The Protection And Mechanism Of GYY4137 Against Oxidative Stress-induced Endothelial Cells Injury

Objectives:Cardiovascular diseases have been the major contributor to mortality in modern days.Endothelial dysfunction,mainly induced by oxidative stress,has been discovered in various cardiovascular diseases.Therefore,inhibition of oxidative stress plays one of most important role in prevention and treatment of cardiovascular diseases.Hydrogen sulfide?H₂S?is a gasotransmitter found after nitric oxide?NO?and carbon monoxide?CO?.H₂S exerts wide physiological function in mammals,including human beings.In cardiovascular system,Cystathionine gamma-lyase?CSE?can catalyze H₂S generation,which contributes to vasodilation,inhibition of smooth muscle cell proliferation and protection against atherosclerosis,making itself the focus in cardiovascular research nowadays.Recent studies showed that H₂S can alleviate oxidative stress in ischemic-reperfusion and heart failure by upgrading transcription factor Nrf2.However,whether H₂S can mediate certain anti-oxidative factor to protect against oxidative stress and apoptosis in endothelium-dependent cardiovascular diseases remains unknown.In this research,the protective effect exerted by H₂S slow-released agent GYY4137against endothelial damage by reducing oxidative stress was studied,as well as its mechanism.Methods and results:EA.hy926 endothelial cells were treated with GYY4137 of different concentrations?12.5?M,25?M,50?M,100?M?for 4 h,which was followed by PBS wash-off step and hydrogen peroxide?H₂O₂,250?M?treatment for 4 h.It was found that morphology of endothelial cells was changed under microscopy.H₂O₂ can impair endothelial cell viability,which can be ameliorated by GYY4137pre-treatment?CCK-8 test kit?.Oxidative stress induced by H₂O₂ can be inhibited and anti-oxidative capability can be improved by GYY4137 as well,tested by DHE staining and SOD test kit.Using free radicals detector to monitor the NO content change in culture medium and found that H₂O₂ treatment can decrease NO level in endothelium,which was improved after treatment with GYY4137.By Hochest33342 staining and flow cytometry detection for duel-staining with ANNEXIN?and PI positive,it was observed that GYY4137 can ameliorate H₂O₂-induced apoptosis.It was found that it lowered more mitochondrial basal activity in H₂O₂treatment group,while GYY4137 pretreatment alleviated the effect using bio-energy detector.H₂S content in cell culture,H₂S formation activity and CSE level were tested with chemical method and western blotting,respectively.No significant change in H₂S content was found after GYY4137 treatment.CSE activity was reduced in H₂O₂ group with or without GYY4137 treatment,While CSE protein level was significant increased after being treated with H₂O₂,which was inhibited by GYY4137.MAPKs?P38,ERK,JNK?phosphorylation as well as total protein levels,IDH2,SOD2,were detected by western blotting.H₂O₂ led to significant increase of phosphorylation level of MAPKs?P38,ERK,JNK?,significant decrease of IDH2and SOD2,which can all be reversed by GYY4137.The mRNA levels of SIRT1-7respectively treated by H₂O₂ and/or GYY4137 were detected by RT-PCR and found that SIRT 1,3 and 4 mRNA expressions significantly decreased after H₂O₂.Among these three proteins,only the mRNA expression of SIRT 3 increased significantly after treated with GYY4137.After knocking down the expression of SIRT3 with siRNA,the protection of GYY4137 against oxidative stress was abrogated.Conclusion:GYY4137 protects endothelial cells against apoptosis and oxidative stress induced by H₂O₂ and maintains normal function of mitochondria by boosting mitochondria basal activity,both of which are realized by activating SIRT3,followed by activation of SOD2 and IDH2,as well as the inhibiton of activation of MAPKs.