The Neuroprotection Of Mesenchymal Stem Cells Mediated By Preventing Mutiple Cell Death Pathway In Vitro Model Of Cerebral Ischemia

BACKGROUND:Ischemic stroke is one of primary causes of death and long-term disability.Despite the high prevalence of ischemic stroke,there are remain limited options for therapy,especially in restoring lost neurological function.Although thrombolysis therapies using of recombinant tissue plasminogen activator(r-tPA)have been recognized to improve ischemic stroke outcome,the narrow time window limits this approach.Rehabilitation could facilitate functional recovery in the patients of ischemic stroke.However,its effects still remain not optimal.Thus,researchers are seeking new methods to treat ischemic stroke to reduce mortality and restore neurological function.Cellular therapy with mesenchymal stem cells(MSCs)is a potential treatment for ischemic stroke.Previous investigations have demonstrated that MSCs protected neuron against apoptosis in experimental model of cerebral ischemia.However,whether MSCs can protect neurons against parthanatos,necroptosis and autophagy in cerebral ischemia model has not been investigated.Objective:To elucidate the specific type of cell death which MSCs protect neurons against after hypoxic-ischemic injury.Method:The whole medulloculture plus differential adherence method was used for these paration,purification and cultivation of bone marrow MSCs.we used oxygen-glucose deprivation(OGD)and N-methyl-D-aspartate(NMDA)toxicity model to mimic a hypoxic-ischemic condition in vitro and established a MSCs-neuron co-culture system using transwell(?)6 well plates with 0.4 ?m polycarbonate membrane to explore the underlying mechanisms of MSCs-induced neuroprotection.Propidium iodide(PI)/Hoechst 33342 double staining was applied for the quantitative measurement of neuronal cell death and assessing the neuroprotective effect of MSCs compairing with the selected inhibitors to parthanatos,necroptosis,apoptosis and autophagy.Immunofluorescence staining,western blot(WB)and real-time PCR were used to determine the influence of MSCs co-culturing on the expression of the mediators of parthanatos-nucleolus apoptosis-inducing factor(AIF)and poly ADP-ribose(PAR),the critical mediators of necroptosis-receptor interacting protein 1(RIP1)and receptor interacting protein3(RIP3),the mediators of apoptosis-cleaved caspase-3 and the mediators of autophagy-Beclinl and light chain3B(LC3B)in OGD and NMDA injured neurons.Result:Co-culturing with MSCs in a transwell co-culture system,the OGD injured neurons were resecued by about 73%-75%and the NMDA injured neurons were resecued by about 68%,which is superior to the effect of any cell death inhibitors.The data from both immunofluorescence staining and western blot analysis demonstrated that,co-culturing with MSCs protected the cortical neurons from the OGD/NMDA-induced parthanatos by preventing the translocation of AIF from the mitochondria to the nucleus;reduced the neuronal necroptosis by down-regulating the expression of RIP1 and RIP3,the two essential kinases in necroptosis;protect neurons against apoptosis by deactivating caspase-3;whilst possessed no significant influence on OGD/NMDA induced neuronal autophagy,according to its failed regulation on Beclin1.These results were further confirmed by real-time PCR.Conclusion:MSCs protects the cortical neurons against OGD/NMDA-injury in vitro via inhibiting apoptosis,parthanatos and necroptosis at the same time but not autophagy,which could provide some evidence to the mechanism explanation on cellular therapy for ischemic stroke with MSCs.Our data presents the new evidence for clinical application of bone marrow MSCs as cellular therapy for ischemic stroke.