Protective Effect Of EGCG On Cell Injury After Subarachnoid Hemorrhage

BackgroundSubarachnoid hemorrhage(SAH)is a serious cerebrovascular disease in which blood enters the subarachnoid space after the rupture of cerebrovascular.Spontaneous SAH is usually caused by rupture of intracranial aneurysms,with a high mortality and disability rate,which brings great burden to families and society.Aneurysm clipping or embolization can avoid re-bleeding,but brain injury after SAH continues,and there is no effective treatment at present.Epigallocatechin Gallate(EGCG),the main active ingredient of tea polyphenols,is an antioxidant.Its protective role in central nervous system diseases has been extensively studied,but its role in SAH has rarely been reported.ObjectiveThe protective effect of EGCG on nerve cell injury after SAH was investigated from the perspective of mitochondrial function and autophagy level.MethodsPC12 cells and Oxyhemoglobin(OxyHb)were used to establish SAH models,EGCG with the concentration of 50?M as an intervention drug,the experiment was divided into Con group,SAH group and EGCG group,48 h after OxyHb treatment was selected as observation point.1.Observe the morphology of PC12 cells by ordinary microscope,MTT assay for cell viability,Western blot for the detection of apoptosis related protein caspase3 expression,To observe the protective effect of EGCG on neurons after SAH.2.Under the fluorescence microscope,the levels of mitochondrial membrane potential(??m)and reactive oxygen species(ROS)in each group were observed by JC-1 kit and DCFH-DA kit,and quantify fluorescence intensity using flow cytometry,reactive ??m with green/red fluorescence ratio,Detection of the expression of mitochondrial division-related protein Drp1 by Western blot,Observation of Mitochondrial Morphology in Cells by fluorescence microscope,To observe the effect of EGCG on mitochondrial function after SAH.3.Western blot was used to detect the expression of autophagy-related proteins Beclin-1 and Atg5,and to explore the effect of EGCG on autophagy after SAH.4.Isolation and extraction of intracellular mitochondria by differential centrifugation,Western blot was used to detect the expression levels of apoptosis-related proteins Bax and Cyt c in mitochondria and cytoplasm respectively.Mitochondria were isolated and extracted by differential centrifugation.The levels of mitochondrial and non-mitochondrial proteins Bax and Cyt c were detected respectively.The anti-apoptotic effect of EGCG through internal mitochondrial pathway was observed.Results1.EGCG inhibits abnormal neuronal proliferation and apoptosis after SAH.Normal PC12 cells have longer dendrites and can cross-link each other to form a network structure,yet the SAH model was established by stimulating PC12 cells with OxyHb,the cells show obvious shrinkage and decrease of dendrites,suggesting that OxyHb may affect the function of cells.The cell viability assay showed that,Compared with con group,cell viability in SAH group was significantly higher(P < 0.01),cell viability in EGCG group was lower than that in SAH group(P < 0.05),and cell viability in EGCG group was lower than that in con group(P < 0.01).The protein level showed that the level of caspase3 protein in SAH group was significantly higher than that in Con group(P< 0.01).The caspase3 protein level in EGCG group was lower than that in SAH group(P< 0.01).2.EGCG protects mitochondrial function after SAH.2.1 EGCG inhibits ??m depolarization after SAH.Fluorescence microscopy showed that red fluorescence in Con group was more than green fluorescence,while red fluorescence in SAH group decreased and green fluorescence increased,and EGCG group recovered to the state of more red fluorescence and less green fluorescence.The quantitative analysis of flow cytometry showed that the mitochondrial membrane potential decreased in the SAH group compared with the Con group(P < 0.01),and the EGCG group restored the intracellular mitochondrial membrane potential to the basal level(P < 0.01 vs SAH group).2.2 EGCG reduces intracellular ROS levels after SAH.Fluorescence microscopy showed that the intracellular green fluorescence intensity of con group was weak,that of SAH group was enhanced,and that of EGCG group was restored to the basic level.The results of flow cytometry showed that the intracellular ROS level in the SAH group was higher than that in the Con group(P < 0.01).Compared with the SAH group,the intracellular ROS level in the EGCG group was decreased(P < 0.05).2.3 The normal morphology of mitochondria is protected by EGCG after SAH.Observation of mitochondrial morphology showed that the long rod-shaped mitochondria in the Con group accounted for the majority and interweaved into a network.In the SAH group,a part of the mitochondria was fragmented,and the long rod-shaped mitochondria decreased,while in the EGCG group,the mitochondria morphology returned to normal level.Compared with Con group,the expression of Drp1 protein in SAH group increased significantly(P < 0.05),while the expression of Drp1 protein in EGCG group decreased significantly(P < 0.05),indicating that EGCG inhibited mitochondrial excessive division after SAH.3.EGCG regulates abnormal autophagy after SAH.Protein level assay showed that Beclin-1 protein expression was increased after SAH compared with Con group(P < 0.01).After EGCG pretreatment,Beclin-1 protein expression level was lower than that of Con group(P < 0.05),indicating that EGCG inhibits the initiation of autophagy after SAH.Compared with the Con group,the expression level of Atg5 protein decreased after SAH(P < 0.01).After EGCG pretreatment,the expression level of Atg5 protein was higher than that of SAH group(P < 0.05),suggesting that egcg may interfere with abnormal autophagy flow after SAH.4.EGCG regulates the levels of apoptotic mitochondrial pathway-related proteins after SAH.4.1 Apoptotic mitochondrial pathway-associated protein levels in mitochondria.The level of Bax protein in mitochondria of SAH group was significantly higher than that of Con group(P < 0.05),and the level of Cyt c protein was significantly lower than that of Con group(P < 0.01).Compared with SAH group,the level of Bax protein in mitochondria of EGCG group was inhibited(P < 0.05).Cyt c protein levels were elevated(P < 0.05).4.2 The levels of apoptotic mitochondrial pathway-related proteins in cytoplasm.The level of Bax protein in the cytoplasm of SAH group was lower than that of Con group(P < 0.01),and the level of Cyt C protein was significantly higher than that of Con group(P < 0.05);compared with SAH group,the level of Bax protein in the cytoplasm of EGCG group was higher(P < 0.05).Cyt C protein level decreased(P < 0.01).ConclusionAfter SAH,EGCG exerts neuroprotective effects by inhibiting abnormal autophagy and improving mitochondrial function,thereby reducing apoptosis.