Pathway And Mechanism Of Improving Survival Rate And Efficacy Of Bone Marrow Mesenchymal Stem Cells In Ischemic Stroke

Background:Stem cell transplantation has always been the focus of many brain damage disease research,but transplantation efficiency is one of the main reasons for limiting the therapeutic effect of stem cells.Hypoxia-Inducible-Factor-1-alpha(HIF-1α)is a relatively sensitive factor in a low oxygen status.Several researches showed that HIF-la could affect proliferation and apoptosis of the tumor cells.However,there is no report about the role of HIF-1α in the transplant efficiency of curing brain stroke by transplanting bone marrow mesenchymal stem cells(BMSCs).In this research,by overexpressing HIF-1α,we want to study the effect of HIF-1α on BMSCs survival,and whether the HIF-1αoverexpressing could improve the transplanting efficiency of BMSCs and the function in curing ischemia stroke.Also we’re going to explore the releated mechanism between the HIF-1α overexpressing in BMSCs and the therapeutic efficiency in stroke model following the BMSCs transplantation.Method:This study is divided into the experiment in vitro and in vivo.In vitro,flow cytometry was used for identification of BMSCs with some of special markers such as CD29,CD90,CD 105,and CD45 mainly.BMSCs were infected with HIF-la green fluorescent protein(HIF-1α-GFP)lentiviral vector and exposed to oxygen-glucose deprivation(OGD).The main condition of OGD here includes the 37℃ thermostat with“50 mL/L CO2 + 950 mL/L N2" of mixture air for 30min in order to balance the pH,and the anaerobic sugar-free culture BMSCs for 2h.The survival rate of BMSCs was detected by methyl thiazolyl tetrazolium(MTT)assay.Flow cytometry and TUNEL were employed to assay the apoptosis.The ultrastructure of autophagy in BMSCs were detected by transmission electron microscopy(TEM).The protein expressions of HIF-1 a,apoptosis-related protein and autophagy-related protein were detected by Western Blot.The signal transduction pathway was blocked by adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)inhibitor Compound C to test whether or not the HIF-1α regulates AMPK and mammalian target of rapamycin(mTOR)signal pathways.In vivo,the HIF-1α overexpressed BMSCs were transplanted into the the striatum of the rats with middle cerebral artery occlusion(MCAO),the volume of infarction was measured by methyl thiazolyl tetrazolium(TTC),the modified neurological severity score(mNSS)was used to evaluate the effect of transplantation,and the western blot was used to test the expression of cytokins.Results:In vitro experiments,BMSCs isolated from Wistar rats were detected with flow cytometry to identify the surface markers of BMSCs,in which the CD59(96.72%),CD90(98.53%)and CD105(98.99%)were positive respectively.But the CD45(0.53%)expression was negative.The results of qRT-PCR showed that HIF-1α-GFP was stably transfected in BMSCs positively,and the mRNA and protein level of HIF-1α were significantly increased compared with the control group.The results of MTT,TUNEL,and flow cytometry showed that the survival rate of BMSCs was significantly decreased and cell apoptosis was significantly induced by OGD exposure,while the up-regulation of HIF-1α expression in BMSCs could significantly enhance the survival of BMSCs,inhibit cell apoptosis.The expressions of Caspase-9 and Bax proteins were up-regulated in BMSCs induced by OGD,and the expressions of Bcl-2 and Bcl-xl were down-regulated.The expression of apoptotic protein could be positively regulated by the overexpression of HIF-1α.The TEM examination of BMSCs showed that autophagosomes and autolysosomes were observed in HIF-1α overexpressed BMSCs,and the overexpression of HIF-1αpromoted the development of autophagy.The overexpression of HIF-1α in BMSCs promoted the expression of LC-Ⅱ and Beclinl,and inhibited the expression of P62.HIF-1α promoted the phosphorylation of AMPK in OGD-treated cells and inhibited the phosphorylation of mTOR,whereas AMPK phosphorylation inhibitor compound C reversed the effects described above.In vivo,compared with BMSCs,a significant increase in the number of GFP-positive BMSCs was observed after HIF-la overexpression,while the cerebral infarct volume was significantly lower,neurological score was better.Otherwise,some of factors such as tumor necrosis factor-a(TNF-α),interleukin 1β(IL1β),and IL-6 were lower,but others including brain-derived neurotrophic factor(BDNF)and vascular endothelial growth factor(VEGF)were significantly higher following the HIF-1α overexpression in BMSCs.Conclusion:The overexpressed HIF-la in BMSCs could enhance the survival and the functional efficiency of the grafted BMSCs themselves in the host after transplantation.Its overexpression plays an important positive role in the repair of brain tissue and the treatment of cerebral infarction,by improving both of the AMPK protein phosphorylation and the autohpagy but by inhibiting both of the mTOR phosphorylation and apoptosis of BMSCs.