Preliminary Study On The Mechanism Of H₂S Inhibiting Coronary Artery Endothelial Cell Injury In Kawasaki Disease

Objective: Although the pathogenesis of Kawasaki disease is unclear,previous studies have shown that H2 S levels was change in Kawasaki disease and H2 S plays a role in a variety of cardiovascular diseases.Therefore,HCAEC(human coronary artery endothelial cells)was used as the research object in the study.In vitro cell experiments were conducted to study the effect of H2 S on HCAEC in the Kawasaki disease microenvironment,and the mechanism of H2 S in coronary artery endothelial cell injury was preliminarily studied.Methods: First,HCAEC was treated with normal / KD serum to simulate the environment in normal and diseased conditions.Then different concentrations of Na HS were added to treat HCAEC with different time.At last the optimal treatment time and concentration of Na HS were determined by CCK8 experiment.HCAEC treated with normal serum was used to as the control,the potential reversal effect of H2 S on Kawasaki disease were determined by comparing the levels of HCAEC oxidative stress,inflammation and vascular injury between KD and KD + H2 S group.HCAEC comes from three groups were used to transcriptomes sequencing.Venn diagram of expressed genes,volcano diagram and heat map of differential genes,GO enrichment analysis and KEGG pathway enrichment analysis of differential genes were made and the differentially expressed gene THRIL was screened.QPCR experiment were use to detect the expression of differential gene screened by transcriptome sequencing.Transfection experiments were use to interfere or over-express the differential genes in HCAEC.Compared with the level of oxidative stress,inflammation and vascular injury to explore the role of the differential gene THRIL in coronary artery endothelial cell injury of Kawasaki disease.Results:1.Through CCK8 cell proliferation assay,the optimal concentration of Na HS on HCAEC was determined to be 100?M and the optimal inbuaction time was 24 h.2.The fluorescence value of HCAEC cells in the KD group was significantly higher than that in the normal group,and the p value was 0.014,indicating a significant difference.After Na HS treatment,the fluorescence value of KD+H2S was significantly lower than that of the KD group(p value was 0.0011).3.Compared with the normal group,the activity of SOD in HCAEC of the KD group was significantly decreased(p value was 0.0034).While compared with KD group,SOD activity in KD+H2S group was increased with a p value was 0.0226,showing significant difference.4.ELISA experiments showed that there was no significant difference in the content of IL-1? in the supernatant of the three groups of cells;the content of IL-6 in the KD group was higher than that in the normal group,and the difference was significant.Compared with the KD + H2 S group,there was no significant difference in the content of IL-6;the content of TNF-? increased in the KD group compared with the normal group,and the difference was significant;the content of TNF-? decreased in the KD + H2 S group compared with the KD group,and the difference was significant.5.Western blotting results showed that the expression of EDN-1,THBD and VWF was the highest in the KD group,significantly different from the normal group.Compared with the KD group,the expressions of the three proteins in the KD + H2 S group were down-regulated,and there were significant differences.6.Transcriptome sequencing results were analyzed and Venn diagram of expressed gene,differential gene volcano diagram,heat map,differential gene GO function enrichment analysis and KEGG pathway enrichment analysis were successfully made.From those reults we can get:(1)The number of genes in HCAEC of the three groups is 12275,12115 and 11313.(2)Compared with the cell in the KD group,there are 165 differential genes,of which 111 genes are down-regulated and 54 genes are up-regulated.Compared with the KD group,the KD + H2 S group has a total of 1257 differential genes.There were 456 genes down-regulated and 801 genes up-regulated.(3)GO function enrichment analysis was performed on the differential genes between the normal group and the KD group,and the results showed that the differential genes were mainly concentrated in biological processes such as vascular system development regulation,vascular endothelial growth factor expression regulation,and protein binding;KD differential genes between the group and the KD + H2 S group mainly exist in biological processes related to immune response,kinase activation regulation,arterial development,cell proliferation and regulation and so on.(4)KEGG pathway enrichment analysis results show that the pathways involved in the differential genes between the normal group and the KD group mainly including the TGF-? signaling pathway,rheumatoid arthritis-related pathway,prion infection-related pathway and tuberculosis-related pathway.The pathways involved in the differential genes between the KD group and the KD + H2 S group mainly including PI3-Akt pathway,cell adhesion molecule pathway,platelet activation pathway,arrhythmogenic right ventricular cardiomyopathy-related pathways and interactions between ECM and receptors.(5)The cluster heat map results showed that there were 11 differential genes between the KD + H2 S group and KD group;there were 2 differential genes between the normal group and KD group.Both the differential genes between KD group and normal group or KD + H2 S group containing THRIL gene.7.QPCR showed that THRIL expression was increased in KD group and decreased in KD+H2S group,which was consistent with transcriptome sequencing results.8.Interference and overexpression experiments of THRIL gene showed that THRIL contributed in KD-induced the inhibition of SOD activity and the induction of inflammatory cytokines and vascular injury factors,while do not contributed in KD-induced the inhibition of cell proliferation and the production of reactive oxygen species.Conclusion: Hydrogen sulfide through attenuates the effect of KD serum on HCAEC by promoting the proliferation of cells,the activity of SOD and the expression of inflammatory factors to play an important role in the treatment of coronary arterial lesions in Kawasaki disease.Transcriptome sequencing screened the differential gene THRIL,THRIL may be a important regulator in KD serum inducing HCAEC cell injury and H2 S may play a role in HCAEC by regulating the expression of THRIL.