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Objective To explore the feasibility of the bilayer chitosan barrier membrane loaded with tannic acid(CS@TA) for guided bone regeneration by exploring the reactive oxygen species(ROS) scavenging ability, biocompatibility, and antibacterial properties. Methods The single-layer chitosan(CS) film was prepared by self-evaporation, and the double-layer CS film was prepared by directional freezing and freeze-drying, and its microstructure was observed by scanning electron microscope. The prepared CS bilayer membrane was grafted with tannic acid(TA) in different proportions, and the interaction between TA and CS bilayer membrane was analyzed by Fourier infrared spectrometer(FITR). The ROS scavenging ability was tested by 1,1-diphenyl-2-picrylhydrazyl(DPPH), and the double-layer membrane of loading TA scavenging efficiency of more than 90% was selected to continue the follow-up experiment. CCK-8 assay and lived dead staining were used to evaluate the survival rate of cell in each groups. MC3T3-E1 cells was adhesion the CS@TA barrier film for studying by SEM. Colony counting was performed to test its antibacterial performance againstEscherichia coli(E.coli) andStaphylococcus aureus(S.aureus). Results One side was the smooth, dense while other side was rough, loose and porous, with a longitudinal ordered porous structure in cross-section of the double-layer membrane of CS@TA. With the addition of TA, the ROS scavenging ability of the double-layer membrane first increased rapidly and then stabilized slowly. The results of CCK-8 and lived and dead cells staining showed that excessive TA addition significantly affected the biocompatibility of the double-layer membrane. The counting results of bacterial dilution coating showed that compared with the double-layer membrane without TA loading, the double-layer membrane had certain antibacterial ability againstE.coliandS.aureuswhen the appropriate amount of TA was added. Conclusion Thus the double-layer with appropriate TA loading has strong ROS scavenging ability, good biological performance, and certain antibacterial ability forE.coliandS.aureus.

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Objective To investigate whether fenoldopam(FNDP)(an agonist of type 1 dopamine receptor) has a protective effect on thoracic aortic aneurysm(TAA) in mice.Methods Three-week-old male C57BL/6J mice were treated with β-aminopropionitrile(BAPN) to induce TAA.The mice were divided into three groups:the control group,the BAPN group,and the BAPN+FNDP group(FNDP injected intraperitoneally).The incidence and survival rate of TAA were recorded.Gross anatomy of the whole aortae was observed.Elastin staining was performed to assess morphological change,while immunohistochemistry was employed to evaluate the expressions of matrix metalloproteinase 2(MMP2),matrix metalloproteinase 9(MMP9) and cluster of differentiation 68(CD68)respectively.Gelatin zymography was conducted to assess MMP2 and MMP9 activity.Reverse transcription-polymerase chain reaction(RT-PCR) was performed to measure the mRNA expression levels of dopamine receptor D1(D1DR),dopamine receptor d2(D2DR),dopamine receptor d3(D3DR),dopamine receptor d5(D5DR),interleukin-1β(IL-1β),interleukin-6(IL-6),tumour necrosis factor-α(TNF-α),monocyte chemoattractant protein-1(MCP-1),alpha-smooth muscle actin(α-SMA) and smooth muscle protein 22-alpha(SM22α).Results Compared to the control group,the BAPN group exhibited significant formation of TAA.Elastic fiber disruption was also observed in the thoracic aortic wall,along with a significant decrease in the mRNA levels of D1DR and D5DR.The BAPN+FNDP group showed a significant reduction in the incidence of TAA formation and the rate of aneurysm rupture compared to the BAPN group.The disruption and rupture of elastic fibers in the thoracic aortic wall were significantly improved in the BAPN+FNDP group.The levels of MMP2 and MMP9 in the thoracic aortic wall significantly decreased,and the enzymatic activity of MMP2 in the serum was significantly reduced.Moreover,macrophage infiltration in the thoracic aortic wall was significantly reduced and the mRNA levels of IL-1β,IL-6,TNF-α and MCP-1 also significantly decreased after FNDP treatment.There was no statistically significant difference in the mRNA levels of α-SMA and SM22α.Conclusion FNDP shows an inhibitory effect on TAA progression in mice,suggesting a potential of FNDP as a therapeutic agent for TAA.

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Objective To investigate the effects and underlying mechanisms of α-mangostin in a spinal cord injury model of microglial cell inflammation. Methods Mouse microglial cell line BV-2 was culturedin vitro, and an inflammation model was established by co-treatment with lipopolysaccharide and adenosine triphosphate(LPS/ATP). The CCK-8 assay was used to test the influence of different concentrations(0, 10, 20, 40, 80 μmol/L) of α-mangostin on cell proliferation vitality under LPS/ATP stimulation to select an appropriate concentration range of α-mangostin; BV-2 cells were divided into Ctrl group, LPS/ATP group, 40 μmol/L α-mangostin group, and intervention groups with different concentrations(10, 20, 40 μmol/L) of α-mangostin(designated as LPS/ATP+10 μmol/L α-mangostin group, LPS/ATP+20 μmol/L α-mangostin group, and LPS/ATP+40 μmol/L α-mangostin group, respectively). ELISA experiments were conducted to detect the levels of pro-inflammatory cytokines interleukin-6/1β/18(IL-6, IL-1β, IL-18) and tumor necrosis factor(TNF-α) in the supernatants of each group, and Western blot was used to detect the expression of NLRP3, ASC, cleaved caspase-1, IL-1β, and the phosphorylation levels of p65(p-p65/p65) in the NF-κB pathway, as well as the expression of p65 in the nuclei of BV-2 cells. Results Compared with the Ctrl group, cell proliferation vitality in the LPS/ATP group was significantly reduced(P<0.05), but low concentrations(10, 20, 40 μmol/L) of α-mangostin significantly improved the inhibitory effect of LPS/ATP on microglial cell proliferation vitality(P<0.05), while a high concentration(80 μmol/L) of α-mangostin exacerbated the damage to microglial cells caused by LPS/ATP(P<0.05). Compared with the Ctrl group, the levels of inflammatory factors IL-6, IL-1β, IL-18, TNF-α, and the expression of NLRP3, ASC, cleaved caspase-1, IL-1β, and the p-p65/p65 ratio in the 40 μmol/L α-mangostin group, as well as the expression of p65 protein in the nuclei, showed no significant changes(P>0.05), whereas these significantly increased in the LPS/ATP group(P<0.05). Compared with the LPS/ATP group, the levels of IL-6, IL-1β, IL-18, TNF-α, and the expression of NLRP3, ASC, cleaved caspase-1, IL-1β, and the p-p65/p65 ratio in the intervention groups, as well as the expression of p65 protein in the nuclei, decreased in a concentration-dependent manner with increasing α-mangostin concentration, with the most significant reduction observed in the LPS/ATP+40 μmol/L α-mangostin group(P<0.01). Conclusion α-mangostin can inhibit the neuroinflammatory response mediated by NLRP3 inflammasome activation in BV-2 cells through the NF-κB pathway.

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Objective To explore the candidate genes and potential molecular mechanisms of anti-neutrophil cytoplasmic antibodies(ANCA)-associated vasculitis by bioinformatics and experimental validation, and to provide a scientific theoretical basis for the treatment of potential inflammatory targets for ANCA-associated vasculitis. Methods The GSE108109 chip data was retrieved from the Gene Expression Omnibus(GEO) database, and the differential genes were processed, analyzed and screened using the R language related program package. Kyoto encyclopedia of genes and genomes(KEGG) and gene ontology(GO) enrichment analysis was carried out using DAVID online network cable, and the interaction network of the protein encoded by the selected genes of inflammatory syndrome was constructed through STRING website. Further endogenous competitive RNA(ceRNA) regulatory network was predicted and constructed through miRWalk and DIANA-LncBase databases, and key genes were screened from the network to draw ROC curve. The renal biopsy samples of patients with ANCA-associated vasculitis confirmed by our hospital were collected as the experimental group, and the renal biopsy samples of IgA nephropathy and micro-adaptive nephropathy were collected as the control group. Immunohistochemical staining was performed on the collected renal biopsy samples, and the average optical density was calculated by semi-quantitative analysis of immunohistochemical staining to further verify the expression of the key genes screened by the bioinformatics analysis. Pearson linear correlation analysis was performed between the average optical density results and the clinical inflammatory data of patients. Results 846 differential genes were screened, of which 444 genes were significantly up-regulated and 402 genes were significantly down-regulated. Through KEGG and GO analysis, important differentially expressed genes related to inflammation regulation were obtained. Among them, CSF1R and TNFRSF1B, two differentially expressed genes never reported in ANCA-associated vasculitis, attracted our attention. At the same time, we constructed multiple ceRNA regulatory axes including KCNQ1OT1-hsa-miR-125a-5p-TNFRSF1B. There were 15 samples of ANCA-associated vasculitis, 6 samples of IgA nephropathy, and 3 samples of micropathological kidney. Immunohistochemical results of renal biopsy specimens showed that the expression of CSF1R and TNFRSF1B in ANCA-associated vasculitis kidney tissue was higher than that in the control group. Pearson correlation analysis of clinical data of patients in ANCA group showed that the expression of CSF1R was positively correlated with the content of neutrophil count(r=0.587), and the expression of TNFRSF1B was positively correlated with the content of serum C-reactive protein(r=0.646). Conclusion Key genes related to inflammatory regulation such as CSF1R and TNFRSF1B were investigated by bioinformatics methods, and a rigorous ceRNA regulatory network was constructed. The expression of CSF1R and TNFRSF1B in ANCA vasculitis was higher than that in the control group through immunohistochemistry. The results provides a scientific theoretical basis for the molecular mechanism of inflammation, and laid a good foundation for new therapeutic targets of ANCA-related vasculitis for inflammation.

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Objective To develop a high modulus and high strength biodegradable silk fibroin GBR membrane to address the issue of maintaining the space for bone regeneration in the repair of osseous defects. Methods After purifying silk fibroin protein, membrane materials were prepared using evaporation-hot pressing method. The physical and chemical properties and biological performance of the membranes were evaluated using stretching tests,in vitrosimulations, and cell co-culturing methods. Results A silk fibroin GBR membrane was successfully fabricated, resulting in a simulated degradation rate of 35.3% after 12 hin vitro. The wet-state elastic modulus reached 45 MPa, while the tensile strength reached 8.39 MPa. Furthermore, the cell survival rate was nearly 100% after 7 days. Conclusion The biodegradable GBR membrane produced in this study possesses high modulus and strength, as well as excellent biocompatibility, offering a promise as a foundation for addressing the bone defect repair and bone space maintenance.

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Objective To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further investgation of the specific role of Spi1-encoded protein PU.1. Methods The Lck-Cre mice were mated with Spi1flox/floxmice to obtain Lck-Cre×Spi1flox/floxmice(T lymphocyte-specific Spi1 knockout mice),and the genotype was determined by polymerase chain reaction(PCR) and agarose gel electrophoresis. Magnetic beads were used to sort out the splenic T lymphocytes, and the knockdown efficiency of PU.1 in T cells was detected by Western blot, quantitative real-time PCR(qPCR) and flow cytometry. Results The Lck-Cre×Spi1flox/floxmouse genotype was stably inherited. Compared with Spi1flox/floxmice, the expression level of PU.1 was significantly reduced in splenic T cells of Lck-Cre×Spi1flox/floxmice. Conclusion In this study, the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology, which provided a reliable animal model for the subsequent experiments of the specific role of PU.1 in T cell-related diseases.

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Objective To investigate the role of lncSIL in transforming growth factor-β1(TGF-β1)-induced alveolar epithelial interstitial transformation(EMT) and its related signaling pathways. Methods Western blot was used to detect the effect of lncSIL silencing on the expression of E-cadherin(E-cad), alpha-smooth muscle actin(α-SMA) and Collagen I(Col I) in the process of EMT induced by TGF-β1. LncSIL interacting proteins were analyzed by RNA pulldown. Western blot was used to detect the effect of overexpression or silencing of lncSIL on the expression of its target gene enhancer of zeste homolog 2(EZH2) and its downstream factors P21 and cyclin-dependent kinase 6(CDK6). Flow cytometry was used to analyze the effect of lncSIL on cell cycle progression. Results After lncSIL silencing, the expression of α-SMA and Col I increased, the expression of E-cad decreased. RNA pulldown assay showed that EZH2 was the target protein that interacted with lncSIL, and the expression of EZH2 increased after silencing lncSIL, the expression of EZH2 downstream gene P21 decreased, CDK6 increased. Flow cytometry showed that the number of cells in S phase significantly increased. When lncSIL was overexpressed, the expression of EZH2 and CDK6 was down-regulated, the expression of P21 was up-regulated, and the number of S phase cells significantly decreased. Conclusion LncSIL inhibits TGF-β1-induced alveolar epithelial cell mesenchymal transition by negatively regulating EZH2/P21/CDK6 signaling pathway to inhibit cell cycle progression.

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Objective To investigate the effect of autophagy activation on cell proliferation in human umbilical vein endothelial cells(HUVECs). Methods HUVECs were treated with rapamycin(Rapa). Western blot assay was performed to examine the expression of protein of microtubule associated protein 1 light chain 3(LC3), Beclin 1 and unc-51-like kinase 1(ULK1). Autophagosomes were detected by transmission electron microscopy(TEM), and autophagy fluorescence was detected by monodansylcadaverine staining(MDC) assay. The effect of autophagy activation on cell proliferation was assessed by CCK-8 assay and EdU assay. Vascular formation experiments were used to detect vasculogenic ability. Results After Rapa treatment, LC3, Beclin1 and ULK1 expressions were enhanced, while the green autophagy fluorescence expression in the experimental group was stronger than that in the control group, and autophagosomes were visible by TEM; CCK-8 and EdU results showed that compared with the control group, the cell proliferation ability was weakened and tubes formation ability was reduced after the activation of autophagy in experimental cells. Conclusion Rapa upregulates autophagy activity in HUVECs to inhibit cell proliferation under certain time.

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Objective To screen the core genes of diabetic kidney disease(DKD) based on bioinformatics, explore the therapeutic targets of DKD, and discuss its possible regulatory mechanism. Methods The expression data matrix of glomerular transcriptome in patients with DKD in GEO database(GSE30528, GSE47183) was extracted, and the differentially expressed genes(DEGs) were screened by bioinformatics methods to identify the core differential genes, and then gene expression and enrichment analysis(GSEA) were conducted to predict effective targets. Results By screening and identifying the mRNA expression matrix of DKD, five core genes were screened out. Among them, C1orf21 and NPHS1 were significantly down regulated, and CD48, COL1A2, and TGFBI were up regulated. NPHS1 and CD48 were significantly related to immune differences, intercellular communication, and cell surface interaction. Through receiver operating characteristic curve(ROC) analysis and GSEA analysis and drug target prediction, it might be of great significance for the treatment of DKD. Conclusion The core genes screened in this study have significant correlation with DKD, which may be used as effective markers for the treatment of diabetes. And then, this study provides a theoretical basis for the treatment and identification of DKD.

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Objective To investigate the effect and mechanism of methyltransferase-like 3(METTL3) on the proliferation, migration, and secretion of inflammatory factors by synovial fibroblasts from rheumatoid arthritis(RA). Methods The expression of METTL3 in synovial tissue(SF) from 25 patients with rheumatoid arthritis and 25 patients with osteoarthritis was detected by RT-qPCR and immunohistochemistry, respectively. The concentration of RNA m6A was detected by ELISA. RA synovial fibroblasts were isolated and cultured, and divided into NC(normal control) group, hi-METTL3(overexpression of METTL3) group, si-METTL3(knock-down METTL3) group, and STM2457(METTL3 specific inhibitor) intervention group. Cell proliferation was detected by CCK-8 method. Apoptosis was detected by flow cytometry. And the concentrations of interleukin-6(IL-6), interleukin-17A(IL-17A), receptor activator of nuclear factor-kappa B ligand(RANKL), and osteoprotegerin(OPG) in the supernatant of cell culture were detected by ELISA. Results Compared with synovial tissue of osteoarthritis, the expression of mRNA m6A and METTL3 in synovial tissue of RA significantly increased(P<0.05). After overexpression of METTL3, the expression of m6A in synovial fibroblasts increased. The proliferation and migration abilities of SF in hi-METTL3 group were significantly improved, and their apoptosis did not change significantly. The secretion of cytokines IL-6 and RANKL of SF in hi-METTL3 group significantly increased, while the OPG significantly decreased(P<0.05). After interfering with METTL3 expression, the expression of m6A in synovial fibroblasts decreased. Cell proliferation and migration of SF in siMETTL3 group significantly decreased. The secretion of cytokines IL-6 and RANKL significantly decreased, and OPG significantly increased(P<0.05). After intervention with METTL3 inhibitor STM2457, the proliferation and migration of synovial fibroblasts were significantly reduced, and the secretion of cytokines IL-6 and RANKL significantly reduced, and OPG significantly increased(P<0.05). There was no significant difference in the expression of IL-17A among each group. Conclusion METTL3 may promote the proliferation and migration of RA synovial fibroblasts, enhance the expression of IL-6 and RANKL, and inhibit the expression of OPG through RNA m6A methylation modification.

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Objective To investigate the effect and mechanism of puerarin on the proliferation, invasion and apoptosis of breast cancer cell HCC1806vianetwork pharmacology, molecular docking andin vitroexperiments. Methods The data of breast cancer and mitochondrial diseases were collected from GEO database, and the differentially expressed genes were analyzed by GEO2R. Overlapping genes between breast cancer and mitochondrial database were analyzed by Venn diagram. GO and KEGG enrichment analyzed the overlapping genes. STRING and Cytoscape analyzed overlapping gene interaction networks and screen core genes. Interaction between puerarin and core genes was docked with autodock. Cell proliferation, apoptosis and invasion capacity were measured by CCK-8, EdU, TUNEL and Transwell experiments, mitochondrial membrane potential was measured by Mito-Tracker experiments and protein expression levels were measured by Western blot. Anti-tumor efficacy of puerarin was analyzed by subcutaneous xenograft of breast cancer and immunohistochemical assayin vivo. Results Among 132 overlapping genes in breast cancer and mitochondrial disease, 10 core differentially expressed genes were selected. Among these core genes, dual serine/threonine and tyrosine protein kinase(DST) was low expressed in breast cancer tissues. Puerarin bound to DST, up-regulated its expression, inhibited the proliferation and invasion of HCC1806 breast cancer cells, promoted breast cell apoptosis, increased the expression levels of apoptosis-related proteins Cleaved-Caspase 3 and Bax, down-regulated the expression of anti-apoptosis protein Bcl-2, and decreased mitochondrial membrane potential.In vivo, puerarin inhibited the growth of breast cancer and up-regulated the expression of DST in tumor tissues. Conclusion Puerarin inhibits the proliferation and invasion of breast cancer cells, promotes the apoptosis of cancer cells and inhibits breast cancer growth.

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Objective To explore the molecular mechanism of lipopolysaccharide(LPS) on the contraction of pregnant uterine smooth muscle at tissue and cellular levels. Methods C57BL/6J mice at 16 days of gestation were randomly divided into control group and LPS group. The mice in LPS group were intraperitoneally injected with 20 μg in LPS solution to establish the model of preterm birth, and the mice in control group were intraperitoneally injected with the same amount of normal saline. Isolated uterine muscle strips were used to detect changes in the contractile function of the tissue, as well as changes in the expression and function of the contraction key signaling molecule TWIK-related K+channel 1(TREK-1). Primary cultured pregnant mouse uterine smooth muscle cells were used to detect the expression of TREK-1 under the regulation of LPS. Results The contractility of mouse uterine tissues was significantly enhanced by LPS, and the protein expression of TREK-1, a key signal for contraction, was significantly reduced, and activation of TREK-1 resulted in a significant down-regulation of the enhanced contractility of mouse uterine tissues in the LPS group. However, there was no significant difference in the expression of TREK-1 protein, which was highly expressed in the smooth muscle of pregnant mice, when LPS acted on the primary uterine smooth muscle cells of pregnant mice. Conclusion Uterine contractility is enhanced in pregnant mice uterine tissues by inhibiting TREK-1 expression and function in response to LPS, and it may be one of the mechanisms by which LPS induces preterm labor. However, the effect of LPS on TREK-1 on mouse pregnant uterine smooth muscle cells may be realized through intercellular signaling and not directly on uterine smooth muscle cells. This further suggests that the animal and histological experiments cannot be completely replaced by isolated cell experiments in the study of inflammatory preterm labor.

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Objective To explore the mechanism of mesenchymal epithelial transformation(EMT) mediated by Wnt/β-catenin signaling pathway in the inhibition of endometrial fibrosis by endometrial mesenchymal stem cells(eMSCs). Methods Eighteen female SD rats were randomly divided into Sham group, Model group and eMSCs group, with 6 rats in each group. Rats in Sham group merely had laparotomy without any treatment. A rat model of intrauterine adhesion(IUA) was established in the Model group and eMSCs group. In eMSCs group, the total amount of eMSCs cell suspension transplanted immediately after model injury was 0.05 ml(2×107cells/ml) per uterus for treatment. Three weeks later, the uterus with unilateral injury was collected for hematoxylin-eosin(HE) staining and Masson staining. Endometrial fibrosis, EMT, Wnt/β-catenin pathway protein expression were analyzed by protein blot. Results In the Model group, the structure of the uterine cavity was destroyed and the number of glands were significantly reduced with a large number of blue collagen fibers were accumulated. However, after eMSCs treatment, the number of endometrial glands increased, and the fibrotic area decreased significantly. Compared with Sham group, the expression levels of type I collagen and α-SMA protein in Model group increased significantly(P<0.05), but decreased significantly in eMSCs group(P<0.05). In the Model group, the expressions of N-cadherin, vimentin and ZEB1 increased significantly, while the expression of E-cadherin decreased. However, in eMSCs group, the changes of protein of the above molecules were completely opposite. Compared with Sham group, the expression of β-catenin and C-myc increased in Model group(P<0.05). Compared with the Model group, the expressions of CyclinE, β-catenin and C-myc increased in eMSCs group(P<0.05). Conclusion eMSCs can promote endometrial repair in IUA rats by inhibiting EMT and endometrial fibrosis, which is partly achieved by activating Wnt/β-catenin signaling pathway.

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Objective To study the effect and mechanism of high glucose on mesothelial-mesenchymal transition(MMT) of peritoneal mesothelial cells(HMrSV5),and the protective effect of pharmacological blocking of signal transducer and activator of transcription 3(STAT3) on rat peritoneal fibrosis(PF) model.Methods The animals were divided into three groups:the sham group,the model group,and the STAT3 inhibitor group.A miniature peritoneal dialysis catheter was implanted under the dorsal skin of rat and the rat peritoneal fibrosis model was induced by daily injection of high glucose dialysate.After 10 weeks,HE staining was used to evaluate the histology of the peritoneum,and the level of transforming growth factor-β1(TGF-β1) in the peritoneum was measured by immunohistochemistry.HMrSV5 was cultured in high glucose and the optimal stimulation concentration of high glucose was determined by Western blot.High glucose was used to stimulate HMrSV5 after successful transfection with siSTAT3 and Western blot was used to measure the protein level of STAT3,p-STAT3,and the key enzymes of glycolysis 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3) and lactate dehydrogenase A(LDHA).Results HE staining showed that administration of STAT3 inhibitor(BP-1-102) could inhibit the thickening of subperitoneal tissue and the proliferation of vessels in HG dialysis rats.The expression of TGF-β1 in the rats peritoneum of the model group was significantly higher than that in the sham group,and the level of TGF-β1 was markedly lower in the STAT3 inhibitor group compared to the model group(P<0.05).Compared to the control group,high glucose induced the up-regulation of α-smooth muscle actin(α-SMA),the down-regulation of E-cadherin and STAT3 activation in HMrSV5(P<0.05).Mesothelial cells treated with high glucose also exhibited high expression of the key enzymes of glycolysis(PFKFB3,LDHA)(P<0.05),and si-STAT3 can effectively inhibit the overexpression of PFKFB3 and LDHA induced by high glucose(P<0.05).Conclusion STAT3 is involved in high glucose-induced HMrSV5 hyperglycolysis and MMT,and targeting STAT3 alleviates peritoneal fibrosis and angiogenesis during peritoneal dialysis treatment in rats.

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Objective To establish anin vitrorenal injury model of amikacin(AKN) and investigate the protective effect and mechanism of vaccarin(VA) in the AKN-inducedin vitrorenal injury model. Methods Human renal tubular epithelial cells(HK-2) were culturedin vitroand incubated with different drugs of AKN or/and VA to determine the optimal drug concentration based on cell viability tested by MTT. The changes in intracellular oxidative stress were assessed using the dihydroethidium(DHE) probe and malondialdehyde(MDA)/glutathione(GSH) assay kits at different time points. Total RNA was extracted, and RT-qPCR was performed to detect the changes in the gene expression of kidney injury molecule-1(KIM-1) and neutropil gelatinase-associated lipocalin(NGAL). Western blot analysis was performed to detect the levels of ferroptosis-related markers solute carrier family 7 member 11(SLC7A11) and glutathione peroxidase 4(GPX4) in HK-2 cell lysis. Results High concentrations of AKN significantly decreased the viability of HK-2 cellsin vitro, with a half maximal inhibitory concentration(IC50) of(5.74±0.47) mmol/L. VA at concentrations of 25-100 μmol/L increased the viability of AKN-stimulated HK-2 cells(P<0.05). After treatment with AKN(4 mmol/L), the mRNA expression levels of KIM-1 and NGAL were significantly higher than those of the negative control(NC) group(P<0.001). VA(50 μmol/L) significantly reduced the mRNA expression levels of KIM-1(P<0.01) and NGAL(P<0.05). The intensity of DHE staining increased after 3 hours of AKN treatment, but the difference was not statistically significant. However, the intensity of DHE staining was significantly higher in the 6-24 hours group compared to the 0-hour group(P<0.01). Furthermore, MDA levels significantly increased, while GSH levels significantly decreased after 6-24 hours of AKN treatment, with statistically significant differences(P<0.05). After 6-24 hours of AKN stimulation, the ferroptosis-related proteins SLC7A11 and GPX4 both significantly decreased(P<0.001). Co-incubation with VA for 24 hours effectively reversed the changes in DHE staining, MDA and GSH levels, as well as the changes of SLC7A11 and GPX4 protein levels(P<0.001). Conclusion In this study, anin vitrorenal injury model was established by stimulating HK-2 cells with high concentrations of AKN, and it was found that VA might alleviate the damage to renal tubular cells caused by AKNviainhibiting oxidative stress related ferroptosis.

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Objective To investigate the role of knockdown of peroxiredoxin-6(PRDX6) in injury and adaptive expression of bile acid transporter in human hepatoellular carcinomas(HepG2) cells induced by rifampicin(RFP). Methods Cells in logarithmic growth phase were uniformly inoculated in six-well plates, and HepG2 cells were transiently transfected with specific PRDX6-siRNA and control-siRNA to construct the knockdown group and control group. After 24 h of induction with 100 μmol/L RFP, Western blot and qRT-PCR were performed to detect the protein and gene expression levels of PRDX6, multidrug resistance protein 1(MDR1), multidrug resistance-associated proteins 2, 3 and 4(MRP2, MRP3 and MRP4), and Na+/taurine taurocholate cotransporter protein(NTCP). Annexin V-FITC/PI double staining assay was used to detect the apoptosis rate of cells in each group; CCK-8 assay was used to detect the changes of cell proliferation in each group; The relative contents of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), indirect bilirubin(IBIL) and total bile acid(TBA) in the supernatant of cell culture medium of each group were detected by kits. Results RFP increased the protein and gene expression levels of MRP2, MRP3, MRP4, MDR1, NTCP and PRDX6 in HepG2 cells(P<0.05), while the protein and gene expression levels of MRP2, MRP3, MRP4, MDR1 and NTCP decreased to different degrees after PRDX6 knockdown(P<0.05). In addition, PRDX6 knockdown resulted in increased apoptosis rate of HepG2 cells(P<0.05), decreased cell proliferation ability(P<0.05), and increased levels of cell injury markers(ALT, AST, TBIL, DBIL, TBA) in cell culture supernatants(P<0.05). Conclusion RFP increased the protein and gene expression of bile acid transporter and PRDX6 to increase in HepG2 cells. However, following knockdown of PRDX6 and treatment with RFP, the protein and gene expression levels of the bile acid transporter decreased and cell injury was aggravated, suggesting that PRDX6 played a protective role in RFP-induced adaptive response in HepG2 cells.

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Objective To develop a CD30-targeted CAR-T cell drug based on the multi-chain chimeric antigen receptor T cells(CAR-T) of the bridging protein DAP12, and to study thein vitroandin vivopreclinical efficacy of CD30 CAR-T on Hodgkin lymphoma tumor cells. Methods Through gene synthesis and molecular cloning techniques, a CAR plasmid targeting CD30 was designed and constructed, and the obtained lentivirus was packaged. The T cells were transfected with the lentivirus, where the multi-chain CAR-T targeting CD30 was the CD30-KIRS2/Dap12-BB group, the single-chain second-generation CAR-T was the CD30-41BBζ group, and the T cells without virus infection were the NTD group. The positive rate of CAR was detected by flow cytometry, the cytotoxicity of the cells was detected by lactate dehydrogenase(LDH) release assay, the secretion level of the cytokine interferon γ(IFN-γ) was detected by enzyme-linked immunosorbent assay(ELISA), and the antitumor activity of CD30 CAR-T in mice was further detected by a mouse xenograft tumor model. Results A comparison was made between the multi-chain CAR-T targeting CD30 and the single-chain second-generation CAR-T. It was found that the antitumor effect of the multi-chain CAR-T was similar to that of the single-chain CAR-T. However, it was worth noting that the IFN-γ secretion level of the multi-chain CAR-T was higher(P<0.001). More importantly, in the mouse tumor model experiment, the multi-chain CAR-T achieved complete tumor regression. Conclusion The multi-chain CAR-T targeting CD30 is superior to the traditional single-chain CAR-T in terms of antitumor activity.

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Objective To investigate the reversal effect and mechanism of cinobufagin(CBG) on cisplatin resistance in human ovarian cancer cells. Methods A2780 cell line and its cisplatin-resistant cell line A2780/DDP are common ovarian cancer cells in clinic, so these two cell lines were selected as the research objects. The cell viability was detected by cell Counting Kit-8(CCK-8) assay, and the cell proliferation ability was detected by plate cloning and 5-ethynyl-2′-deoxyuridine(EdU) assay. Hoechst staining was used to observe cell apoptosis. Cell scratch test and Transwell test were used to evaluate cell migration and invasion ability. Western blot and quantitativereverse transcription PCR(RT-qPCR) were used to detect the protein and mRNA expressions of phosphatidylinositol 3-kinase/protein kinase(PI3K/AKT) signaling pathway and epithelial-mesenchymal transition(EMT). Results Compared with A2780 cells, the drug resistance indexes of A2780/DDP cells were 5.636, 5.864, 5.695, respectively. After treatment of A2780/DDP cells with CBG(2, 4, 6 mg/ml), the reversal resistance indexes were 1.617, 2.570, 3.461, respectively. CBG treatment significantly increased the level of apoptosis and inhibited the proliferation, migration and invasion of the cells in a concentration-dependent manner(P<0.05).Western blot results showed that compared with A2780 cells, the relative ratio of P-PI3K/PI3K and P-AKT/AKT protein levels, as well as the protein expression of N-cadherin, Vimentin, and Snail were higher in the control group(A2780/DDP) cells, while the protein expression of E-cadherin was lower(tP-PI3K/PI3K=8.115,tP-AKT/AKT=17.62,tN-cadherin=6.126,tVimentin=4.001,tSnail=17.333,tE-cadherin=4.620,P<0.01); As the dose of CBG increased, the protein expression levels of P-PI3K, P-AKT, N-cadherin, Vimentin, and Snail in drug-resistant cells decreased, while the protein expression level of E-cadherin increased(F P - PI3K =268.5,F P - AKT =190.5,F N - cadherin =24.02,F Vimentin =57.65,F Snail =87.24,F E - cadherin =135.8,P<0.05). qRT-PCR results showed that with the increase of CBG concentration, the mRNA expression levels of PI3K, AKT, N-cadherin, Vimentin and Snail decreased, while the mRNA expression level of E-cadherin gradually increased(F PI3K =101.1,F AKT =558.3,F N - cadherin =86.97,F Vimentin =105.9,F Snail =85.71,F E - cadherin =80.96,P<0.01). Conclusion CBG can reverse cisplatin resistance of ovarian cancer A2780/DDP cell line, and its mechanism may be related to the regulation of PI3K/AKT signaling pathway and inhibition of EMT by CBG.

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Objective To analyze the incidence characteristics and trends in pulmonary tuberculosis in the Hotan prefecture, before and after the epidemic, and to provide a reference basis for the formulation and evaluation of tuberculosis prevention and control measures in the Hotan prefecture. Methods The Hotan prefecture's pulmonary tuberculosis incidence data was collected between 2015 and 2021. Joinpoint regression(JPR) model and Interrupted Time Series(ITS) model were established to explore the incidence trend of pulmonary tuberculosis, as well as the impact of COVID-19 prevention and control measures in Xinjiang on the incidence trend in Hotan, respectively. Furthermore, an analysis of variations in incidence among different age and gender subgroups was carried out. Results The results of the JPR model showed that from 2015 to 2021, the reported incidence rate of pulmonary tuberculosis in the Hotan prefecture initially increased and then decreased, with a turning point appearing in December 2018. The incidence rate in males was slightly higher than that in females, and the turning point and incidence trend were consistent with the overall trend. Among all age subgroups, those ≥60 age group had the highest incidence rate, with the trend also showing an initial increase followed by a decrease. A turning point in the incidence rate for the under 18 age group appeared in June 2021, yet the trend was not statistically significant(P>0.05). The turning points in the 19-59 age group and in those aged ≥60 were consistent with the overall trend. The results of the ITS model showed that the incidence rate of pulmonary tuberculosis in the Hotan prefecture significantly decreased since January 2020, dropping from 319.28 per 100 000 in 2019 to 155.88 per 100 000 in 2021, a decrease of 51.16% year-on-year, with a monthly average reduction of 0.049 per 100 000. Conclusion In 2018, Xinjiang province integrated tuberculosis screening into the universal health checkup for the entire population, which led to the identification of numerous cases of tuberculosis. In the Hotan prefecture, the reported incidence of pulmonary tuberculosis peaked in December 2018 and then started to decline. Under the impact of COVID-19 isolation measures in Xinjiang, the reported incidence rate showed a notable decrease starting in January 2020. Reiterating preventive measures and remaining watchful for the possible appearance of latent tuberculosis patients is crucial as the pandemic fades.

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Objective To investigate the factors influencing the pregnancy outcomes during frozen-thawed embryo transfer(FET) cycles in patients with polycystic ovary syndrome(PCOS). Methods A retrospective analysis was conducted on patients' data from 882 FET cycles. According to the pregnancy outcome, the patients were divided into non-implantation group(Group A), abortion group(Group B1) and live birth group(Group B2). Clinical data and laboratory parameters were compared among the three groups, and ordered Logistic regression analysis was used to study the factors influencing pregnancy outcomes after FET. Patients were also divided into four groups(C1-C4) based on the number of high-quality embryos obtained(0-3, 4-6, 7-10, ≥11), and their clinical data and laboratory parameters were compared. Results The clinical pregnancy rate, live birth rate, and miscarriage rate in the 882 treatment cycles were 71.09%(627/882), 61.68%(544/882), and 13.24%(83/627), respectively. Single-factor analysis showed significant differences in body mass index(BMI), infertility type, human chorionic gonadotropin(hCG) day estradiol(E2) level, number of retrieved oocytes, and number of high-quality embryos among Groups A, B1, and B2(P<0.05). Further multiple Logistic regression analysis revealed that BMI(OR=1.046,95%CI:1.001-1.093,P=0.044) and a history of previous pregnancy(OR=1.417,95%CI:1.030-1.950,P=0.032) were independent risk factors for successful FET in PCOS patients, while an increased number of high-quality embryos was an independent protective factor for successful pregnancy. Based on the results of Group B2, compared to Group A,OR=0.920,95%CI:0.880-0.962,P=0.000;compared to Group B1,OR=0.923,95%CI:0.862-0.988,P=0.022.Compared with the other three groups(C1-C3), the total amount of gonadotropin(Gn) in the C4 group was the lowest and the number of oocytes obtained was the highest(P<0.05). Multiple comparisons showed that Group C4 had lower BMI, follicle-stimulating hormone(FSH), very low-density lipoprotein(vLDL) levels, a higher luteinizing hormone and follicle-stimulating hormone(LH/FSH) ratio compared to Group C1(P<0.05). Group C4 had lower fasting insulin(FINS) and homeostasis model assessment of insulin resistance(HOMA-IR) levels compared to Group C3, and higher high-density lipoprotein-cholesterol(HDL-C) and apolipoprotein A1(Apo A1) levels compared to Groups C2 and C3(P<0.05). Conclusion BMI,the history of previous pregnancy and the number of high-quality embryos were both independent factors for predicting pregnancy outcomes in PCOS patients undergoing FET cycles. Patients with a higher number of high-quality embryos have a higher clinical pregnancy rate during FET cycles.

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Objective To investigate the value of methyltransferase-like protein 16(METTL16) in the clinical diagnosis and prognostic prediction of multiple myeloma(MM) patients. Methods The expression level and prognostic potential of each gene involved in N6-methyladenosine(m6A) modification in MM were respectively analyzed in the databases of the Multiple Myeloma Research Foundation(MMRF) and the Genotype-Tissue Expression Project(GTEx). Bone marrow specimens from 26 patients with initial diagnosis of MM and 19 patients with MM after treatment with standard regimens and peripheral blood specimens from 24 normal subjects were collected respectively, and the expression levels of m6A genes were determined by qRT-PCR. The correlation between METTL16 expression and various laboratory and clinical indexes was analyzed: hemoglobin(Hb), white blood cell count(WBC), platelet count(PLT), blood creatinine(Scr), serum calcium(Ca2+), β-microglobulin(β-MG), bone destruction, ISS stage, type, and overall survival(OS) in the patients with primary diagnosis. The expression levels of interleukin(IL)-4, IL-6, IL-10, IL-18 and chemokine ligand 2(CCL2), CCL3, CCL4 in the specimens were further examined and their correlation with the expression of METTL16 was investigated. Results Database analysis suggested that METTL16 expression was significantly higher in MM patient samples compared with normal controls, which was associated with poor prognosis and had certain diagnostic value. qRT-PCR results showed that the expression level of METTL16 in the bone marrow of patients with initial diagnosis of MM was significantly higher than that of treated patients and normal controls. Its expression was positively correlated with hemoglobin, leukocytes and stage, and its expression was positively correlated with CCL4 expression. Conclusion METTL16 expression was significantly elevated in patients with MM, and its expression level was correlated with anemia, more bone destruction and worse stage, which might indicate a poor prognosis. The significant correlation between the expression of METTL16 and CCL4 suggests that METTL16 may play a corresponding pathogenic role through the relevant pathway. METTL16 will have significant clinical value in the management of MM.

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Objective To investigate the risk factors associated with the presence of multiple Lugol-voiding lesions(LVLs) in patients with early esophageal cancer and the correlation with alcohol dehy-drogenase 1B(ADH1B) and aldehyde dehydrogenase 2(ALDH2) polymorphisms. Methods Patients who underwent endoscopic submucosal dissection due to early esophageal cancer were divided into group with multiple LVLs and group without multiple LVLs based on their endoscopic features. Their clinical data and the genotype of ADH1B and ALDH2 were collected and SPSS 27.0 was used to statistically analyze the above data. Results A total of 83 subjects were included in the study, 23 had multiple LVLs, most of them were seen in males, alcohol drinkers, and smokers with smoking index≥1 000, and multivariate analysis showed that alcohol consumption was an independent risk factor for it(OR=6.215,P=0.008). The gene polymorphism of ADH1B and ALDH2 and their interactions did not have any significant correlation with multiple LVLs. However, among alcohol drinkers, there was a 12-fold increased risk of multiple LVLs in patients carrying the ALDH2 A allele and drinking ≥50 g per day compared to those carrying the ALDH2 GG genotype and drinking<50 g per day(P=0.045). Conclusion Alcohol consumption is an independent risk factor of multiple LVLs of the esophageal mucosa in patients with early esophageal cancer, and heavy alcohol consumption in carriers of the ALDH2 A allele will significantly increase the risk of multiple LVLs, and such patients should be closely followed up with endoscopy.

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Objective To explore the difference of serum inflammatory factors in patients with first episode schizophrenia,patients with relapse episode schizophrenia and healthy people,and the correlation between serum inflammatory factors with negative symptoms in patients with schizophrenia,so as to provide reference for clinical intervention.Methods A total of 86 patients with first episode schizophrenia(first episode group),80 patients with relapse episode schizophrenia(relapse episode group) and 82 healthy people(control group) were included in the study.The difference of serum inflammatory factors among the three groups and the correlation between serum inflammatory factors with negative symptoms were analyzed.Results There were significant differences in serum interleukin(IL)-1β and IL-16 levels among the three groups(P<0.05).The analysis and comparison between the two groups showed that the serum IL-1β in first episode group was significantly higher than that in relapse episode group and control group(P<0.05),serum IL-16 in first episode group and relapse episode group was significantly higher than that in control group(P<0.05).Serum IL-1β was negatively correlated with PANSS general psychopathological scale factor score in first episode group(P<0.05),and serum IL-16 was positively correlated with PANSS negative symptom scale factor score in relapse episode group(P<0.05).IL-16 level might be an independent risk factor affecting the onset of first episode group and relapse episode group(P<0.05).Conclusion There are differences in serum levels of IL-1β and IL-16 between patients with schizophrenia and healthy people.Serum IL-16 levels in patients with relapse episode schizophrenia are associated with negative symptoms.IL-16 level may be an independent risk factor for schizophrenia.

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Objective To explore the risk factors for intracardiac thrombosis in dilated cardiomyopathy(DCM) patients and to construct, validate, and evaluate a nomogram prediction model based on these factors. Methods 88 patients diagnosed with DCM and complicated with intracardiac thrombus, and 544 patients without intracardiac thrombus were included. The participants were randomly divided into training and validation sets at a ratio of 7 ∶3. Using both univariate and multivariate Logistic regression analyses, independent risk factors for intracardiac thrombosis in DCM patients were identified. A nomogram prediction model was constructed using R software. The model's validity and performance were assessed using the receiver operating characteristic(ROC) curve, the Hosmer-Lemeshow goodness-of-fit test, calibration curve, and decision curve. Results The binary Logistic regression analysis showed that age, atrial fibrillation, left ventricular end-diastolic diameter(LVEDD), brain natriuretic peptide(BNP), and β-blockers were independently associated with intracardiac thrombosis in DCM patients. Based on these five factors, a nomogram was constructed and validated. The area under the ROC curve for the training set was 0.823(95%CI: 0.760～0.887) and 0.803(95%CI: 0.705～0.901) for the validation set, indicating a good discriminative ability. The Hosmer-Lemeshow test results for the calibration curve were(χ2=6.679,P=0.572) for the training set and(χ2=2.588,P=0.958) for the validation set, indicating a good fit between predicted and observed outcomes. The decision curve showed a high net clinical benefit in the threshold range of 0.05～0.92. Conclusion Based on age, atrial fibrillation, LVEDD, BNP, and β-blockers, the nomogram prediction model exhibits good discriminative and calibration abilities, and high clinical benefit. It can effectively guide clinicians in early intervention of risk factors, reducing the risk of intracardiac thrombosis in DCM patients.