The Effect Of Astragaloside Iv On Oxidative Stress Induced Apoptosis Of Umbilical Cord Mesenchymal Stem Cell

Objective:Myocardial infarction?MI?patients suffering from myocardial cell necrosis and scar formation due to interrupted coronary blood flow.Traditional methods such as drugs,interventions and surgical operations can effectively alleviate symptoms and pain and reduce mortality,but can't fundamentally regenerate myocardial tissue,eventually leading to heart failure and even deathwhich seriously affect human health.In recent years,stem cell therapy has brought the new dawn for the treatment of MI because of their high proliferation and multi-directional differentiation potential.Umbilical cord mesenchymal stem cells?UC-MSCs?are easy to be isolated and amplified and have very low immunogenicity and tumorigenicity allowing them to become one of the ideal seed cells for the treatment of MI.However,stem cells implanted into infarcted myocardium face oxidative stress and hypoxic-ischemic microenvironmentcausing to death of most implantedcells which can'tadequately exert the therapeutic effection.Therefore,how to effectively increase the survival ability of stem cells in ischemic microenvironment isbecoming one of the important issues to solve the problem of low curative effect on stem cell therapy in MI.Many emerging studies have shown that Astragaloside IV?AS-IV?has significant effect of anti-oxidative stress,anti-apoptosis,anti-fibrosis and angiogenesis effects in cardiovascular diseases.Therefore,we focuse on the low survival rate of stem cell inMI and investigated whether AS-IV could inhibit the UC-MSC apoptosis induced by oxidative stress which will lay important basis for the further study of mechanism of AS-IV and the promotion of the clinical application of stem celltreatment of MI.Results:1.The isolation,culture and amplification of UC-MSCs.UC-MSCswere isolated and cultured by tissue explants adherent method.After about 1 week,UC-MSCs crawled out of the adherent tissues.After 8¹0 days of culture,UC-MSCsbegan to proliferate rapidly?Passage 0?.When thecell fusion rate reached about 90%,UC-MSCs were digested by trypsinase and passaged to obtain fibroblast-like cells showing uniform distribution and spiral growth?Passage 1?.Passage 1 cells were cultured and amplified to obtain passage 2 or3 which also presented uniform distribution form and spiral growthstatus.2.Flow cytological detection of surface specific antigen of UC-MSCs.Flow Cytometer was used to test the surface specific antigen of passage 3UC-MSCs showing positive expression of CD90,CD73,CD44?greater than95%?and negative expression of HLA-DR,CD34,CD90?less than 5%?.3.Cell activity detection of UC-MSCs.Flow cytometric analysis of apoptotic cells by 7-AAD indicated no significantly difference of apoptotic cells between blank-control group and 7-AAD group?<2%?.Doubling times were calculated by growth curve exhibited no marked change among different passages of UC-MSCs?passage1,passage2 and passages3?.4.The adverse effect of AS-IV and its solvent-0.1%DMSO on the growth of umbilical cord mesenchymal stem cellsweredetected.UC-MSC s weretreatedwithdifferent concentrations of AS-IV?0?10?25?50?100?M?and 0.1%DMSO for 24 hours.Then,the flow cytometric analysi s by Annexin?-FITC and propidine iodide?PI?stainingand CCK 8 assa y demonstrated no significant difference in the percent of survival cells comparing to control group.5.H₂O₂ induced UC-MSC apoptosis in does-and time-dependent manner.UC-MSCs were treated with a series of concentrations of H₂O₂?0,50,100,200?M?for 6 h.The results of Annexin?-FITC and propidine iodide?PI?staining indicated increased rate of early apoptic cells?Annexin V+/PI^<sub>?,late apoptotic cells?Annexin V+/PI+?and necrotic cells?Annexin V-/PI+?and reduced proportion of survival cells to about 85%?Annexin V-/PI-?.Western blottling detection of cleaved-caspase3 protein in different time course?0,1,3,6 h?stimulated by 200?MH₂O₂ showed significantly increased level in 6h groupthanother groupswhich were normalized to?-actin protein.6.AS-IV inhibited H₂O₂-induced UC-MSC apoptosis.UC-MSCs were pretreated with different concentrations of AS-IV?0?10?25?50?100?M?for24h before exposure to 200?MH₂O₂for 6h.Cell apoptosis was determined by Annexin?-FITC and propidine iodide?PI?staining and detection of mRNA expression level ofcaspase 3 by qRT-PCR.Annexin?-FITC and PI analysisindicated that 50?MAS-IV could significantly inhibit early apoptotic cells?Annexin V+/PI-?and late apoptotic cells?AnnexinV+/PI+?and demonstratedelevated proportion of survival cells?Annexin V-/PI-?.qRT-PCR analysis of exhibited decreased mRNA level of caspase3.Conclusions:Based on the establishment of oxidative stress damage cell model,we found that AS-IV can significantly inhibit the apoptosis of UC-MSCs induced by H₂O₂which provides a new strategy for the treatment of MI.