Effects Of BAG-1 Gene Silencing On HSP70 Expression And Cell Apoptosis In SH-SY5Y Cells Induced By Oxygen Glucose Deprivation/reoxygenation

ObjectiveTo investigate the effect of lentivirus mediated BAG-1 gene silencing on oxygen glucose deprivation/reoxygenation(OGD/R)damage in human neuroblastoma cells(SH-SY5Y)and the expression of HSP70.MethodsUsing BAG-1 RNAi lentiviral infect the logarithmic phase of SH-SY5 Y cells to suppress the expression of BAG-1.Fluorescence microscope was used to observe the cell fluorescence.In order to ensure the efficiency of infection,we adopted puromycin screening cells.Then cells were divided into four groups: normal cell group without lentivirus infection,lentivirus control group with lentivirus containing EGFP,BAG-1 RNAi-? group and BAG-1 RNAi-? group with lentivirus containing EGFP and different RNAi target sites.After 72 h of recombination lentivirus infection,Western Blot was used to detect the effect of the BAG-1 gene silencing.For determine the best OGD time to simulate the ischemia-reperfusion injury,four groups of cells were treated with OGD 4 h,8 h,12 h,24 h and reoxygention 24 h respectively and CCK-8 was used to detect the viability of cells;The activity difference among the four groups was also be compared.Then cell apoptosis after OGD 8 h and reoxygenation 24 h was measured by flow cytometry;the expression of HSP70 protein and transcription level of m RNA was detected by Western Blot and RT-q PCR respectively.The comparison between the groups of samples was made by one-way ANOVA,and the LSD-t test was used for futher multiple comparisions.Results1.After recombination lentivirus infection 72 h,fluorescence was observed under fluorescence microscope and the efficiency was more than 75%.Stable express cells were obtained by puromycin selection.Western Blot showed that the interference effect of BAG-1 RNAi-? group was better than that of BAG-1 RNAi-? group.2.According to the results of CCK-8,SH-SY5 Y cells challenged with OGD for 8 h and reoxygention for 24 h was used as the ischemia-reperfusion injury model.We can get that,under this condition,the BAG-1 RNAi-? group which absorbance value lower than that of the other three groups [A:(0.591�0.091)vs.(0.937�0.118),(0.902�0.106)and(0.910�0.139),all P(27)0.01].3.The cell apoptosis detected by flow cytometry showed that the rate of apoptosis in BAG-1 RNAi-? group was significantly higher than the other three groups[Apoptosis(%):(34.633�3.464)vs.(14.833�3.753),(19.933�6.490),(16.400�1.179)all P(27)0.01].4.There was no statistical difference in the expression of HSP70 protein and m RNA among the four groups.Conclusions1.The cell viability can be affected by oxygen-glucose deprivation/ reoxygenation injury,and SH-SY5 Y cells challenged with OGD for 8 h and reoxygention for 24 h injury can be used as the model of ischemia reperfusion injury neural cells.2.BAG-1 may be have a protective effect on SH-SY5 Y cells from OGD/R injury.3.There was no change in the expression of HSP70 after BAG-1 gene silencing,which provides the experimental basis for the further research of BAG-1/HSP70 interaction in ischemic cerebrovascular disease.