| Objective:To clarify the relationship between chloride channel protein(CLCN3)and inflammatory factors in sepsis,to investigate the effect of chloride channel protein on inflammatory factor release through NF-κB pathway using bioinformatics methods,in vitro cellular assays and clinical trials in sepsis patients,and to provide a basis for chloride channel protein as a a potential therapeutic target for sepsis.Methods:1.We searched the GEO database by typing "sepsis" in the search box and obtained the dataset GSE28750,which contained all the gene expression information of 10 sepsis patients and 20 healthy volunteers,and analyzed and compared the differential genes.The signaling pathways of the differential genes in sepsis inflammation were analyzed by KEGG enrichment.Finally,another dataset GSE65517 was selected to validate sepsis differential genes.2.5 ml of peripheral blood was collected from 20 patients who met the diagnostic criteria of sepsis and 10 healthy check-ups among patients admitted to the Emergency Intensive Care Unit of the First Affiliated Hospital of Nanchang University during 2022.9-2023.3,as experimental group(sepsis patients)and control group(healthy check-ups),respectively.mononuclear macrophages were extracted,and the expression of chloride channels and inflammatory factors IL-6,TNF-ɑ were detected by q-PCR,TNF-ɑ expression,and the expression of CLCN3 and IL-6 and TNF-ɑ were analyzed by spearman correlation.3.In vitro mononuclear cell model of sepsis was constructed and divided into LPS group,LPS+DIDS group and control group.The LPS group was stimulated with LPS for 6,12 and 24 hours,and the cells were extracted at 6,12 and 24 hours,respectively,and the expression of CLCN3,IL-6 and TNF-ɑ was detected by q-PCR technique,and graphs were made by Graphpad Prism9 software.The LPS+DIDS group stimulated the cell model with chloride channel protein blocker DIDS for 6 h.The expression of CLCN3,IL-6,TNF-ɑ was detected by q-PCR technique,and the expression of CLCN3 and NF-κB was detected by Werstern Blot technique,and the graphs were made by Graphpad Prism9 software.The control group was left untreated.To further investigate the relationship between CLCN3 and inflammation,THP-1 cells were transfected with synthetic CLCN3-specific small interfering RNA(Si-CLCN3)and divided into LPS group,L+S group,and control group,and the expression levels of CLCN3,IL-6,and TNF-ɑ were detected by q-PCR technique.Results:1,3800 differential genes were screened by raw letter analysis,1963up-regulated genes and 1837 down-regulated genes were screened,CLCN3 was the up-regulated gene among the differential genes,and the pathogenesis of sepsis was found to be highly correlated with NF-κB,MAPK and other signaling pathways by KEGG enrichment analysis.2.In the clinical study of sepsis patients,the q-PCR results suggested that the expression levels of CLCN3 and inflammatory factors IL-6 and TNF-ɑ were higher in the sepsis group than in the healthy physical examination patients,with significant differences(P<0.05),and the expression of CLCN3 was positively correlated with the expression levels of IL-6 and TNF-ɑ(R2=0.58,P=0.0004;R2= 0.35,P=0.0005),suggesting that CLCN3 may be involved in sepsis inflammation release.3.In an in vitro sepsis cell model,q-PCR results suggested that the expression of CLCN3,IL-6,and TNF-α also increased at 6h,12 h,and 24 h with the prolongation of LPS stimulation time,with significant differences between groups(P<0.05).Blocking CLCN with DIDS,q-PCR results suggested that the expression of CLCN3 decreased in parallel with IL-6 and TNF-ɑ expression;Werstern Blot results suggested that the expression of CLCN3 changed in the same direction as NF-κB expression.Silencing CLCN3 gene with Si-CLCN3,q-PCR results suggested that the expression of CLCN3 decreased with the expression of IL-6 and TNF-ɑ.It is suggested that CLCN3 may promote the release of inflammatory factors through the NF-κB pathway.Conclusion:CLCN3 promotes sepsis inflammatory factor release through NF-κB signaling pathway. |
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