| ObjectiveTo investigate the effects of 3-MA and chloroquine on autophagy,apoptosis and necrosis of brain cells after cerebral ischemia reperfusion（CIR）in rats,and to study their related factors（Atg5,Atg7,Atg16L1,Beclin1,LC3,Caspase3,Caspase9,PARP1,PI3K,TGF-β,TNF-ɑ）.MethodsRats were divided into four groups:sham group,cerebral ischemia reperfusion model group,55μg of 3-methyladenine（3-MA）,CQ 0.6mg·kg-1 group（CQ）,16 rats in each group.Except the Sham group,the other groups were all treated with a line embolization method to make the middle cerebral artery occlusion（MCAO）.3-MA group and CQ group were given 3-MA 55μg,CQ 0.6mg·kg-1 respectively.Then,neurological deficit score,TTC staining and brain water content were used to judge whether the model was made successfully.TUNEL staining and HE staining were used to detect apoptosis and necrosis,respectively.The levels of TGF-β,TNF-ɑwere detected by ELISA.The expressions of Atg5,Atg7,Atg16L1,Beclin1,LC3,Caspase3,Caspase9,PARP1,PI3K were detected by Western blotting,and expression of Beclin1,Caspase3,Caspase9,PARP1 protein were detected by immunofluorescence.ResultsThe score of neurological deficit was 0 in Sham group and 1 to 3 in other groups.TTC staining showed that except for Sham group,all other models were successful,the brain water content,the highest in 3-MA group,Sham group was the lowest.Electron microscopy showed no significant autophagy,apoptosis and necrosis markers in Sham group,while other groups had different levels of autophagy,apoptosis and necrosis markers.TUNEL staining showed that there was significantly increased apoptotic cells in Model,3-MA and CQ group.HE staining showed no significant necrosis in Sham group,necrosis occurred in other groups,of which 3-MA group was the highest.The result of ELISA showed that TGF-βwas the highest in model group,TNF-ɑwas the highest in 3-MA group.Western blotting and immunofluorescence showed that autophagy and apoptosis-related factors in Sham group were lower than those in Model group（P<0.05）,followed by CQ group and lowest in 3-MA group.ConclusionsIn cerebral ischemic reperfusion rats,autophagy was inhibited by 3-MA,which may inhibit the expression of Caspases by high expression of TNF-α,and the apoptosis of brain cells was slightly improved,the necrosis was significantly increased,and the area of cerebral infarction was increased;and chloroquine inhibition of autophagy,may reduce the expression of Caspases caused by brain cell apoptosis and necrosis improved,the area of cerebral infarction reduced. |
