Effect And Mechanism Of Hydrogen Sulfide On Liver Injury In Hyperglycemia And Hyperlipidemia Mice

Background:Hyperglycemia and hyperlipidemia are important risk factors for liver injury.Hydrogen sulfide(H2S),as a gas signaling molecule,plays an important role in the physiological and pathophysiology processes of liver.It has been reported that H2S can play a role in antagonizing ischemia-reperfusion,acetaminophen and carbon tetrachloride-induced liver injury in rodent.However,its precise role in liver injury caused by hyperglycemia and hyperlipidemia remains unknown.Objective:This study was designed to investigate the effect of H2S on liver injury in hyperglycemia and hyperlipidemia mice and to elucidate its underlying mechanisms.Methods and Results:In vivo,8-week-old healthy male LDLr-/-mice were injected with streptozotocin(STZ,60 mg/kg/day)for 5 consecutive days to construct a hyperglycemic model.When mice fasting blood glucose>300 mg/dL,hyperglycemic mice were administered the hydrogen sulfide donor GYY4137(50 mg/kg/day)keep on a high fat diet for 4-weeks.The changes of H2S levels in plasma were detected by the chemical methods.The changes of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in plasma were detected by the kit.We weighed body weights and liver weights of the mice.The changes of hepatic steatosis and lipid deposition were observed by hematoxylin(HE)and oil red staining.DHE staining was used to detect the change of ROS content in liver tissue.Western blot was used to detect the changes of Nrf2 signaling pathway in liver tissue of mice with hyperglycemia and hyperlipidemia.The results showed that the level of H2S was recuced,while the level of ALT and AST was significantly higher in mice with hyperglycemia and hyperlipidemia compared with control mice,meaning the severe injury in the liver.However,H2S donor GYY4137 increased H2S levels in plasma,and decreased ALT and AST level in hyperglycemia and hyperlipidemia mice,which means the degree of liver damage was reduced with the treatment of H2S donor GYY4137 in high glucose and hyperlipidemic mice,and improved liver function.H2S donor GYY4137 had no significant improvement in the liver weight and the ratio of liver weight to body weight in hyperglycemia and hyperlipidemia mice,Compared with control mice,the liver of vacuolar degeneration and lipid deposition were severe in mice with hyperglycemia and hyperlipidemia,while H2S donor GYY4137 did not significantly improve the liver tissue vacuolar degeneration and lipid deposition.H2S donor GYY4137 decreased the level of ROS by promoting nuclear translocation of Nrf2 and up-regulating the Nrf2-regulated downstream antioxidant protein y-glutamylcysteine catalytic subunit(GCLC),heme oxygenase 1(HO-1)and NAPDH quinone oxidoreductase 1(NQO1)in the liver of hyperglycemia and hyperlipidemia.In vitro,C57BL/6J(Wildtype,WT)and Nrf2-/-mouse primary liver parenchymal cells were extracted and treated with High-gluose(HG,25mmol/L)and oxidized low density lipoprotein(ox-LDL,50ug/mL)for simulating hyperglycemia and hyperlipidemia induced liver injury,while H2S group was pretreated with GYY413750 umol/L(G50)or 100 umol/L(G100)for 15 min.The change of ROS levels was detected by DHE staining.Western blot was used to detect the changes of Nrf2 signaling pathway.The results showed that H2S donor GYY4137 promoted Nrf2 nuclear translocation in HG and ox-LDL-treated mouse primary liver parenchymal cells from C57BL/6J mice.Moreover,we found that H2S donor GYY4137 decreased the level of ROS,and up-regulated the expression of Nrf2 target gens in HG and ox-LDL-treated mouse primary liver parenchymal cells from C57BL/6J mice but not Nrf2-/-mice.Biotin-Switch method was used to detect Keapl S-sulfhydration.The interaction between Keapl and Nrf2 was observed by immunoprecipitation method.Our results showed that H2S donor GYY4137 increased the level of Keapl S-sulfhydration and induced Nrf2 separation from Keap1 in HG and ox-LDL-treated mouse primary liver parenchymal cells from C57BL/6J mice.To further demonstrate the specific site of Keapl S-sulfhydration,we constructed Keapl-specific cysteine site mutant plasmids,transfected into HepG2 cells.The results showed that the site of Keap1 S-sulfhydration was mainly the cysteine site at position 151,and H2S donor GYY4137 failed to promote dissociation of Nrf2 from Keapl and Nrf2 nuclear translocation after Keapl Cysl51 mutant plasmids in HG and ox-LDL-treated HepG2 cells.Conclusion:The above results indicate that H2S plays a protective role in liver injury induced by hyperglycemia and hyperlipidemia,which may be related to inhibition of oxidative stress via promoting the dissociation of Keapl and Nrf2 by altering the level of Keap1 S-sulfhydration at Cys151,activating Nrf2 signaling pathway.