Roles Of Endogenous Hydrogen Sulfide In Hypoxia/sd-induced Apoptosis Of Mesenchymal Stem Cells And Its Probable Mechanism

Background:Acute myocardial infarction(AMI) has been a leading cause of morbidity and mortality in human. Current therapy including drug treatment, percultaneous coronaryintervention(PCI) and coronartery by pass graft(CABG) mainly aim at saving the viable cardlomyoeytes, improving the function of survival cardiomyoeytes and reversing ventricular remodeling. The ability to regenerate damaged myocardium presents a major challenge in the treatment of myocardium infarction. With the development of stem cells and tissue engineering, transplantation of stem cells has been proposed a strategy for cardiac repair following myocardial damage.Bone marrow mesenchymal stem cells(MSCs), the early cell of mesoderm, which can keep the potent plastieity of differentiate to multi-tissue have the advantage of can be isolated easily from a variety of sources, genetiestability, no immunologic and ethical concerns. So they were considered as ideal candidate donor cells for stem cell therapy and target cells for gene transfer. However, Poor cell viability after transplantation seriously limited the therapeutic efficiency of MSCs. Mounting evidence has demonstrated that MSCs undergo apoptosis soon after transplantation, which significantly reduces their effectiveness in tissue repair and regeneration. Thus,identifying the mechanisms relative to MSCs apoptosis and promoting the survival of transplanted MSCs may be vital for successful utilization in cell therapy.Hydrogen sulfide(H2S), a poisonous gas used as chemical reagent with an odour of rotten eggs, has recently been proposed to be the third signaling gastransmitter besides nitric oxide and carbon monoxide. H2 S has wide biological effects and plays very important roles in regulating multiple organ’ functions, such as central nervous system, respiratory system and cardiovascular system. In mammalian tissues, endogenous H2 S from L-cysteine is catalyzed primarily by two enzymes, cystathionine gamma-lyase(CSE) and cystathionine beta-synthase(CBS). Accumulating evidence suggests that both exogenously applied H2 S and inhibition of endogenous H2 S production have been shown to regulate cell apoptosis in various systems such as cardiovascular, Nervous, respiratory and pancreatic systems. Recently, the finding from Xie et al suggests that H2 S preconditioning protects MSCs against hypoxia and serum deprivation(hypoxia/SD)-induced apoptosis in vitro and enhances the efficacy of MSCs transplantation in a rat model of acute myocardial infarction. However, the production of endogenous H2 S in MSCs and its regulatory effect on MSCs apoptosis have not been completely understood.In the present study, we established an apoptosis model induced by hypoxia/SD to determine whether H2 S could be endogenously generated by MSCs, and investigate the role of endogenous H2 S in hypoxia/SD-induced apoptosis of MSCs. Our finding indicate that H2 S generation pathway exists in MSCs and inhibition of endogenous CSE/H2 S system contributes to hypoxia/SD-induced apoptosis of MSCs.Objective: Endogenous hydrogen sulfide is recognized as a new signaling gastransmitter which take part in the regulation of physiology and diseases in multiplesystems. The aim of this study was to explore the effect of endogenous hydrogen sulfide on hypoxia and serum deprivation(hypoxia/SD)-induced apoptosis in MSCs and its probable mechanism.Methods:(1) MSCs were isolated, purified and expanded in vitro, by western blots we determined the intracellular protein levels of cystathionine gamma-lyase(CSE) and cystathionine beta-synthase(CBS).(2) To culture MSCs and develop the model of hypoxia indueed apoptosis of MSCs in vitro. by using flow cytometry analysis, the N,N-dimethyl-p-phenylenediamine sulphate(NNDPD) method, western blots, we studied the effects of hypoxia/SD-induced cell apoptosis on endogenous H2 S production, cystathionine gamma-lyase(CSE) expression and activity in MSCs.(3) To construct CSE lentivirus vector and transfect with MSCs, by using flow cytometry analysis, the N,N-dimethyl-p-phenylenediamine sulphate(NNDPD) method, western blots, we studied the effects of CSE overexpression on hypoxia/SD-induced decrease of endogenous H2 S generation and apoptosis in MSCs. Meantime we investigated the effects of PAG, a blocker of CSE activity on hypoxia/SD-induced decrease of endogenous H2 S generation and apoptosis in MSCs.(4) To construct CSE lentivirus vector and transfect with MSCs, Western blots were utilized to determine the effect of CSE overexpression on the protein levels of Cyt c、Bcl-2、Bax、GRP78、CHOP and p Akt、Akt in MSCs after hypoxic/SD treatment.Results:(1) Under basal conditions, CSE, but not CBS, was detected in MSCs, The activity of H2 S synthesis in MSCs was significantly inhibited by pretreatment with PAG prior to the application of L-cysteine. On the other hand, pretreatment with AOAA had no effect on the level of H2 S production.(2) An apoptosis model induced by hypoxia/SD was successfully set up, treatment of MSCs with hypoxia/SD significantly induced the apoptosis of MSCs in a time-dependent manner, and after exposure of MSCs to hypoxia/SD the content of H2 S in culture supernatant was time-dependentlydecreased. At the same time, after treatment of MSCs with hypoxia/SD, CSE expressions were decrease in a time-dependent manner. Consistent with the results of CSE expression, the activity of CSE in MSCs was decreased by treatment with hypoxia/SD in a time-dependent manner.(3) CSE overexpression was mediated by Lentiviral transduction in MSCs. Western blot analysis showed that CSE expression in the MSCs infected by p LV-Zs Green-CSE lentivirus(CSEMSCs) was upregulated by more than 2.4-fold compared with MSCs infected by Plv-Zs Green lentivus(GFPMSCs) or untransduced MSCs(NormMSCs) Afterward, the modified MSCs were treated under hypoxic/SD for 12 h. we found that CSEMSCs had a significant lower proportion of apoptosis compared with NormMSCs or GFPMSCs groups under hypoxic/SD for 12 h. Furthermore, the content of H2 S in culture supernatant was significantly increased in the CSEMSC group compared with the NormMSCs or GFPMSCs under hypoxic/SD for 12 h. However, cell apoptosis under hypoxic/SD were significantly deteriorated by pretreating MSCs with the CSE inhibitor PAG(5 mmol/ l). CSE inhibitor PAG(5 mmol/ l) had no effect on cell apoptosis. Furthermore, PAG(5 mmol/ l) not only reduced H2 S generation but also exacerbated the inhibition of H2 S generation elicited by hypoxic/SD for 12 h.(4) Western-blot analysis revealed CSEMSCs decreased cytochrome c accumulation in the cytosol but increased the protein levels of Bcl-2 compared with the NormMSCs or GFPMSCs under hypoxic/SD for 12 h. Furthermore, we found that CSEMSCs had a significant lower protein levels of Bax、GRP78、CHOP compared with NormMSCs or GFPMSCs groups under hypoxic/SD for 12 h. However, exposure of CSEMSCs to hypoxic/SD for 12 h significantly increased the phosphorylation of Akt compared with NormMSCs or GFPMSCs, the CSE inhibitor PAG(5mmol/L) could inhibit the phosphorylation of Akt in CSEMSCs under hypoxic/SD for 12 h.Conclusions:(1) MSCs can generate H2 S and CSE is the main enzyme involved in the generation of H2 S in MSCs.(2) Endogenous H2 S plays an important role in regulatinghypoxia/SD-induced apoptosis in MSCs.(3) Endogenous H2 S regulates hypoxia/SD-induced MSCs apoptosis through inhibition of the the mitochondrial apoptotic pathway, endoplasmic reticulum stress(ERS) pathway and activation of PI3K/Akt.