Objective:To analyze the target signaling pathway of histone H3K27 methylation-induced podocyte injury, verify the regulatory effect of histone H3K27 methylation on podocyte injury in focal segmental glomerulosclerosis (FSGS) mice through target signaling pathway, and explore the mechanism of abnormal methylation of histone H3K27-induced podocyte injury in FSGS mice.Methods:(1) Cell experiments: primary cultured immortalized mouse podocytes MPC5 were cultured in vitro, and divided into control group, adriamycin (ADR) group, ADR+GSK-J4 (histone demethylase, KDM6B inhibitor) group, ADR+coumarin A1 (C-A1, JAK2 agonist) group and ADR+GSK-J4+C-A1 group. The transmission electron microscope was used to observe ultrastructure of podocytes. Immunofluorescence was used to detect the protein expression of H3K27me3 and nephrin in podocytes. The whole genome sequence of podocytes was obtained, the differentially expressed genes were screened, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for enrichment analysis. Real time-quantitative PCR and Western blotting were used to detect the gene and protein expression of JAK2-STAT3 signaling pathway in podocytes respectively. Enzyme-linked immunosorbent assay was used to detect interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1). (2) Animal experiments: EZH2 gene knock out ( EZH2podKo)-FSGS (tail vein injection of ADR) mouse models were established, and divided into EZH2ctrl+control group ( n=20), EZH2ctrl+FSGS ( n=20), EZH2podKo+control group ( n=30) and EZH2podKo+FSGS group ( n=30). HE staining was used to observe the morphology of kidney tissues. Immunohistochemistry was used to detect the H3K27me3 protein expression in podocytes. Real time-quantitative PCR and Western blotting were used to verify the gene and protein expression of JAK2-STAT3 signaling pathway respectively. Enzyme-linked immunosorbent assay was used to detect IL-6, MCP-1, α-SMA and TGF-β1 of kidney tissues. Results:(1) Cell experiments: Compared with control group, the nucleus shrank and ruptured, the cytoplasm showed vacuole, and mitochondria and endoplasmic reticulum swelled in ADR group, which verified that the podocyte injury model of ADR nephropathy was successfully established. Compared with control group, the protein expression level of H3K27me3 in ADR group was significantly lower ( P<0.05). Compared with ADR group, the protein expression level of H3K27me3 in podocytes in ADR+GSK-J4 group was significantly higher ( P<0.05), and there were 502 increased genes and 443 decreased genes. GO enrichment analysis showed that the differentially enriched peaks were mainly in ribonucleoprotein complex biogenesis, ribosome biogenesis, establishment of protein localization to organelle, and involved in regulation of receptor signaling pathway via JAK-STAT and receptor signaling pathway via JAK-STAT. Differential expressed genes were Irf1, Tnfrsf1a, S ocs1, Notch1, Gadd45a, Hes1 and Socs3, involving in the regulation of JAK-STAT signaling pathway. KEGG enrichment analysis showed that the differentially enriched peaks were mainly in amyotrophic lateral sclerosis, and the target genes were Mcl1, Egfr, Socs1, Cdkn1a, Pdgfa and Socs3, involving in the regulation of JAK-STAT signaling pathway. Compared with ADR group, the mRNA and protein expression levels of JAK2 and STAT3 in the ADR+GSK-J4 group were significantly lower, and the downstream inflammatory factors of IL-6, MCP-1 and α-SMA were significantly higher (all P<0.05). Compared with ADR group, the protein expression level of nephrin in ADR+GSK-J4 group was higher ( P<0.05), and the protein expression level of nephrin in ADR+C-A1 group was lower ( P<0.05). Compared with ADR+GSK-J4 group, the protein expression level of nephrin in ADR+GSK-J4+C-A1 group was lower ( P<0.05). (2) Animal experiments: Compared with EZH2ctrl+FSGS group, EZH2podKo+FSGS group showed obvious renal tissue damage, matrix hyperplasia in mesangial area with massive homogeneous substance deposition, apoptosis and necrosis of renal tubular epithelial cells, obvious thickening and extensive fusion of glomerular epithelial cells, basement membrane collapse, and compression and narrowing of capillary structure. Compared with EZH2ctrl+FSGS group, the protein expression level of H3K27me3 in EZH2podKo+FSGS group was significantly lower, and the mRNA and protein expression levels of JAK2 and STAT3, and the levels of IL-6, MCP-1, α-SMA and TGF-β1 were higher (all P<0.05). Conclusions:Abnormal methylation modification of H3K27 leads to change of target gene expression, activation of JAK2-STAT3 signaling pathway, podocyte injury, glomerulosclerosis and renal tubular injury, participating in the development of FSGS.

Objective:To analyze the target signaling pathway of histone H3K27 methylation-induced podocyte injury, verify the regulatory effect of histone H3K27 methylation on podocyte injury in focal segmental glomerulosclerosis (FSGS) mice through target signaling pathway, and explore the mechanism of abnormal methylation of histone H3K27-induced podocyte injury in FSGS mice.Methods:(1) Cell experiments: primary cultured immortalized mouse podocytes MPC5 were cultured in vitro, and divided into control group, adriamycin (ADR) group, ADR+GSK-J4 (histone demethylase, KDM6B inhibitor) group, ADR+coumarin A1 (C-A1, JAK2 agonist) group and ADR+GSK-J4+C-A1 group. The transmission electron microscope was used to observe ultrastructure of podocytes. Immunofluorescence was used to detect the protein expression of H3K27me3 and nephrin in podocytes. The whole genome sequence of podocytes was obtained, the differentially expressed genes were screened, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for enrichment analysis. Real time-quantitative PCR and Western blotting were used to detect the gene and protein expression of JAK2-STAT3 signaling pathway in podocytes respectively. Enzyme-linked immunosorbent assay was used to detect interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1). (2) Animal experiments: EZH2 gene knock out ( EZH2podKo)-FSGS (tail vein injection of ADR) mouse models were established, and divided into EZH2ctrl+control group ( n=20), EZH2ctrl+FSGS ( n=20), EZH2podKo+control group ( n=30) and EZH2podKo+FSGS group ( n=30). HE staining was used to observe the morphology of kidney tissues. Immunohistochemistry was used to detect the H3K27me3 protein expression in podocytes. Real time-quantitative PCR and Western blotting were used to verify the gene and protein expression of JAK2-STAT3 signaling pathway respectively. Enzyme-linked immunosorbent assay was used to detect IL-6, MCP-1, α-SMA and TGF-β1 of kidney tissues. Results:(1) Cell experiments: Compared with control group, the nucleus shrank and ruptured, the cytoplasm showed vacuole, and mitochondria and endoplasmic reticulum swelled in ADR group, which verified that the podocyte injury model of ADR nephropathy was successfully established. Compared with control group, the protein expression level of H3K27me3 in ADR group was significantly lower ( P<0.05). Compared with ADR group, the protein expression level of H3K27me3 in podocytes in ADR+GSK-J4 group was significantly higher ( P<0.05), and there were 502 increased genes and 443 decreased genes. GO enrichment analysis showed that the differentially enriched peaks were mainly in ribonucleoprotein complex biogenesis, ribosome biogenesis, establishment of protein localization to organelle, and involved in regulation of receptor signaling pathway via JAK-STAT and receptor signaling pathway via JAK-STAT. Differential expressed genes were Irf1, Tnfrsf1a, S ocs1, Notch1, Gadd45a, Hes1 and Socs3, involving in the regulation of JAK-STAT signaling pathway. KEGG enrichment analysis showed that the differentially enriched peaks were mainly in amyotrophic lateral sclerosis, and the target genes were Mcl1, Egfr, Socs1, Cdkn1a, Pdgfa and Socs3, involving in the regulation of JAK-STAT signaling pathway. Compared with ADR group, the mRNA and protein expression levels of JAK2 and STAT3 in the ADR+GSK-J4 group were significantly lower, and the downstream inflammatory factors of IL-6, MCP-1 and α-SMA were significantly higher (all P<0.05). Compared with ADR group, the protein expression level of nephrin in ADR+GSK-J4 group was higher ( P<0.05), and the protein expression level of nephrin in ADR+C-A1 group was lower ( P<0.05). Compared with ADR+GSK-J4 group, the protein expression level of nephrin in ADR+GSK-J4+C-A1 group was lower ( P<0.05). (2) Animal experiments: Compared with EZH2ctrl+FSGS group, EZH2podKo+FSGS group showed obvious renal tissue damage, matrix hyperplasia in mesangial area with massive homogeneous substance deposition, apoptosis and necrosis of renal tubular epithelial cells, obvious thickening and extensive fusion of glomerular epithelial cells, basement membrane collapse, and compression and narrowing of capillary structure. Compared with EZH2ctrl+FSGS group, the protein expression level of H3K27me3 in EZH2podKo+FSGS group was significantly lower, and the mRNA and protein expression levels of JAK2 and STAT3, and the levels of IL-6, MCP-1, α-SMA and TGF-β1 were higher (all P<0.05). Conclusions:Abnormal methylation modification of H3K27 leads to change of target gene expression, activation of JAK2-STAT3 signaling pathway, podocyte injury, glomerulosclerosis and renal tubular injury, participating in the development of FSGS.

Objective To investigate the relationship between serum levels of vitamin D(VD)and brain derived neurotrophic factor(BDNF)and restless leg syndrome(RLS)in maintenance hemodialysis patients.Methods A total of 168 end-stage renal disease patients admitted to the hospital for maintenance hemodialy-sis treatment from May 2021 to May 2022 were regarded as the observation group.According to whether they had concurrent RLS,they were separated into concurrent RLS group and non concurrent RLS group.In addi-tion,121 healthy volunteers who came to the hospital for physical examination were regarded as the control group.Enzyme-linked immunosorbent assay(ELISA)was applied to detect the levels of VD and BDNF in ser-um,multivariate Logistic regression was applied to analyze the influencing factors of RLS in maintenance he-modialysis patients.Receiver operating characteristic(ROC)curve was plotted to analyze the predictive value of single and combined detections of VD and BDNF for RLS in maintenance hemodialysis patients.Results Compared with the control group,the serum levels of VD and BDNF in the observation group were reduced(P<0.05).Compared with the non concurrent RLS group,the continuous dialysis time and serum ferritin level in the non concurrent RLS group were increased,while serum VD and BDNF levels were reduced(P<0.05).Multivariate Logistic regression analysis showed that VD and BDNF were protective factors for RLS in maintenance hemodialysis patients,while continuous dialysis time and ferritin were risk factors for RLS in maintenance hemodialysis pa-tients(P<0.05).The area under the curve of the combined prediction of VD and BDNF for RLS in mainte-nance hemodialysis patients was 0.889,which was superior to those of single detection(Zcombination-VD=3.748,Zcombination-BDNF=2.245,P<0.05),and the sensitivity of the combined detection was 86.11%,and the specificity was 79.55%.Conclusion The levels of VD and BDNF in serum are protective factors for RLS in maintenance hemodialysis patients.The combination of the two could effectively predict RLS in maintenance hemodialysis patients.

The stenosis and embolization of internal fistula vessels directly affect the clinical treatment effect of maintenance hemodialysis patients, and the study of the mechanism of internal fistula stenosis has become a research hotspot in recent years. Previous studies mainly focused on the hemodynamics and pathophysiology of blood vessel wall, and there were few studies on molecular biology and its related signaling pathways. This paper reviews the hemodynamics of the vascular pathway of internal arteriovenous fistula (AVF), the pathophysiological mechanism, molecular biology, and changes in various signaling pathways of AVF dysfunction at home and abroad, in order to provide references for the study of AVF dysfunction.

This study aims to establish a rat model of renal ischemia reperfusion injury (RIRI) to observe the alterations in the expression of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway following various exosome treatments. Additionally, differential miRNA expression analysis will be conducted to elucidate the molecular mechanisms underlying the effects of exosomes derived from ischemic preconditioned (IPC) renal tubular cells in mitigating RIRI in rats. Initially, ten SD rats were subjected to bilateral nephrectomy under general anesthesia to prepare primary renal tubular cells. The second-generation renal tubular cells were then subjected to the following treatments for 12 hours: normoxia (38% O 2, 5% CO 2), hypoxia (1% O 2, 5% CO 2), and hypoxia plus inactivation (heated at 65 ℃ for 30 minutes). Following these treatments, exosomes were extracted, yielding normoxic exosomes, IPC exosomes, and inactivated exosomes, respectively. A subsequent cohort of 50 SD rats was randomly divided into five groups: Sham group, RIRI group, RIRI + normoxic exosome group (NC group), RIRI + IPC exosome group (IPC group), and RIRI + inactivated exosome group (INA group). RIRI model was established in the latter four groups. Twenty-four hours after RIRI modeling, the NC, IPC, and INA groups received intravenous injections of 200 μg of normoxic exosomes, IPC exosomes, and inactivated exosomes via the tail vein, respectively. Six days later, venous blood samples were collected, and both kidneys were excised to observe renal function, histopathological changes in kidney tissue, and alterations in the PI3K/AKT/mTOR signaling pathway among the five groups. Furthermore, differential miRNA expression analysis [ P<0.05, |log 2(Fold Change)|≥1] was conducted between the NC and IPC groups to investigate the changes in the miRNA expression profile. Subsequently, GO analysis and KEGG pathway enrichment analysis were performed. The results revealed that: (1) Compared with the Sham group, the RIRI and INA groups exhibited elevated levels of serum creatinine and urea nitrogen (all P<0.01). Histopathological examination of kidney tissues showed substantial inflammatory cell infiltration in the interstitium accompanied by varying degrees of edema, degenerative swelling of tubular structures, necrosis, and detachment of tubular epithelial cells. Notably, the number of TUNEL-positive cells was significantly increased, while the number of Ki67-stained positive cells was markedly decreased. Additionally, the mRNA and protein expression of PI3K/AKT/mTOR signaling pathway in RIRI group and INA group were down-regulated. (2) Compared to the NC group, the IPC group demonstrated lower levels of serum creatinine and urea nitrogen (both P<0.01). Notably, there was a significant decrease in the accumulation of inflammatory cells in the renal interstitium, and tissue edema was markedly improved. Moreover, the number of TUNEL-positive cells was reduced, while the number of Ki67-stained positive cells was significantly increased. Additionally, the mRNA and protein expressions of PI3K, PDK1, AKT, and mTOR were all up-regulated (all P<0.05). (3) Compared to the NC group, 56 miRNAs were up-regulated and 42 miRNAs were down-regulated in the IPC group. The target genes of GO enrichment analysis were PIK3C2A, PIK3CA, PIK3CB, PIK3CD, PIK3C2G, AKT1, mTOR, Rheb, and KEGG enrichment analysis revealed significant enrichment in PI3K/AKT signal pathway and mTOR signal pathway. In conclusion, this study reveals that during the course of RIRI, exosomes derived from IPC renal tubular cells induce differential miRNA expression in kidney tissues, resulting in enhanced expression of the PI3K/AKT/mTOR signaling pathway, which plays a pivotal role in mitigating RIRI in rats.

Purpose To investigate the expression of chiti-nase-3-like protein 1(CHI3L1)in esophageal squamous cell carcinoma(ESCC)and its correlations between CHI3L1 and molecules associated with the Wnt/β-catenin signaling pathway.Methods Paraffin-embedded tissue specimens from 119 ESCC patients and 60 normal esophageal tissues were retrospectively selected.Immunohistochemistry was used to detect the protein expression levels of CHI3L1 and Wnt/β-catenin signaling path-way related molecules in the above tissues,and the ectopic pro-tein expression levels of CHI3L1,TCF4,Cyclin D1,APC andβ-catenin in ESCC patients'tissues and normal esophageal tis-sues were compared.The survival of ESCC patients was followed up.Results Compared with normal esophageal tissue,the pos-itive rates of CHI3L1,TCF4,Cyclin D1 and β-catenin in ESCC were higher(63.0％,69.7％,68.1％and 67.2％,respec-tively),and the positive rates of APC were lower(41.2％).The expression levels of CHI3L1,TCF4,Cyclin D1 and β-cate-nin were positively correlated with each other(P＜0.05),but negatively correlated with APC(P＜0.05).CHI3L1 protein was associated with ESCC tumor volume,invasion depth,lymph node metastasis,and pTNM stage(P＜0.05).High expression of CHI3L1,TCF4,Cyclin D1 or abnormal high expression of β-catenin protein was associated with poor prognosis in ESCC pa-tients,suggesting shortened survival time(Log-rank=10.121,Log-rank=34.896,Log-rank=34.923,Log-rank=4.082,all P＜0.05).Conclusion In ESCC,the high expression of CHI3L1 is closely related to the activation of Wnt/β-catenin sig-naling pathway,suggesting that CHI3L1 may participate in the occurrence and development of ESCC through abnormal activa-tion of Wnt/β-catenin signaling pathway.

Purpose To investigate the expression of chiti-nase-3-like protein 1(CHI3L1)in esophageal squamous cell carcinoma(ESCC)and its correlations between CHI3L1 and molecules associated with the Wnt/β-catenin signaling pathway.Methods Paraffin-embedded tissue specimens from 119 ESCC patients and 60 normal esophageal tissues were retrospectively selected.Immunohistochemistry was used to detect the protein expression levels of CHI3L1 and Wnt/β-catenin signaling path-way related molecules in the above tissues,and the ectopic pro-tein expression levels of CHI3L1,TCF4,Cyclin D1,APC andβ-catenin in ESCC patients'tissues and normal esophageal tis-sues were compared.The survival of ESCC patients was followed up.Results Compared with normal esophageal tissue,the pos-itive rates of CHI3L1,TCF4,Cyclin D1 and β-catenin in ESCC were higher(63.0％,69.7％,68.1％and 67.2％,respec-tively),and the positive rates of APC were lower(41.2％).The expression levels of CHI3L1,TCF4,Cyclin D1 and β-cate-nin were positively correlated with each other(P＜0.05),but negatively correlated with APC(P＜0.05).CHI3L1 protein was associated with ESCC tumor volume,invasion depth,lymph node metastasis,and pTNM stage(P＜0.05).High expression of CHI3L1,TCF4,Cyclin D1 or abnormal high expression of β-catenin protein was associated with poor prognosis in ESCC pa-tients,suggesting shortened survival time(Log-rank=10.121,Log-rank=34.896,Log-rank=34.923,Log-rank=4.082,all P＜0.05).Conclusion In ESCC,the high expression of CHI3L1 is closely related to the activation of Wnt/β-catenin sig-naling pathway,suggesting that CHI3L1 may participate in the occurrence and development of ESCC through abnormal activa-tion of Wnt/β-catenin signaling pathway.

Objective:Clamping bilateral renal arteries with refined surgical methods to establish the rat renal ischemia-reperfusion injury (RIRI) model, and study the protective mechanism of ischemic preconditioning renal (IPC) tubular cell-derived exosomes in RIRI.Methods:25 female Sprague Dawley (SD) rats were divided into sham group, model group, inactivated group, normoxic group, IPC group. In the sham operation group, after bilateral renal arteries were dissociated, the back incision was disinfected and closed. The model group established RIRI model; RIRI models were established in inactivated group, normoxia group and IPC group, and then 200 μg of inactivated exosomes, normal exosomes and IPC exosomes were injected into the caudal vein 24 hours after operation. Serum creatinine (Scr) and urea nitrogen (BUN) levels were detected. The pathological changes of renal tissue were observed under light microscope. Transmission electron microscopy (TEM) was used to observe the shape and size of renal tubular exosomes. Nanoparticle tracking analysis (NTA)was used to detect the concentration and size of renal tubular exosomes.Results:Compared with the sham group, the Scr and BUN levels in the model group were significantly elevated ( P<0.01). Renal pathological changes in the model group showed damaged of the tubular structure, necrosis and shedding of tubular epithelial cells, and a large number of inflammatory cells accumulated in the renal interstitial tissue with varying degrees of edema. Compared with the inactivated group, the Scr and BUN levels significantly decreased in the normoxic group and IPC group ( P<0.01). Renal pathological changes in the normoxic group and IPC group showed that the renal tubular cell necrosis alleviated, inflammatory was reduced, the improved edema. Compared with the normoxic group, the Scr and BUN levels in the IPC group were further reduced ( P<0.01). Renal pathological changes in the IPC group showed that the inflammatory cells were significantly reduced, the cell edema was significantly improved, and the cell apoptosis was significantly reduced. Conclusions:Clamping bilateral renal arteries with refined surgical methods is the main and optimal way to build a rat model of RIRI. IPC tubular cell-derived exosomes have protective and repair effects on RIRI.

Objective:To establish a mouse model of intra-jugular arteriovenous fistula (AVF) to screen differentially expressed genes in the process of intimal stenosis of AVF for investigating the abnormal expression signaling pathways and the mechanisms.Methods:Forty-six male C57BL/6 mice were randomly divided into AVF group ( n=23) and sham-operated group ( n=23). The AVF group underwent internal jugular arteriovenous fistuloplasty, and the sham-operated group separated the right external jugular vein and common carotid artery and then sutured the incision. The whole-genome sequences of mice with AVF stenosis were determined by transcriptomic reversible chain terminator and synthetic sequencing. The microarray data set was established, and the Benjamini & Hochberg method of gene microarray data analysis was applied to screen the differentially expressed genes. The differentially expressed genes were screened by R-language enrichment analysis. Then, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) were performed. The subcellular localization of the differentially expressed genes was performed by BUSCA software. The protein network interaction of differentially expressed genes was analyzed by using STRING database and Cytoscape software. Results:In the AVF group, 21 mice were successfully modeled and 2 mice failed. Therefore, there were 21 mice in the AVF group and only 21 mice in the sham-operated group. This mouse internal jugular AVF model was innovated using the continuous-interrupted suture method, which improved the success rate of modeling this model. The differential gene sequencing analysis showed that there were 2 514 differentially expressed genes in the AVF process, including 1 323 up-regulated genes and 1 191 down-regulated genes. GO functional enrichment analysis showed that the differential genes were mainly enriched in metabolic process, activation, redox, mitochondria and so on. KEGG pathway enrichment analysis showed that the differential genes were enriched in metabolism, energy substance synthesis, diabetes, oxidative stress and so on. Statistical analysis of subcellular localization showed that the differences were mainly in mitochondrial proteins (24.24%), cytoplasmic proteins (17.51%), nuclear proteins (13.13%), cell membrane proteins (11.45%), and extracellular proteins (10.77%).Conclusions:Mitochondrial oxidative stress injury may be involved in the pathological damage process of endothelial proliferation stenosis in the AVF.

Objective:To study the clinical effect of vacuum sealing drainage in the treatment of chronic traumatic subcutaneous hematoma.Methods:Patients of chronic traumatic subcutaneous hematoma who were admitted to the Department of Burns and Plastic Surgery of Jiaozhou Central Hospital of Qingdao from June 2018 to June 2021 were included and randomly divided into the control group and the experimental group according to the random number table method. The subcutaneous hematoma was incised for debridement, and the blood clots, exudates, necrotic tissues, and pseudosynovium in the cavity were removed. The control group was treated with vaseline oil gauze filling drainage and dressing change method, and the experimental group was treated with vacuum sealing drainage and dressing change method. During the treatment, the closing time of subcutaneous hematoma cavity was observed and compared between the two groups.Results:A total of 42 patients with chronic traumatic subcutaneous hematoma were enrolled, 21 in the control group and 21 in the experimental group, including 11 males and 10 females in the control group, aged (46.2±12.4) years; 13 males and 8 females in the experimental group, aged (44.3±10.6) years. After treatment, all the subcutaneous hematoma spaces were closed in the 42 patients. The closing time of subcutaneous hematoma cavity in the experimental group was (15.52±1.69) days, which was significantly shorter than that in the control group (24.14±2.57) days. There was a significant difference between the two groups ( P<0.01). Conclusions:After incision and debridement of chronic traumatic subcutaneous hematoma, vacuum sealing drainage and dressing change can actively and fully drain the exudate in the cavity, residual blood clots, necrotic tissues, and pseudosynovium, promote the growth of new granulation tissue, which is more conducive to the closure of the cavity of subcutaneous hematoma, and shorten the clinical treatment cycle.