Astragaloside-IV Protect PC12 Cells Through Inhibition Of Endoplasmic Reticulum Stress By Preventing The MPTP Opening

Objective To investigate Astragaloside IV exerts the mitochondrial protective effect of PC12 cells and its possible signal transduction mechanism through endoplasmic reticulum stress.Methods(1)Rat adrenal pheochromocytoma cells(well differentiated)were routinely cultured.To investigate the effects of different concentrations of endoplasmic reticulum stress inducer 2-Deoxy-D-glucose(2-DG)on endoplasmic reticulum stress(ERS)and mitochondrial membrane potential(mitochondrial membrane potential,??m),cells were randomly divided into control group and 2-DG group(50,100,150,200 ?M).The expressions of ERS related protein GRP 78 and GRP 94 were detected by Western Blot assay.The changes of TMRE fluorescence intensity were observed by confocal microscopy to determine the optimum concentration of 2-DG.(2)The cells were randomly divided into control group and different concentrations of Astragaloside IV group(25,50,75,100 ?M).Astragaloside IV on PC12 cell phosphorylation of(glycogen synthase kinase-3?,GSK-3?)GSK-3? expression to determine the optimal concentration of Astragaloside.(3)Explore Astragaloside IV on the impact of ERS,the experiment is divided into control group,within the 2-DG group,Astragaloside IV add 2-DG group,Astragaloside group.The expression of ERS related GRP 78,GRP 94 and IRE1,p-PERK,ATF6 and the phosphorylation level of GSK-3? were detected by Western Blot.The changes of TMRE red fluorescence intensity were observed by confocal microscopy.Results(1)GRP 78 and GRP 94 significantly increased expression in 50 ?M 2-DG compared with the control group,has statistical significane(P<0.05).Thus,50 ?M 2-DG was used in thefollowing experiments.(2)The expression of p-GSK-3? in 50 ?M Astragaloside increased most significantly,and has statistical significane(P<0.05).Therefore,Astragaloside in the follow-up experiment 50 ?M was used.Compared with(3)the control group,the expression of GRP 78,GRP 94 and IRE1,p-PERK,ATF6 in 2-DG group was significantly increased,the expression of p-GSK-3? was significantly reduced,the fluorescence intensity of TMRE was significantly decreased,??m decreased,m PTP opening,and the difference was significantly(P<0.05).Astragaloside could significantly inhibit the expression of GRP 78,GRP 94,IRE1,p-PERK,and p-GSK-3? by 2-DG,and enhanced the fluorescence intensity and increased the mitochondrial membrane potential and prevented the mPTP opening,the significant difference(P<0.05).However,Astragaloside may not significantly inhibit ATF6 expression,the difference was not significant(P>0.05).Conclusion 2-DG may induce ERS and Astragaloside can promote the expression of phosphorylated GSK-3?.Astragaloside IV may inhibit ERS via GRP78,GRP94,IRE1,PERK,inactivate GSK-3? and prevent m PTP from opening up and protect PC12 cells,However,it may not play a role in inhibiting endoplasmic reticulum stress through the ATF6 pathway.