The Study Of Astragaloside ? Inhibits Apoptosis Of PC12 Cells Induced By Hypoxia/hypoglycemia And Reoxygenation Through Activating Autophagy

Cerebrovascular disease is a common disease that seriously threatens human health,especially in the elderly over the age of 50 have a higher incidence,morbidity and mortality,60-80% of it is ischemic cerebrovascular disease.Cerebral ischemia-reperfusion injury is an important pathophysiological process of ischemic cerebrovascular disease,and apoptosis is one of the main mechanisms of ischemia reperfusion injury.Autophagy,the important process of catabolism,widely exists in eukaryotic cells and plays an important role in cerebral ischemia reperfusion injury.Moderate cell autophagy can inhibit apoptosis and relieve ischemia anoxic injury,and inhibiting autophagy will make the injury worse.Chinese medicine astragalus membranaceus,promoting blood circulation to remove blood stasis and relieving swelling and pain,is widely used in the treatment of ischemic cerebrovascular disease.Astragalus membranaceus injection can inhibit the neuronal apoptosis and relieve ischemia reperfusion injury in rats proveded by our previous work.Astragaloside IV is one of the main active components of astragalus membranaceus.Whether it could inhibit apoptosis to play a protective role? And whether its mechanism and the effect on regulation of autophagy is closely linked? All of these are unclear.Therefore,this research subjects for PC12 cells induced by hypoxia/hypoglycemia and reoxygenation,widely used in the study of nervous system diseases in vitro,to simulated neurons ischemia-reperfusion injury in in vitro.Discussing the mechanism of astragaloside IV to play a protective role by observing its effect of apoptosis and autophagy on PC12 cells induced by hypoxia/hypoglycemia and reoxygenation.The First Part The effects of astragaloside IV on apoptosis of PC12 cells induced by hypoxia/hypoglycemia and reoxygenationObjective:To observe the effects of astragaloside IV on apoptosis of PC12 cells induced by hypoxia / hypoglycemia and reoxygenation.Methods:PC12 cells were randomly divided into 4 groups: normal control group,model group(hypoxia/hypoglycemia and reoxygenation group),astragaloside IV group and vehicle group(astragaloside IV solution group).Model group,astragaloside IV group and vehicle group were exposed to reoxygenation(12h)after 3h of oxygen and glucose deprivation,and astragaloside IV 100?mol/L(final concentration)was added into cells at the same time.Using inverted microscope to observe the morphological changes of PC12 cells and MTT method to detect the activities of PC12 cells,and using Annexin V-FITC/PI assay to measure the apoptosis of PC12 cells.Results:Compared with normal control group,cells becomed round or swollen and its cellula processes were retracted or disappeared in model group;a large number of apoptotic cells also can be observed,whose nucleus were shrinkaged,fragmented or deep-stained.The activities of model group were decreased markedly than those in normal control group(P<0.05),while the apoptotic rates of model group were increased obviously than those in normal control group(P<0.05).Compared with model group,a good cell growth state can be observed and cellula processes also can be observed significantly in astragaloside IV group.The activities of astragaloside IV group were increased than those in model group(P<0.05),while the apoptotic rates of astragaloside IV group were decreased than those in model group(P<0.05).There was no obvious difference between vehicle group and model group(P>0.05).Summary:Astragaloside IV can reduce the damage of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation,increase cell activity,inhibit cell apoptosis,and then play a protective role.The Second Part The study of astragaloside IV inhibits apoptosis of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation through activating autophagyObjective:To observe the effects of astragaloside IV on autophagy and apoptosis of PC12 cells induced by hypoxia / hypoglycemia and reoxygenation,and investigate the mechanism of anti-apoptosis effect of astragaloside IV.Methods:The PC12 cells were randomly divided into 7 groups: normal control group,model group,vehicle group,astragaloside IV group,3-MA+astragaloside IV group(autophagy inhibitor +astragaloside IV group),3-MA group(autophagy inhibitor group),RAPA group(autophagy activator group).Except normal control group,all other 6 groups were exposed to reoxygenation(12h)after 3h of oxygen and glucose deprivations,and corresponding drugs were added into the cells at the same time(the final concentration of astragaloside IV was 100?mol/L,the final concentration of 3-MA was 5mmol/L,the final concentration of RAPA was 250nmol/L).Using transmission electron microscope to observe the morphological changes of autophagosome and TUNEL staining to measure apoptosis.Western blot were used to detect the protein expression of LC3-II,Beclin1,Caspase3.Results:Compared with normal control group,the numbers of autophagosome and the expression of LC3-II and Beclin1 increased in model group(P<0.05),and the expression of Caspase3 and the apoptotic index also increased at the same time(P<0.05).Compared with model group,the numbers of autophagosome and the expression of LC3-II and Beclin1 increased obviously(P<0.05),and the expression of Caspase3 and the apoptotic index decreased markedly in astragaloside IV group and RAPA group(P<0.05);While the numbers of autophagosome and the expression of LC3-II and Beclin1 decreased(P<0.05),and the expression of Caspase3 and the apoptotic index increased in 3-MA group(P<0.05);There was no obvious difference between vehicle group and model group(P>0.05).Compared with astragaloside IV group,the numbers of autophagosome and the expression of LC3-II and Beclin1 decreased(P<0.05),and the expression of Caspase3 and the apoptotic index increased in 3-MA+astragaloside IV group and 3-MA group(P<0.05).Summary:Astragaloside IV can inhibit apoptosis of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation through activating autophagy,its mechanism is related to up regulation of LC3-II and Beclin1 expression and down regulation of Caspase3 expression.Conclusion:1 Astragaloside IV can obviously reduce the damage of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation,increase cell activity,inhibit cell apoptosis,and then play a protective role.2 Astragaloside IV can inhibit apoptosis of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation through activating autophagy,its mechanism is related to up regulation of LC3-II and Beclin1 expression and down regulation of Caspase3 expression.