| Objective To observe the effect of astragaloside IV（ASI）on oxygen glucose deprivation（OGD）-induced neural stem cells（NSCs）apoptosis in vitro,and explore the possible mechanisms of ASI by micro RNA-199a-5p.Methods 1.Neural stem cells were isolated and cultured from the whole brain of 14-day sprague-dawley rat embryos in vitro,and NSCs was identified by immunofluorescence.OGD model,OGD was treated NSCs at different time（2,4,6,8,10 h）,and the viability of cells was detected by TBS and CCK-8 method.2.CCK-8 was used to detect the cell viability after different concentrations of ASI（1,10,25,50,100,200μM）under different time（24,48,72 h）on NSCs that were damaged by OGD（8 h）.3.Brd U immunofluorescence was used to detect cell proliferation and Annexin V-FITC/PI double staining was used to detect cell apoptosis.4.q RT-PCR was used to detect the changes of miR-199a-5p expression in OGD-induced the damage of NSCs with ASI（50μM）treat.5.Cell transfection:miR-199a-5p antagomir was transfected into NSCs by lipofectamine 2000and transfection efficiency was detected by q RT-PCR.6.The experimental group includes:Control、Model、OGD+miR-199a-5p antagomir、OGD+miR-199a-5p antagomir NC、OGD+ASI、OGD+miR-199a-5p antagomir+ASI、OGD+miR-199a-5p antagomir NC+AS.7.Indexes detection:CCK-8 method was used to detect cell activity,cell proliferation was detected by Brd U immunofluorescence and cell apoptosis was detected by Annexin V-FITC/PI staining,q RT-PCR was used to detect the m RNA expression of HIF-1 and caspase-3,western blot were used to detect HIF-1α,cleaved caspase-3/caspase-3 protein expression.Results 1.The whole brain of 14-day sprague-dawley rat embryos was isolated and cultured in vitro and cultured a large number of NSCs with good growth and high purity.2.The results of CCK-8 and TBS showed that OGD was time dependent on NSCs damage at different time,and the cell activity decreased to 58%after 8 h,which was used for future experiments.3.Compared with the model group,the protective effect of ASI on OGD induced NSCs damage was time and concentration dependent,and 50μmol·L-1 at 72 h was best（P<0.01）.4.Annexin V-FITC/PI staining assay showed that compared with the model group,ASI（50μM）can significantly inhibit the apoptosis of NSCs under OGD（P<0.01）,Brd U immunofluorescence showed that ASI can promote the proliferation of NSCs under OGD（P<0.05）.5.After OGD injury,the expression of miR-199a-5p in NSCs decreased significantly（P<0.01）,compared with the model group,ASI could significantly increase the expression of miR-199a-5p（P<0.05）.After transfection of miR-199a-5p antagomir,it could significantly inhibit the expression of miR-199a-5p（P<0.01）,and significantly inhibit the activity and proliferation,increase the apoptosis of NSCs by OGD induced injury（P<0.05）.6.q RT-PCR and western blot results showed that the m RNA and protein expression of HIF-1α,the protein of cleaved caspase-3 was down-regulated（P<0.01 or P<0.05）in the ASI（50μM）group after NSCs under OGD,and the function was reversed after transfection of miR-199a-5p antagomir.Conclusion ASI can inhibit the apoptosis of NSCs induced by OGD and promote the survival and proliferation of NSCs,the mechanism may be related with up regulation of miR-199a-5p expression and the inhibition of the expression of HIF-1αand cleaved caspase-3. |
