Study Of Hydrogen Sulfide On Diabetes-accelerated Atherosclerosis

Background:Hydrogen sulfide(H2S)has been shown to have powerful anti-oxidative and anti-inflammatory properties which may explain its regulatory effect in multiple cardiovascular functions.However,its precise role in diabetes-accelerated atherosclerosis remains unclear.Objective:To test the hypothesis that exogenous H2S can reduce diabetes-accelerated atherosclerosis and its mechanism underlying,which is partially dependent on regulation of Keapl-Nrf2 signaling pathway.Methods and Results:In vivo,diabetes was induced by injection of streptozotocin(STZ)into LDLr-/-and LDLr-/-Nrf2-/-mice.After STZ administration,diabetic mice were administered the hydrogen sulfide donor GYY4137(133 ?mol/kg/day,i.p.)and kept on a high fat diet for 4 weeks.Mice were housed in metabolic cages to metabolic analysis.Plasma lipid levels were measured enzymatically using commercial kits.Diabetic LDLr-/-mice fed a HFD had lower body weights,higher plasma total cholesterol,triacylglycerols,urinary output,water intake and food intake,and these effects were unaffected by treatment with H2S.The entire aorta and histological section were stained with Oil Red O(ORO)to measure aortic lesion area.Superoxide(O2·-)generation in aorta was measured by the O2·-sensitive fluorescent dye Dihydroethidium(DHE),as well as mRNA levels of Nrf2 target genes and proinflammatory molecules,measured by real-time PCR and immunofluorescence detection.As expected,H2S reduced aortic atherosclerotic plaque formation with reduction in O2·-generation and the adhesion molecules in STZ-induced LDLr-/-mice but not in LDLr-/-Nrf2-/-mice.In vitro,mouse peritoneal macrophages were isolated from C57BL/6 or Nrf2-/-mice.Macrophages,HUVECs or EA.hy926 endothelial cells were incubated with oxidized low density lipoprotein(ox-LDL,50 ?g/mL),D-glucose(25 mmol/L),in the absence or presence of different concentrations of GYY4137(50,100 ?mol/L)for 24 h.Foam cell formation assay was performed with the ORO staining.Superoxide generation was measured by DHE.Intracellular localization of Nrf2 was determined by immunofluorescence and western blot analysis.The expression of Nrf2 target genes and proinflammatory molecules were detected both by real-time PCR and western blot.EA.hy926 endothelial cells were transfected with siRNA against Nrf2 and detected the above indicators.We found that H2S inhibited foam cell formation,decreased O2·-generation,as well as increased Nrf2 nuclear translocation and consequently heme oxygenase-1(HO-1)expression up-regulation in high glucose plus ox-LDL-treated primary peritoneal macrophages from wild-type but not Nrf2-/-mice.H2S also decreased O2·-and adhesion molecules levels,increased Nrf2 nuclear translocation and HO-1 expression which were suppressed by Nrf2 knockdown in high glucose plus ox-LDL-treated endothelial cells.HO-1 knockdown by siRNA or inhibition by ZnPP(HO-1 inhibitor)abrogated H2S-mediated suppression of ROS production and foam cell formation induced by HG plus ox-LDL in endothelial cells and macrophage.Immunoprecipitation experiments were performed to determine the association of Nrf2 and Keap1 proteins.Keap1 S-sulfhydration was detected with"Tag-Switch" method.H2S increased S-sulfhydration of Keapl,induced Nrf2 dissociation from Keap1,enhanced Nrf2 nuclear translocation and inhibited O2·-generation which were abrogated after Keapl mutated at Cys151 in endothelial cells.Conclusion:H2S attenuates diabetes-accelerated atherosclerosis,which may be related to inhibition of oxidative stress via Keapl sulfhydrylation at Cys151 to activate Nrf2 signaling.This may provide a novel therapeutic target to prevent atherosclerosis in the context of diabetes.