Hydrogen Sulfide Protected Human Umbilical Vein Endothelial Cells Against High Glucose-induced Injuries And Inflammation By Inhibiting P38 MAPK/Necroptosis Pathways

World Health Organization issued a global diabetes report that the adult prevalence of diabetes in China closed to 10%in 2016.Diabetic vascular disease is the most common complication of diabetes,which pose enormous economic burden to the country and patients.Therefore,it is urgent to explore the pathogenesis of diabetic vascular disease and seek the effective therapeutic strategies and drugs for preventing and treating diabetic vascular complication.Objective(1)To explore the role of p38 MAPK/necroptosis pathways in HG-induced injuries and inflammation in HUVECs.(2)To explore whether exogenous H2S can protect HUVECs against the HG-induced injuries and inflammation by regulating p38 MAPK/necroptosis pathways.Method1.The cell counting kit 8(CCK-8)was used to determinate the cell viability of HUVECs.2.The expression levels of receptor-interacting protein 3(RIP3,the indication of necroptosis),cleaved caspase-3,caspase-9 and p38 MAPK were analyzed by western blot assay.3.Immunofluorescence was used to analyze the expression level of p3 8 MAPK.4.Intercellular ROS level was assessed by DCFH-DA staining followed by photofluorography.5.MMP was assessed by rhodamine 123(Rh123)staining followed by photofluorography.6.Morphological changes were assessed by Hoechst 33258 staining followed by photofluorography.7.Enzyme-linked immunosorbent assay(ELISA)was used to measure the level of inflammatory factors,including IL-1β,IL-6,IL-8 and TNF-α.8.RNA interference assay was performed to knock down the expression of RIP3.9.All data were expressed as mean±SEM and analyzed by SPSS20.0 version.Comparison between groups was assessed by one-way analysis of variance(ANOVA).The difference was considered to be statistically significant at P<0.05.Results1.p38 MAPK/necroptosis pathways contributed to HG-induced injuries and inflammation in HUVECs.1.1 High glucose(40 mmol/1,HG)up-regulated p-p38 MAPK expression level in HUVECs.1.2 HG increased RIP3 expression level in HUVECs.1.3 A siginificant decrease of cell viability in HUVECs was induced by HG in a dose-dependent manner.1.4 Nec-1(the inhibitor of necroptosis)and RIP3-siRNA,as well as SB203580(the inhibitor of p38 MAPK),attenuated the HG-induced cytotoxicity in HUVECs.1.5 Nec-1 and RIP3-siRNA,as well as SB203580 ameliorated HG-induced ROS generation in HUVECs.1.6 Nec-1 and RIP3-siRNA,as well as SB203580 alleviated HG-induced dissipation of MMP in HUVECs.1.7 Nec-1 and RIP3-siRNA,as well as SB203580 inhibited HG-induced secretion of inflammatory factors,including IL-1β,IL-6,IL-8 and TNF-a in HUVECs.1.8 There is a negative interaction between necroptosis and apoptosis in the HG-treated HUVECs.1.8.1 HG up-regulated the expression level of cleaved caspase-3 in HUVECs.1.8.2 Nec-1 upregulated the HG-induced increase in the expression levels of cleaved caspase-3 and caspase-9 in HUVECs.1.8.3 The inhibitory of apoptois,Z-VAD-FMK,upregulated the HG-induced increase in the expression level of RIP3 in HUVECs.1.9 There is a positive interaction between p3 8 MAPK and necroptosis pathway.2.H2S protected HUVECs against HG-induced injuries and inflammation by inhibiting p38 MAPK/necroptosis pathways.2.1 H2S down-regulated the HG-induced increase in expression of p-p38 MAPK in HUVECs.2.2 H2S alleviated the HG-induced increase in expression of RIP3 in HUVECs.2.3 H2S protected HUVECs against the HG-induced cytotoxicity through inhibiting p38 MAPK/necroptosis pathways,leading to an increase of cell viability.2.4 H2S reduced the HG-induced apoptosis in HUVECs.2.5 H2S protected HUVECs against the HG-induced reactive oxygen species(ROS)generation through inhibiting p38 MAPK/necroptosis pathways.2.6 H2S protected HUVECs against the HG-induced dissipation of mitochondrial membrane potential(MMP)through inhibiting p38 MAPK/necroptosis pathways.2.7 H2S protected HUVECs against the HG-induced inflammation through inhibiting p38 MAPK/necroptosis pathways.Conclusion1.p38 MAPK/necroptosis pathways contributed to HG-induced injuries and inflammation in HUVECs.2.H2S protected HUVECs against high glucose-induced injuries and inflammation by inhibiting p38 MAPK/necroptosis pathways.