in the turquoise module. GO and KEGG analyses revealed that upregulated common genes were abundant in cell division and tumor-related pathways, while downregulated common genes were involved in cellular metabolism and metabolism-related pathways. ADIPOQ, UBE2C, and PRC1 were screened out as biomarkers which might distinguish dedifferentiated and well-differentiated liposarcoma. Dedifferentiated liposarcoma patients with low ADIPOQ levels displayed a significantly shorter distant recurrence-free survival than those with high ADIPOQ levels.
Conclusion: ADIPOQ, UBE2C, and PRC1 are potential differentiation-related biomarkers in liposarcoma tissues. ADIPOQ has the potential to be a novel prognostic biomarker for patients with dedifferentiated liposarcoma.
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Background: High mobility group box 1 (HMGB1) participates in regulating autophagy and cell apoptosis in the cytoplasm and plays a role in various kidney diseases. However, no study has focused on the role of the HMGB1/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB, also called AKT) signaling pathway in kidney stone formation. The aim of this study was to elucidate the role of HMGB1 via the PI3K/AKT axis in calcium oxalate monohydrate (COM)-induced kidney stone formation, which might lead to novel research targets for the treatment of kidney stones.
Methods: The human kidney-2 (HK-2) cell line was cultured and treated with si-HMGB1, si-negative control (NC), or PI3K agonist 740 Y-P (1 μM). In vitro kidney stone models were established using COM and calcium oxalate (CaOx) crystals. HMGB1, AKT and PI3K levels in each group of cells were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. Flow cytometry was utilized to measure the rate of cellular apoptosis.
Results: CaOx crystal adhesion was enhanced on the surface of the HK-2 cells stimulated by COM and CaOx, and HMGB1 expression was upregulated. The proportion of apoptotic cells was elevated in HK-2 cells after stimulation with CaOx and COM. Knockdown of HMGB1 inhibited CaOx crystal-cell adhesion and reduced the apoptosis rate in COM-treated HK-2 cells. Activation of the PI3K/AKT pathway counteracted the impact of HMGB1 knockdown on the adhesion of CaOx crystals to cells.
Conclusions: Inhibiting the HMGB1/PI3K/AKT axis could reduce the proportion of apoptosis and improve crystal-cell adherence.
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Background: Brain ischemia-reperfusion (IR) injury leads to reactive astrogliosis (the reaction of astrocytes) and the disruption of the blood-brain barrier (BBB) integrity, which are closely related to IR-induced neuronal damage or death (loss) in vulnerable brain regions. The aim of this study was to investigate and compare differences between mild and severe IR injury in neuronal death, reactive astrogliosis, and BBB leakage in the striatum using a gerbil model of transient forebrain ischemia.
Methods: To examine mild and severe IR-induced neuronal death and reactive astrogliosis, seven gerbils at each time point in each group were allocated. The gerbils received 5-min (for mild IR) and 15-min (for severe IR) ischemia by bilateral common carotid artery occlusion. IR-induced modifications in motor behavior were examined through tests conducted for spontaneous motor activity and motor coordination. Histopathological changes in striatal cells were examined through histochemical staining. Additionally, immunohistochemistry and double immunofluorescence were performed to study reactive astrogliosis and BBB leakage following IR.
Results: IR-induced neuronal death (loss) was observed in the striatum. Neuronal loss was more severe in gerbils with 15-min ischemia than in gerbils with 5-min ischemia. More abundant neurons died in the lateral field than in the medial field of the striatum. Reactive astrogliosis was more apparent in the lateral field than in the medial field, and the reaction was considerably more severe in the gerbils with 15-min ischemia than in those with 5-min ischemia. In particular, astrocyte endfeet around the blood vessels were more damaged (destroyed) and immunoglobulin G leakage in the parenchyma was more severe in the gerbils with severe IR than in those with mild IR.
Conclusions: IR-induced neuronal death in the gerbil striatum was more apparent in the lateral field than in the medial field of gerbils with severe IR than in those with mild IR, which might be closely related to the degree of reactive astrogliosis and BBB disruption and dependent on ischemic duration and the vulnerable region.
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Background: Nasopharyngeal carcinoma (NPC) is a common malignancy originated from the nasopharynx superior mucosal epithelium. Fibroblast growth factor 2 (FGF2) has been proved to play a vital role in various cancers. The aim of this study is to explore the role of FGF2 in NPC development.
Methods: RT-qPCR was applied to examine the mRNA level of FGF2 in NPC tissues and cells. The protein expression of FGF2, Ki-67, and mitochondrial apoptosis-related proteins [Bax, apoptotic protease activating factor-1 (Apaf-1), capase-7, caspase-9, and caspase-3] were determined by western blot. Cell proliferation, cell viability, the invasive ability and the apoptotic rate of NPC cells were measured using 5-Ethynyl-2′-deoxyuridine (EdU) assay, Cell Counting Kit-8 (CCK-8) assay, transwell invasion assay, and flow cytometry, respectively. In xenograft murine NPC model, the mice were divided into sh-NC and sh-FGF2 groups in the first animal experiment. Moreover, sh-NC, sh-FGF2, and sh-FGF2+sh-Apaf-1 groups were established in the second animal experiment. Tumor weight was calculated, and determination of Ca+ intracellular concentration in tumor tissues from mice was carried out using Fura-2 pentakis/acetoxymethyl (Fura-2/AM).
Results: The elevated FGF2 levels were verified in NPC tissues and cells (p < 0.05), and knockdown of FGF2 suppressed cell invasion and proliferation, and enhanced cell apoptosis in NPC cells (p < 0.05). The results from the in vivo experiment demonstrated that FGF2 knockdown dramatically hampered tumor growth (p < 0.05), and increased mitochondrial apoptosis (p < 0.05) and Ca+ intracellular concentration (p < 0.05) in mice. In addition, we found that Apaf-1 knockdown reversed the effects of FGF2 downregulation on tumor growth, mitochondrial apoptosis, and Ca+ intracellular concentration (p < 0.05) in NPC mice.
Conclusions: FGF2 knockdown exerted the inhibitory impacts on the development of NPC through regulating mitochondrial apoptosis.
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Background: The use of disease screening is limited in colorectal cancer (CRC) because of its unique location, and CRC patients are usually in advanced stages at the time of diagnosis. B-Raf proto-oncogene (BRAF)-V600E mutation is observed in tumors, and BRAF-V600E-mutant metastatic CRC is an aggressive subgroup. Droplet digital polymerase chain reaction (ddPCR) is a specific method for detecting circulating tumor DNA (ctDNA) mutations. This study used BRAF detection on cancer tissue as a standard for verifying the accuracy and heterogeneity of ddPCR in the plasma of CRC patients.
Methods: A total of 147 CRC patients were recruited and their clinical data were obtained. Plasma samples were collected from patients to determine cell-free DNA (cfDNA) concentration and ddPCR, and cancer tissues were used for immunohistochemistry (IHC) assay. The effect of BRAF-V600E on patient prognosis was analyzed by Kaplan-Meier curve.
Results: cfDNA was markedly increased in patients with CRC metastasis and was positively correlated with the size of non-metastatic tumors. The patients with BRAF-V600E mutation had a lower 5-year survival rate than those without the mutation, suggesting that BRAF-V600E mutation is a potential target for CRC humoral biopsy. Overall, there were some significant differences concerning the detection rates of IHC and ddPCR and the main distribution of ddPCR in multi-detected cases and stage III cases (p < 0.001).
Conclusion: This study shows the feasibility of using ddPCR to detect BRAF-V600E in the plasma of CRC patients and the differences between ddPCR and IHC, providing support for the establishment of a CRC detection technique.
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Background: Hypermethylation of the suppressor of cytokine signaling 3 (SOCS3) promoter has been found in most malignant tumors, but the correlation between acute lymphoblastic leukemia (ALL) and SOCS3 methylation has rarely been explored. Here, we mainly explored the role of SOCS3 methylation in ALL disease progression.
Methods: Children with ALL were divided into low-level and high-level groups according to the methylation level of the SOCS3 gene. The logistic regression model was used to analyze the relationship between SOCS3 gene methylation level and the prognosis of ALL. Kaplan-Meier analysis was employed to assess overall survival (OS) and cumulative incidence of relapse (CIR). Bisulfite sequencing polymerase chain reaction (PCR) and flow cytometry were used to detect the changes in methylation level of the SOCS3 gene, and analyze the cell cycle and apoptosis in ALL cell lines before and after treatment with a methylase inhibitor. The Cell Counting Kit-8 assay and western blotting were used to detect the changes in cell proliferation and the protein expression levels of SOCS3, cleaved caspase-3, and p53 upregulated modulator of apoptosis (PUMA).
Results: The methylation rate of the SOCS3 gene was 20.7% in the newly diagnosed group, 31.0% in the relapse group, 15.0% in the complete remission group, and almost nonexistent in the control group. Univariate Cox regression analysis showed that the high level of SOCS3 gene methylation was significantly associated with OS. The CIR in the high level group was significantly higher than that in the low-level group. After demethylation treatment, SOCS3 gene expression was significantly upregulated in ALL cells. Demethylation significantly inhibited the proliferation and arrested the cell cycle in the gap (G)0/G1 phase of ALL cells. SOCS3 demethylation may promote cell apoptosis by inducing the expression of apoptosis-related proteins such as cleaved caspase 3 and PUMA.
Conclusions: The results of our study indicate that the level of SOCS3 methylation differs in patients with different stages of ALL, and is related to the relapse rate. SOCS3 methylation can be used as one of the early diagnostic and prognostic indicators of ALL. In ALL cells, SOCS3 demethylation significantly inhibited cell proliferation and viability. Thus, regulating the methylation status of SOCS3 gene may provide a new approach for the treatment of ALL.
Clinical Trial Registration: This trial is registered with the Chinese Clinical Trial Registry, ChiCTR-IPR-14005706.
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Background: Asthma is a chronic disease of the airways which involves airway remodeling and inflammation. Stomatin like protein-2 (STOML2) regulates immunity and inflammation. This study aimed to elucidate the functional role of STOML2 in childhood asthma and the mechanisms by which it acts.
Materials and Methods: Airway smooth muscle cells (ASMCs) treated with platelet-derived growth factor-BB (PDGF-BB) were used to mimic airway hyper-reactivity in vitro. STOML2 expression in serum from patients with asthma and in PDGF-BB-induced ASMCs was evaluated by quantitative real-time PCR (qRT-PCR) and western blot assays. Cell proliferation was detected by cell counting kit-8 (CCK-8) and colony formation assays. Cell migration and invasion were measured by wound healing and transwell assays. Enzyme-linked immunosorbent assay (ELISA) was applied to analyze the expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in PDGF-BB-induced ASMCs. The levels of the nucleotide-binding oligomerization domain-like receptor (NLR) containing pyrin domain 3 (NLRP3) inflammasome were assessed by western blot assay.
Results: STOML2 was decreased in the serum of children with asthma and in PDGF-BB-induced ASMCs (p < 0.01). Overexpression of STOML2 reduced the proliferation, migration, and invasion of PDGF-BB-induced ASMCs (p < 0.01). Increased STOML2 suppressed the secretion of inflammatory cytokines in PDGF-BB-induced ASMCs (p < 0.05). In addition, STOML2 overexpression inhibited the activation of the NLRP3 inflammasome.
Conclusions: STOML2 regulated the inflammation and airway remodeling of ASMCs induced by PDGF-BB through inhibiting NLRP3, which might provide a novel direction for the treatment of asthma.
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Background: Myocardial ischemia-reperfusion injury (MIRI) is damage caused by restoring coronary blood supply after myocardial ischemia. Recombinant mouse methyltransferase-like protein 4 (METTL4), a N6-methyladenin (6mA) methylase-related protein, is associated with cardiovascular disease. However, the role and mechanism of METTL4 in MIRI have yet to be clarified. This study aims to explore whether METTL4 regulates MIRI by mitochondrial DNA 6mA modification.
Methods: We first constructed a MIRI rats' model and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model from mouse cardiomyocytes (HL-1). The 6mA level of mitochondrial DNA, METTL4 expression, and mitochondrial DNA (mtDNA) copy number were confirmed using Dot blot, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) in the rat and cell models, respectively. Functionally, cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8) and flow cytometry, respectively. In addition, the levels of adenosine triphosphate (ATP) and mitochondria-related genes were monitored by enzyme-linked immunosorbent assay (ELISA) and qRT-PCR.
Results: Our results showed that mitochondrial DNA 6mA and METTL4 levels were higher in MIRI model rats and OGD/R HL-1 model cells (p < 0.001). Then, we showed that knockdown of METTL4 induces proliferation and attenuates apoptosis of OGD/R HL-1 cells (p < 0.01, p < 0.001). We also found that METTL4 silencing reduces mitochondrial DNA 6mA modification and increases mitochondrial DNA (mtDNA) copy in OGD/R HL-1 cells (p < 0.001). The results indicated that silencing METTL4 reduces ATP and increases the levels of mitochondria-related genes in OGD/R HL-1 cells (p < 0.01, p < 0.001).
Conclusions: METTL4 silencing reduces mitochondrial DNA 6mA modification, increases mitochondrial DNA copy number, improves mitochondrial dysfunction, and inhibits apoptosis. Therefore, inhibition of METTL4 might be a therapeutic strategy for MIRI.
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Backgrounds: Mesenchymal stem cells-extracted Exosomes (Ex) have now been proposed as a novel approach for neurologic diseases. We aimed to explore the potential neuroprotective role of Ex extracted from adipose tissue stem cells in a Rotenone (Rot) Parkinsonian rat model.
Methods: For this work, thirty-six rats were allocated into 3 groups: control, Rotenone (1.5 mg/kg/day intra-peritoneal injection) and Rot + Ex (single intra-venous injection of 800 μg protein suspended in 1 mL phosphate buffered saline in the rat tail vein). Behavioral and motor assessment of the rats at the different study groups was evaluated using catalepsy test, rotarod test and open field test. The Enzyme-linked immunosorbent assay (ELISA) study included assessment of the Substantia Nigra Pars Compacta (SNPc) & striatum levels of catalase, Nuclear factor-erythroid 2-related factor 2 (Nrf2), caspase-3 and Tumor protein 53 (P53). An immunohistochemical study was performed for evaluating the expression of Tyrosine Hydroxylase (TH), α-synuclein, Ionized calcium-binding adapter molecule 1 (Iba-1), Microtubule-associated protein 1A/1B-light chain 3 (LC3), P62 and Glial fibrillary acidic protein (GFAP). The expression of Micro-RNA 7 and Micro-RNA 21 was also measured.
Results: Ex significantly (p < 0.05) improved the Rot-induced deterioration in motor functions. Ex also modulated SNPc & striatum levels of the oxidative stress markers catalase and Nrf2 and reduced the apoptotic proteins caspase-3 and P53. Ex also up-regulated microRNA miRNA-7 and down-regulated miRNA-21 expression. They also ameliorated the Rot-induced histopathological nigrostriatal degeneration, alleviated α-synuclein deposition and restored dopaminergic neurons in SNPc & striatum as seen by TH immunostaining. In addition, Ex also mitigated Rot-induced microglial & astrocytic activation and modulated the autophagy markers LC3 and P62.
Conclusion: Ex treatment exhibited neuroprotective potential against Rotenone-induced nigrostriatal neurodegeneration and synucleinopathy, presumably through anti-apoptosis, autophagy modulation and amelioration of oxidative stress.
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Background: Lung adenocarcinoma (LUAD) is a form of non-small cell lung cancer. Patients develop drug resistance during long-term treatment, which is associated with T790M mutations. This study aimed to assess the correlation between T790M mutation in advanced LUAD and clinical features after epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance, and its effect on median progressive free survival (mPFS) and median overall survival (mOS).
Methods: A total of 164 patients diagnosed with advanced LUAD who developed resistance to EGFR-TKI therapy were recruited in this study. These patients received EGFR-TKI therapy at the Zhejiang Rongjun Hospital, China from May 2020 to March 2022. Based on the presence or absence of the T790M mutation, they were divided into two groups: the T790M (–) (n = 70) and T790M (+) (n = 94). A droplet digital polymerase chain reaction (ddPCR) was performed, and subsequently, the EGFR mutation was evaluated using the ∆Ct value (Ct mutation-CT external control). The correlation between T790M mutation and clinical characteristics following EGFR-TKI resistance was determined. Moreover, the impact of T790M mutation on prognosis, as well as on median progressive free survival (mPFS) and median overall survival (mOS), was also observed.
Results: The carcinoembryonic antigen (CEA) levels, history of smoking, Eastern Cooperative Oncology Group (ECOG) score and the pattern of disease progression after developing drug resistance were identified as the main risk factors for positive T790M mutation. Under 50% probability of survival, the patients with negative T790M mutations had mPFS of 14.0 months and mOS of 21.0 months, whereas patients with positive T790M mutations exhibited mPFS of 38.0 months and mOS of 41.0 months. There were statistically significant mPFS and mOS in patients with T790M-positive mutations compared to the patients with T790M-negative mutations (p < 0.001).
Conclusion: The progression of the disease, smoking history, ECOG score and the level of CEA were independent risk factors for T790M mutation in patients with advanced LUAD. Furthermore, these factors also exhibited an impact on both mPFS and mOS of the patients.
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Background: BH3 interacting domain death agonist (BH3) proteins are key mediators of autophagy. B-cell lymphoma-2 (Bcl-2) family proteins inhibit autophagy by binding to Beclin-1. Studies have shown that blocking the binding of Beclin-1 and Bcl-2 with simulated BH3 can promote autophagy, and this strategy has been applied in the treatment of malignant tumor diseases. In this study, we screened specific RNA aptamers to block the binding of Beclin-1 and Bcl-2 by the systematic evolution of ligands by exponential enrichment (SELEX) method to promote autophagy, which provides a new therapeutic approach for disease research.
Methods: Recombinant BH3 protein was obtained and validated using a prokaryotic expression system. Recombinant BH3 protein was immobilized on carboxyl-conjugated magnetic beads, incubated with RNA libraries in SHMCK buffer under different conditions for 16 rounds, and then screened for RNA fittings that bind specifically to BH3 proteins. After screening, the sequence and secondary structure of the adaptors were analyzed.
Results: We obtained recombinant BH3 protein of roughly 35KD under the optimized conditions. The main body of RNA aptamers can be divided into two families according to the homology tree analysis after 16 rounds of screening by magnetic bead coupling of target proteins and RNA aptamers. The homology of the two families is 77%, and the secondary structures of the two families are dominated by combinations of multiple stem-loops of different sizes, so it is assumed that aptamer bodies may bind to BH3 proteins through stem-loop topologies. We selected ligands #1 and #7 from both families to further demonstrate the affinities between the ligands and the proteins. According to affinity tests, the No. 1 RNA ligand and the BH3 protein exhibited substantial interaction (p < 0.05).
Conclusion: In the study, we developed an innovative RNA aptamers screening method using carboxylated magnetic beads in SELEX technology to screen RNA aptamers specifically binding to BH3 protein. We determined the affinity of RNA aptamers to the BH3 protein using the enzyme-linked immunosorbent assay (ELISA) principle. This study broadens the scope of protein targets that may be screened using SELEX technology and offers a novel technique for determining the binding affinity of RNA aptamers and targets.
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Background: Gefitinib is a first-line treatment option for advanced non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations. However, its effectiveness is limited due to unknown resistance mechanisms. The emergence of long non-coding RNAs (lncRNAs) has unveiled new insights into EGFR-tyrosine kinase inhibitor resistance. This study aimed to investigate the effects of LINC00968 and LINC00511 on NSCLC cells treated with gefitinib.
Methods: We conducted high-throughput sequencing and enrichment analysis to screen potential target lncRNAs, and the expression levels of these lncRNAs were validated in cells using quantitative real-time PCR (qRT-PCR). The effects of LINC00968 overexpression or LINC00511 knockdown in gefitinib-treated cells on cell viability, proliferation, invasion, and apoptosis were determined using the Cell Counting Kit-8, clonogenesis, transwell, flow cytometry, and immunofluorescence. Additionally, western blotting was performed to detect alterations in the expression of drug-resistant proteins.
Results: Transcriptome sequencing and verification in cell lines revealed that LINC00968 exhibited low expression levels in NSCLC cells, while LINC00511 exhibited high expression levels (p < 0.05). Overexpression of LINC00968 and knockdown of LINC00511 enhanced gefitinib-induced proliferation inhibition, invasion inhibition, and apoptosis in NSCLC cell lines (p < 0.05). In the si-LINC00511 and LINC00968 group, drug resistance-related genes, including multidrug resistance-associated protein 1, P-glycoprotein, low-density lipoprotein receptor-related protein 1, and hypoxia-inducible factor-1α, were upregulated. Furthermore, knockdown of LINC00511 and overexpression of LINC00968 elevated N-Cadherin expression while inhibiting E-Cadherin (p < 0.05).
Conclusion: Our findings suggest that LINC00968 and LINC00511 regulate the sensitivity of NSCLC cells to gefitinib, potentially representing promising targets for improving the clinical efficacy of gefitinib.
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Background: Sepsis and mild cognitive impairment (MCI) are two distinct clinical conditions that often interact in their progression. However, the potential shared pathological mechanisms of these two diseases are yet to be explored. This study aims to investigate differentially expressed genes (DEGs) in sepsis and cognitive dysfunction using biological data methods. The goal is to delve into their shared genetic characteristics and underlying molecular mechanisms, providing innovative ideas for the treatment of sepsis in combination with MCI.
Methods: Gene expression profiles for sepsis (GSE131761) and cognitive dysfunction (GSE63060) were accessed from the gene expression omnibus (GEO) database. The data were analyzed using R software (version 4.3.2, R Core Team, Vienna, Austria) to identify DEGs between sepsis and MCI. Furthermore, annotation of the biological pathways was performed using the gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis. Additionally, the STRING database and Cytoscape software were employed to construct a protein-protein interaction (PPI) network and screen the key genes. Finally, the hub genes were validated using external datasets (GSE46955, GSE63061, GSE137342).
Results: It was found that 36 DEGs were shared between sepsis and MCI. Furthermore, biological processes were significantly enriched within key pathways such as cellular respiration, oxidative phosphorylation, aerobic respiration, and ATP synthesis coupled electron transport. In terms of cellular components, these DEGs were enriched in the inner mitochondrial membrane protein complex, respiratory chain complex, mitochondrial respirasome, and mitochondrial protein-containing complex. Regarding molecular function, they were enriched in structural constituent of ribosome, Nicotinamide adenine dinucleotide (NADH) dehydrogenase activity, and oxidoreductase activity. Furthermore, the KEGG analysis enriched these genes in Parkinson's disease, oxidative phosphorylation, thermogenesis, neurodegeneration, and prion disease. The PPI network analysis revealed 10 hub genes, including cytochrome c oxidase subunit 7C (COX7C), NADH:ubiquinone oxidoreductase subunit B3 (NDUFB3), ubiquinol-cytochrome c reductase complex III subunit VII (UQCRQ), cytochrome c oxidase subunit 7A2 (COX7A2), NDUFA4 mitochondrial complex associated (NDUFA4), ATP synthase peripheral stalk subunit F6 (ATP5PF), NADH:ubiquinone oxidoreductase subunit A1 (NDUFA1), SRA stem-loop interacting RNA binding protein (SLIRP), cytochrome c oxidase copper chaperone COX17 (COX17) and small nuclear ribonucleoprotein polypeptide G (SNRPG). The validation results with external datasets showed that reduced efficiency of oxidative phosphorylation was a common mechanism of sepsis and MCI.
Conclusion: This study identified pathological mechanisms between sepsis and MCI. Moreover, this study identified key genes and their regulatory pathways, and their core mechanisms might be linked to oxidative phosphorylation, providing a theoretical basis for understanding their genetic linkage, and offering innovative ideas for future research.
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Background: Sepsis-associated encephalopathy (SAE) is characterized by a diffuse or multifactorial cerebral dysfunction caused by sepsis without central nervous system (CNS) infection. Gut dysbacteriosis plays a critical role in the development of SAE. However, the underlying mechanisms of gut microbiota in the progression of SAE remain unclear. The current study investigated how gut microbiota and interleukin (IL)-17 affect SAE rat models using fecal microbiota transplantation (FMT) and Y320 (IL-17 inhibitor). Additionally, we examined the link between gut microbiota and IL-17 in SAE.
Methods: The cecal ligation perforation (CLP) induced sepsis rat model was established and treated with fecal microbiota transplantation (FMT) or IL-17 inhibitor (Y320). 16S rDNA genes isolated from rat Fecal Flora were analyzed using high-throughput sequencing. Simple non-postural somatomotor functions were assessed using the pinna reflex and corneal reflex tests. The simple postural somatomotor function was evaluated using the tail flexion reflex test. However, complex postural somatomotor functions were evaluated using righting reflex and escape response tests. Furthermore, the Blood-Brain Barrier Permeability was assessed. The whole blood monocytes were isolated, and the T cell proportion was evaluated using flow cytometry. The levels of inflammatory cytokines (IL-7, IL-1β, tumor necrosis factor-α (TNF-α), and IL-10) in hippocampus tissue were determined using their respective enzyme-linked immunosorbent assay (ELISA) kits. Additionally, brain injury and apoptosis rate were evaluated using Hematoxylin-Eosin staining and TdT-mediated dUT PNick-End Labeling (TUNEL) assay. Protein expression levels were assessed using western blot analysis and immunofluorescence assay.
Results: The brain tissue injury, expressions of inflammatory factors, levels of apoptosis, and autophagy in the hippocampus of the SAE group were higher compared to the Sham group. Furthermore, in both the SAE+FMT and SAE+Y320 groups, brain damage was improved, inflammatory factors in the hippocampus were reduced, and apoptosis and autophagy in the hippocampus were downregulated compared to the SAE+NC group (p < 0.05). Additionally, the imbalance of T helper cell 17 (Th17)/Treg was regulated to suppress IL-17 in the hippocampus following FMT or Y320 treatment (p < 0.01).
Conclusions: Reducing IL-17 levels by restoring gut microecological homeostasis might be a potential strategy to prevent and treat SAE.
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Background: Diabetic cardiomyopathy (DCM) is a condition that is characterized by impairment in both systolic and diastolic cardiac functions. Inflammation, apoptosis, interstitial fibrosis, and oxidative stress are the primary ultrastructural alterations in DCM. LCZ696 which has been reported to protect against DCM, contains two molecular components, is a novel anti-heart failure medication that combines a neprilysin inhibitor and an angiotensin receptor blocker. This study aimed to examine the effect of LCZ696 on heart function in a rat model of diabetic heart disease, with a particular emphasis on the Protein Kinase B (Akt)/Forkhead box O1 (FoxO1) signaling pathway.
Methods: We induced diabetes in male Wistar rats by streptozotocin injection and high-fat diet feeding. The rats were randomly divided into four groups: control group, diabetic group (DM), low-dose LCZ696 treatment group (LCL), and high-dose LCZ696 treatment group (LCH). LCZ696 was administered daily at a dose of 30 mg/kg or 60 mg/kg for 16 weeks. Serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were measured and cardiac function was assessed by echocardiography. Biochemical analyses included blood glucose, serum triglycerides, cholesterol, creatinine, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and other inflammatory cytokines. Myocardial apoptosis and fibrosis were assessed by histopathologic analysis. Western blot analysis was performed to determine the expression of relevant proteins and factors in the Akt/FoxO1 signaling pathway.
Results: Compared with the control group, the DM group showed significant cardiac dysfunction (left ventricular ejection fraction (LVEF) 51.7 ± 3.3 vs. 81.2 ± 1.8, p < 0.05), significantly increased levels of caspase-3 protein associated with myocardial apoptosis (p < 0.05), significantly decreased levels of B-cell lymphoma 2 (Bcl-2) expression (p < 0.05), and alterations in cardiac structure. Treatment with LCZ696 effectively improved cardiac function in diabetes mellitus rats (LVEF improved from 51.7 ± 3.3 in DM group to 62.5 ± 4.1 in DM+LCL group, p < 0.05; and to 75.6 ± 4.8 in DM+LCH group, p < 0.05), significantly downregulated caspase-3 protein expression related to myocardial apoptosis (p < 0.05), and significantly upregulated Bcl-2 expression (p < 0.05), with a more pronounced effect observed with high-dose LCZ696 treatment. Furthermore, LCZ696 treatment activated the Akt/FoxO1 signaling pathway, and inhibited myocardial inflammation (downregulated TNF-α and IL-6 expressions, p < 0.05).
Conclusions: LCZ696 can protect the myocardium from damage and ameliorate cardiac function in a rat model of diabetic heart disease. These findings suggest that LCZ696 could be a potential therapeutic agent for DCM.
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Background: Eimeria columbae (E. columbae) is a disease that threatens domestic pigeon health and productivity due to resistance and the side effects of treatments. Therefore, there is a pressing need to explore alternative therapies to manage E. columbae infection. This study investigates the possibility of bile application as an alternative treatment to assess its anticoccidial efficacy and determine the concentration with the most substantial effect on oocysts and sporozoites.
Methods: We subjected samples of oocysts to several concentrations of bile in a solution containing 2.5% potassium dichromate at 12.5%, 25%, 50%, 75%, and 100% to assay oocyst vitality during the course of 24, 48, 72, and 96 h. In addition, we used 125, 250, 500, 750, and 1000 μg/mL concentrations to determine anti-sporozoite efficacy after 12 and 24 h.
Results: The infrared analysis of the bile showed many bioactive components associated with plant secondary metabolites. Furthermore, the phenol and flavonoid totals were estimated. The results showed statistically significant differences at p < 0.05 in the 100% and 75% concentrations whereby sheep bile liquid could inhibit E. columbae oocysts by approximately 94% and 81%, respectively. In addition, bile also demonstrated maximum inhibition of E. columbae sporozoite vitality (92%) at a dose of 1000 g/mL and minimum inhibition of 8% at a concentration of 125 g/mL. The sporulation percentage was highly negatively correlated with the exposure time (R = –0.97, p < 0.0001; R = –0.80, and p < 0.001) at 100% and 75% concentrations, respectively. Thus, inhibiting oocysts was found to prevent infection and enhance the birds' health, which contributes to improving the quality of the product, both quantitatively and qualitatively.
Conclusions: The overall results of the current study suggest that sheep bile possesses inhibitory properties for oocyst sporulation, meaning that it could be employed in coccidia management.
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Backgrounds: Acute cerebral infarction significantly impacts the quality of life and prognostic outcomes for affected individuals. Inflammation is crucial in the pathological and physiological processes following acute cerebral infarction. Investigating the correlation of CXC chemokine ligand 13 (CXCL13), monocyte chemoattractant protein-1 (MCP-1), and CC chemokine ligand 11 (CCL11) with the severity and prognosis of acute cerebral infarction patients may contribute to the identification of new biomarkers and pathophysiological mechanism, thereby enhancing treatment and rehabilitation.
Methods: We enrolled 150 patients with acute cerebral infarction and 100 healthy controls. Patients were categorized into mild (National Institutes of Health Stroke Scale (NIHSS) score <7), moderate (7≤ NIHSS score <15), and severe (NIHSS score ≥15) subgroups based on their NIHSS scores. According to the modified Rankin scale (mRS) score, patients were further divided into good prognosis (mRS score ≤2) and poor prognosis (mRS score >2) subgroups. Serum concentrations of CXCL13, MCP-1, and CCL11 were compared. Multivariate logistic regression was used to analyze risk factors for moderate and severe neurological impairment and poor prognosis in acute cerebral infarction patients. Spearman rank correlation analysis was used to explore the correlation of CXCL13, MCP-1, and CCL11 with NIHSS and mRS scores.
Results: In the study group, concentrations of CXCL13, MCP-1, and CCL11 were significantly higher, with the severe subgroup > moderate subgroup > mild subgroup and poor prognosis subgroup > good prognosis subgroup, compared to the control group (p < 0.05). Elevated concentrations of CXCL13, MCP-1, and CCL11 emerged as independent risk factors for moderate and severe neurological impairment and poor prognosis in acute cerebral infarction patients (p < 0.05). Serum CXCL13, MCP-1, and CCL11 demonstrated positive correlations with NIHSS and mRS scores in acute cerebral infarction patients (p < 0.05).
Conclusion: CXCL13, MCP-1, and CCL11 likely play a role in the onset and progression of acute cerebral infarction. Their concentrations positively correlate with NIHSS and mRS scores, providing insights into the degree of neurological impairment and prognosis in affected patients.
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Background: Tyrosine kinase inhibitors (TKIs) have been used as first-line therapy drugs for the treatment of Philadelphia chromosome-positive (Ph+) leukemia, although some patients develop drug resistance. The present study investigates the role of glycogen synthase kinase 3 (GSK3) in Ph+ leukemia cells treated with TKIs.
Methods: K562 and Ku812 leukemia cells were treated with TKIs (imatinib or dasatinib) and GSK3 inhibitors (SB216763 and lithium chloride). The cell counting kit-8 (CCK-8) was employed to evaluate cell viability, while cell apoptosis was quantified by flow cytometry. The expression of apoptotic proteins was assessed using Western blot. The expressions of GSK3α and GSK3β in K562 cells were knocked down using lentivirus plasmids. TOP/FOP flash assay was performed to detect the transcriptional activity of Wnt/β-catenin.
Results: SB216763 and lithium chloride increased cell viability and decreased cell apoptosis induced by imatinib (IM) and dasatinib (DA) in K562 and Ku812 cells. Knockdown of GSK3α and GSK3β inhibited apoptosis and promoted the viability of K562 cells induced by TKIs. Knockdown of GSK3α and GSK3β also reversed the decrease in TOP/FOP ratio induced by IM and DA.
Conclusion: GSK3 promotes TKI-induced apoptosis in Ph+ leukemia cells, and may be related to the inhibition of the Wnt/β-catenin pathway. GSK3α and GSK3β were both involved in Ph+ leukemia, indicating that GSK3 is a tumor suppressor.
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Background: Recent studies have shown that endothelial progenitor cells (EPCs) and bone marrow stromal cells (BMSCs) may interact functionally through the secretion of factors or direct cell-to-cell contact. This interaction is crucial for regulating their migration, proliferation, and differentiation. Particularly during osteogenic differentiation, the Wnt/β-catenin signaling pathway has been proven to be an important regulatory mechanism. This study aimed to elucidate the mechanisms underlying the promotion of BMSCs differentiation by EPCs of bone marrow, providing new insights for the clinical treatment of osteonecrosis of the femoral head (ONFH).
Methods: Rabbit BMSCs and EPCs were isolated, and immunofluorescence staining was employed to confirm markers of both cell types. The experimental group involved the co-culture of EPCs and BMSCs, while the control group consisted of BMSCs cultured independently. Osteogenic differentiation was assessed by alkaline phosphatase and Alizarin Red staining. The expression levels of Bone Morphogenetic Protein 2 (BMP2), Osteocalcin (OCN), Osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and β-catenin were measured using Western blotting and qualitative reverse transcription polymerase chain reaction (qRT-PCR).
Results: Positive expression levels of differentiation 29 (CD29) and CD90 confirmed successful BMSC isolation, while kinase insert domain receptor (KDR) and CD31 positivity confirmed EPC isolation. The experimental group exhibited a significant increase in alkaline phosphatase activity and mineralized nodules compared the control. EPCs significantly upregulated the expression of osteogenic-associated proteins BMP2, OCN, OPN, and RUNX2 in BMSCs (p < 0.01). Notably, the gene and protein expression of Wnt/β-catenin signaling pathway-related molecules were upregulated in the experimental group compared to the control (p < 0.01). siRNA-mediated silencing of the β-catenin in BMSCs resulted in the downregulation of osteogenic-related genes (BMP2, OPN, OCN, and RUNX2) and reduced β-catenin expression (p < 0.001). Additionally, osteogenic-related proteins (BMP2, OCN, OPN, and RUNX2) were downregulated (p < 0.05, p < 0.01, p < 0.001), accompanied by reduced β-catenin synthesis (p < 0.01). Treatment with 4-Ethyl-5-methyl-5,6-dihydro-[1,3]dioxolo[4,5-j]phenanthridine (HLY78) reversed the inhibitory effect of siRNA on β-catenin, promoting BMSC osteogenic differentiation.
Conclusions: These findings emphasize the role of the EPC-BMSC interaction and the Wnt/β-catenin signaling pathway in enhancing osteogenic differentiation. Our study provides novel insights into the potential clinical use of EPCs for ONFH treatment.
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Background: Due to its antioxidant and anti-inflammatory effects, hydrogen-rich water has been used to reduce inflammatory responses in many inflammatory diseases. However, its efficacy in treating peri-implantitis remains unclear. This study aims to investigate the efficacy of hydrogen-rich water in reducing inflammatory responses in experimental peri-implantitis.
Methods: A ligature-induced experimental peri-implantitis model was established using three Beagle dogs (n = 24 implants). The implants were divided into two groups: a hydrogen-rich water-treated group (n = 12 implants), and a non-treated control group (n = 12 implants). The clinical indices, including the gingival index (GI), modified sulcus bleeding index (mSBI), and probing depth (PD), were examined. Hematoxylin and eosin (H&E) staining was used to assess the inflammatory cell infiltrate in the peri-implant tissue. Additionally, three host-derived pro-inflammatory cytokines, interleukin-1 (IL-1), interleukin-6 (IL-6), and matrix metalloproteinase-8 (MMP-8) were assessed using enzyme-linked immunosorbent assay (ELISA). Furthermore, microbial community profiles were analyzed using high-throughput sequencing (HTS) technology.
Results: In terms of the GI, the differences between the control and treated groups at 0, 2, and 4 weeks were not statistically significant (all p > 0.05). However, the GI value in the treated group at 6 weeks was significantly lower than that in the control group (p = 0.005). As for the PD, the differences between the control and treated groups at 0, 2, 4, and 6 weeks were not statistically significant (all p > 0.05). Regarding the mSBI, the difference between the control and treated groups at 0 week was not statistically significant (all p > 0.05), while the mSBI value in the treated group at 2, 4, and 6 weeks was significantly lower than that in the control group (p = 0.008, p = 0.005, p = 0.001). Compared to the non-treated group, the hydrogen-rich water-treated group showed a significantly lower number of inflammatory cells, lower pro-inflammatory cytokines, IL-1, IL-6, and MMP-8 levels (p < 0.05). Additionally, there was a significantly decreased relative abundance of pathogenic bacterial species including Porphyromonas gingivalis, Fusobacterium nucleatum, Fusibacter, and Fretibacterium (p < 0.05).
Conclusions: The use of hydrogen-rich water for treating peri-implantitis showed promise and effectiveness within the scope of this study.
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Objective: In women, breast cancer (BC) is the most common malignant tumor, accounting for 7–10% of the incidence of various malignant tumor throughout the body. The purpose of this study was to investigate the role of γδ1T cells-derived interleukin (IL)-17D in BC and its molecular mechanism.
Methods: The γδ1T cells were isolated from human BC tissues using magnetic beads and identified by flow cytometry. MDA-MB-231 cells or cells co-cultured with γδ1T cells were injected subcutaneously into nude mice at a dose of 5 × 106 cells/mouse to construct an animal model of BC. Enzyme-Linked Immunosorbent Assay (ELISA), Western blot, cell cloning, and scratch test were used to detect malignant biological behavior of BC cells. Observation of mammary carcinoma pathology in nude mice was conducted using hematoxylin-eosin (HE) staining.
Results: In in vitro cell experiments, co-immunoprecipitation demonstrated the presence of binding relationships between IL-17D and CD93 as well as between Src and Casitas B-spectrum lymphoma (Cbl). γδ1T-derived IL-17D targeted activation of the breast cancer CD93 receptor and promoted Src-mediated phosphorylation of Cbl, which in turn induced Rho family activation, ultimately promoting MDA-MB-231 cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT), enhancing the malignant biological behavior of BC cells. In in vivo experiments, γδ1T promoted an increase in tumor size, volume, and weight, as well as T-cell infiltration and ki-67 expression in cancerous tissues in nude mice, promoting EMT, and thus, malignant progression of BC.
Conclusions: γδ1T-derived IL-17D promotes BC progression through activation of the CD93/Cbl/Rho molecular axis.
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Background: Ovarian cancer (OC) is a highly malignant disease, and due to its high recurrence rate, high chemotherapy resistance, and lack of effective early diagnostic methods, effective targets for early screening, treatment, and prognosis prediction are necessary for research in this field. This study aimed to investigate the mechanism of periplakin (PPL) in malignant development of ovarian cancer and provide references for the treatment of ovarian cancer.
Methods: We performed a The Cancer Genome Atlas (TCGA) analysis of RNA seq data from clinical tumor sample, and analyzed PPL expression levels in tumors and adjacent tissues in Clinical Proteomic Tumor Analysis Consortium (CPTAC) data. We obtained information related to PPL and patient prognosis in clinical tumor samples of ovarian cancer from the TCGA database, including the expression level of PPL in various epithelial cell cancers and the correlation between PPL expression and patient survival. Then, we analyzed the relationship between PPL expression and ovarian cancer development. Also, we collected clinical tumor samples and used the enzyme-linked immunosorbent assay (ELISA) to detect the difference in PPL expression between tumor tissue and adjacent tissues. We constructed a stable knockdown SKOV3 ovarian cancer cell line with PPL. The western blot (WB) technique was used to detect the expression level of PPL and the protein kinase A (AKT) in the experimental group and control group after downregulation. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was utilized to detect the changes in the growth and proliferation of SKOV3 ovarian cancer cell lines in the control (shctrl) and experimental (shPPL#1, shPPL#2, and PPL Flag) groups after knocking down PPL. The control (BALB/c shctrl) and the experimental (BALB/c shPPL#1 and BALB/c shPPL#2) groups were used to detect the effect of knocking down PPL on the growth of ovarian cancer cells in situ transplanted into mouse models.
Results: Among various human tumors, the majority of people suffer from ovarian cancer. We found that the relative expression level of PPL gene in ovarian cancer is high. Analysis of CPTAC data showed an increase in PPL expression levels in ovarian cancer tissues compared to normal tissues. Knocking down PPL significantly affects the growth of ovarian cancer cells and inhibits the proliferation and migration of ovarian cancer cells. PPL knockdown significantly alters the phosphorylation level of AKT in the SKOV3 ovarian cancer cell line. The results of orthotopic transplantation were similar to those in cell lines. The tumor weight and volume in the experimental group were smaller than those in the control group. Knocking down PPL could reduce the p-AKT activity of transplanted mice.
Conclusions: PPL plays a crucial role in the proliferation and migration of ovarian cancer tumors. It regulates the phosphorylation of AKT and affects the proliferation and migration of ovarian cancer cells. Targeting PPL may serve as a new therapeutic target in ovarian cancer patients.
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Background: Hyperoxic acute lung injury (HALI) is a pathological condition characterized by lung damage resulting from the inhalation of high concentrations of oxygen. The incidence of this disease has been increasing annually in clinical practice. Therefore, in-depth research into the pathogenesis of HALI is of great significance. This study aims to reveal the underlying regulating mechanism of mouse double minute 2 (MDM2)/p53 on Type II Alveolar Epithelial Cells (AEC II) apoptosis in HALI through in vivo and in vitro experiments.
Methods: AEC II cells were isolated, purified, and cultured from healthy male Sprague Dawley (SD) rats. Subsequently, these cells were infected with an adenovirus expressing MDM2/p53 to establish a model of HALI. The proliferation activity of AEC II cells was assessed using the cell counting kit (CCK)-8 assay, and the expressions of MDM2, p53, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, and Caspase-9 mRNA were measured through real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Additionally, cell apoptosis was evaluated through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry (FCM). Arterial blood gas analysis was performed to measure the oxygenation index (OI) and respiratory index (RI). Early apoptosis was detected by measuring mitochondrial membrane potential (MMP), and expressions of MDM2, p53, Bcl-2, Bax, Caspase-3, and Caspase-9 proteins were examined through Western blot analysis. Finally, lung tissue pathology was assessed using the hematoxylin-eosin (HE) staining method.
Results: In H2O2-induced AEC II cells, an increase in apoptosis and a significant decrease in cell viability were observed, accompanied by a notable decrease in MDM2 expression and an evident increase in p53 expression (p < 0.05). Either over-expression of MDM2 or interference with p53 significantly reduced H2O2-induced AEC II apoptosis (p < 0.05), while interfering with MDM2 or over-expressing p53 significantly promoted H2O2-induced AEC II apoptosis (p < 0.05). Over-expression of MDM2 led to a significant reduction in the expressions of p53, Bax, Caspase-3, and Caspase-9, as well as a decrease in the RI and wet-to-dry weight ratio in the HALI model (p < 0.05). It also resulted in an increase in the OI and Bcl-2 expression in the HALI model (p < 0.05), alleviated the pathological state of lung tissue, and restored mitochondrial transmembrane potential in the HALI model (p < 0.05). The p53 molecular inhibitor, Pifithrin-α, significantly promoted the reduction of RI and wet-to-dry weight ratio and increased OI in the HALI model (p < 0.05). Moreover, the Pifithrin-α inhibitor was found to improve lung tissue structure in the HALI model.
Conclusion: MDM2 plays a dual role in preventing the apoptosis of AEC II and protecting lung tissue by inhibiting p53 expression.
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Background: Crush syndrome or what is also known as traumatic rhabdomyolysis, is the leading cause of death following extrication from structural collapse due to earthquakes. Rhabdomyolysis is one of the most common reasons for acute kidney injury (AKI). The present study was designed to investigate the potential curative effect of wheat grass (WG) in acute kidney injury induced by glycerol (rat model of crush syndrome).
Methods: Following a 24-hour period of water deprivation, male rats were randomly divided into 4 groups (6 rats each): The first group received an intramuscular (IM) injection of an equivalent volume of 0.9% saline into the hind limbs in divided dosages. The second group received IM injection of a single dose of 50% v/v glycerol in 0.9% saline (10 mL/kg), in equally divided doses to both hind limbs. The third group was injected a single dose of 50% v/v glycerol in 0.9% saline (10 mL/kg), into the hind limbs in divided dosages; then, the animals were administered WG (75 mg/kg, p.o.) twice per day for 3 successive days. The fourth group received only WG (75 mg/kg, p.o.) twice per day for 3 successive days. At the end of the experiments, animals were sacrificed; blood and kidneys were collected. Interleukin-6 (IL-6), interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), glutathione S-transferase (GST), and catalase (CAT) were measured using enzyme-linked immunosorbent assay (ELISA). Superoxide dismutase (SOD), serum creatinine (SCr), blood urea nitrogen (BUN), and creatine kinase (CK) were estimated using assay kits. Nuclear factor kappa B (NF-κB) was measured using immunohistochemical analysis. Using thiobarbituric acid reactive substances (TBARS), the renal level of malondialdehyde (MDA) was determined. Histopathological examination was also performed.
Results: The levels of BUN, SCr, the rhabdomyolysis marker CK, the oxidative damage marker MDA, and the inflammatory markers IL-6, IL-18, and NF-κB as well as the tubular injury markers KIM-1 and NGAL are increased in glycerol administration group. All these markers were significantly attenuated in those animals when treated with WG. Also, treatment with WG significantly improved GST, SOD, and CAT activities in glycerol-treated animals. In addition, histopathological changes induced by glycerol in renal tissue were highly improved in animals given WG.
Conclusions: The current findings demonstrate that WG has the ability to attenuate acute kidney injury secondary to rhabdomyolysis induced by glycerol in rats by modulating oxidative stress, as well as inflammatory and rhabdomyolysis markers.
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Background: The frequency of neoplastic disorders, pancreatic ductal adenocarcinoma (PDAC), is increasing annually. PDAC is an aggressive cancer and can be partly managed with systemic chemotherapy, as it is usually diagnosed at advanced stages. Unfortunately, only 23.8% PDAC patients are clinically sensitive to gemcitabine (GEM) treatment. Therefore, this study aimed to understand the etiology and pathobiology of chemoresistance in PDAC, deciphering the molecular mechanisms underlying PDAC progression.
Methods: The expression levels of two important genes were investigated in the clinical tissue's samples of gemcitabine-sensitive and gemcitabine-resistant PDAC patients. Furthermore, lentivirus was used to knock down and overexpress target genes, and RNA sequencing was utilized to further investigate the downstream regulators. Additionally, plate cloning, flow cytometry, and in vivo tumorigenicity were used to demonstrate the involvement of these key genes in gemcitabine resistance in PDAC. Eventually, the downstream regulators of gemcitabine resistance of PDAC was assessed using western blot (WB) and enzyme-linked immunosorbent assay (ELISA), respectively, aiming to evaluate the impacts of phosphatidylinositol 3-kinase and protein kinase B (PI3K/AKT) pathway-related proteins and Interleukin 6 (IL6) inflammatory factor.
Results: The expressions of monoacylglycerol lipase (MGLL) and Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) were higher in gemcitabine-resistant PDAC patients than in gemcitabine-sensitive PDAC patients (p < 0.05). In vitro experiments revealed that both overexpression and silencing of these genes altered the capacity to establish gemcitabine resistance (p < 0.05). RNA sequencing results indicated that these two genes combinedly regulate the expression of IL6 and demonstrated that they regulate drug resistance by activating the PI3K/AKT signaling pathway (p < 0.05). Moreover, in vivo experiments validated the involvement of MGLL and YWHAZ in improving gemcitabine resistance in pancreatic cancer (p < 0.05).
Conclusions: This study demonstrates that MGLL and YWHAZ co-regulate the resistance to gemcitabine via the IL6/PI3K/AKT axis in PDAC. Therefore, MGLL/YWHAZ is a promising therapeutic target for the treatment of PDAC.
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Background: Melatonin (MT) has been recognized for its neuroprotective effects. Studies have proved that polarization of microglia from a classic proinflammatory state (M1) to an anti-inflammatory state (M2) is a therapeutic approach for treating ischemic stroke (IS). Therefore, this study aims to investigate the potential mechanism of MT on microglia polarization after IS.
Methods: The rat model of middle cerebral artery occlusion (MCAO) was established according to the Longa wire embolization method. BV2 microglial IS model was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. After MT intervention, Quantitative Real-Time PCR (qRT-PCR) and western blot were used to analyze mitogen-activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) pathway-related proteins and polarization markers expression, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to measure the inflammatory factors. Hematoxylin-eosin (HE) and 2,3,5-triphenyl tetrazolium chloride (TTC) staining were used to observe the pathological changes and the volume of cerebral obstruction in rat brains, respectively. The markers of microglial polarization were evaluated by immunofluorescence staining. Cell Counting Kit-8 (CCK-8) and flow cytometry were used for the detection of viability and apoptosis.
Results: In vivo studies confirmed that the expression of MAPK/ERK pathway proteins (p-ERK1/ERK1 and p-ERK2/ERK2) and proinflammatory factors inducible nitric oxide synthase (iNOS), interleukin-1beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) upregulated, and the levels of anti-inflammatory factors macrophage mannose receptor (CD206) and interleukin-10 (IL-10) downregulated in MCAO rats. MT treatment significantly reduced the volume of cerebral infarction in MCAO rats, inhibited the increase of proinflammatory markers level, and increased anti-inflammatory markers level. In vitro studies confirmed that M1 polarization was reduced, but M2 polarization was increased in MT-treated BV2 microglia. In addition, MT reduced the expression of p-ERK1/ERK1 and p-ERK2/ERK2, increased cell proliferation activity, and restrained apoptosis. Meanwhile, the addition of MAPK/ERK pathway inhibitors produced similar results to MT treatment. Activation of the MAPK/ERK pathway weakened the effect of MT on M2 polarization of microglia and promoted IS brain injury.
Conclusions: MT alleviated IS-induced inflammation response, apoptosis, and M1 polarization and increased cell viability by inhibiting the activation of the MAPK/ERK pathway, thereby indicating that MT is a potential and novel therapeutic drug for IS.
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Background: Glioblastoma (GBM) are the most prevalent malignant brain tumors with extensive morphological and genetic heterogeneity. The objective of this study was to investigate the role of the oncogene Ribonuclease P/MRP Subunit P25 (RPP25) in GBM progression and its underlying molecular mechanisms.
Methods: Glioma cell lines were used to evaluate the effects of RPP25 on ferroptosis-related proteins, oxidative stress levels, and reactive oxygen species (ROS) content. In addition, GBM mouse models were established, including an RPP25 overexpression group (OE-RPP25) and an RPP25 overexpression with integrin beta-8 (ITGB8) knockdown group (OE-RPP25+sh-ITGB8). Tumor volume and weight were measured to assess tumor growth. The levels of ferroptosis and oxidative stress were also evaluated using lipid peroxidation and ROS content assays.
Results: It was observed that RPP25 overexpression significantly elevated both tumor volume and weight (p < 0.05) compared to the control group. However, when ITGB8 was knocked down in the OE-RPP25 group, tumor volume and weight were reduced. Furthermore, RPP25 overexpression upregulated the expression of ITGB8, hypoxia-inducible factor (FIH1), and glutathione peroxidase 4 (GPX4), while ITGB8 knockdown reversed these effects. Additionally, RPP25 overexpression suppressed the expression of apoptosis-related proteins B-cell lymphoma-2 associated X (Bax) and Caspase-3 and increased (p < 0.05) the expression of anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). However, ITGB8 knockdown promoted apoptosis in tumor cells by counteracting these effects. Moreover, RPP25 overexpression inhibited ferroptosis and oxidative stress, while ITGB8 knockdown reversed these effects, promoting lipid peroxidation and glutathione depletion.
Conclusions: These findings suggest that RPP25 plays a crucial role in GBM progression by upregulating the ITGB8/transforming growth factor-beta (TGF-β)/Smad4 axis, inhibiting ferroptosis and oxidative stress, and suppressing apoptosis. These findings provide important insights into the interaction between RPP25 and ITGB8 and their potential applications in GBM therapy. Targeting the RPP25-ITGB8 axis may represent a promising strategy for the treatment of GBM by modulating ferroptosis and apoptosis pathways. The findings also suggest that ferroptosis and oxidative stress may be potential targets for GBM therapy.
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Background: Cyclooxygenase-2 (COX-2) is an enzyme responsible for inflammatory responses and is associated with pain, fever, pyretic diseases, and tumor development. The present study aimed to identify new compounds that could be developed as selective inhibitors for COX-2 isoenzyme by using in-silico approaches. This study was conducted to search for potential anti-inflammatory compounds.
Methods: The physiochemical and drug-like properties of natural compounds were determined by SWISSADME and toxicity by using ProTox-II. In-silico docking was performed to determine the affinity of various compounds with a target protein. Molecular dynamic analysis of the protein was carried out using Normal mode analysis (NMA) with internal coordinates normal mode analysis server (iMODS). Non-protein contacts were visualized using Protein Contacts Atlas. Protein-protein interactions (PPIs) were estimated using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). The free binding energies of the docked protein-ligand complexes were determined using Fast Amber Rescoring (farPPI). Prospective binding pockets in the protein were identified using DoGSiteScorer. Amino acid variation between the query and the database sequence were determined using BlastP analysis.
Results: All the compounds in the present study followed Lipinski's rule of five. ProTox-II analysis revealed that syringic acid, eugenol, and gingerenone A were inactive for all toxicities, whereas isosilychristin was active for immunotoxicity. Isosilychristin was found to be the most effective compound with the highest binding affinity of –10.1 kcal/mol. Molecular dynamic analysis provided insights about the mobility, flexibility and stability of the protein molecule. Protein Contact Atlas analyzed the network of residue-residue interactions at the atomic level. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) provides insights about protein-protein interactions (PPIs), extent of their similarities and functional associations. ShinyGO findings revealed that ovarian steroidogenesis was the predominant pathway. Homology modelling revealed that 96.90% of the residues were found to lie in the Ramachandran favored region. Protein Structure Analysis (ProSA) determined the overall quality of a protein molecule, with a Z-score of –8.91. Molecular Mechanics/Generalized Born Surface area (MM/GBSA) approach described stronger binding affinities between the protein and the ligand molecules. The findings obtained from DoGSiteScorer revealed binding pockets properties with COX-2/isosilychristin complex having the highest surface-volume ratio. BlastP findings revealed that our query sequence showed 100% similarity with prostaglandin G/H synthase 2 (accession number NP_000954.1) and with Crystal Structure of Aspirin Acetylated Human Cyclooxygenase-2 (5IKQ_A and 5F1A_A).
Conclusions: The findings of this study revealed that isosilychristin could be a promising inhibitor to target COX-2.
