The Effects Of Pin1 In Mitochondrial Apoptotic Pathway And The Neuron Damage Induced By Oxygen Paradox At Different Time

Objective To observe the expression change of Pin1 in embryonic cerebral cortical neurons induced by oxygen paradox in different time and effect on neurons form and function.And find the may function and characteristic of Pin1 in the neuron mitochondrial apoptosis pathway,when neurons damage induced by oxygen paradox.Methods Rat embryonic cerebral cortical neurons were primary cultured and identificated by Nissl staining and Microtubule-associated protein 2(MAP2).They were randomly devided into control group;oxygen-glucose deprivasion/reoxygenation groups,that is OGD1h/R6 h group(M6h),OGD1h/R12 h group(M12h);Pin1inhibitor groups:that is Juglone 6?mol/L(M6h+J6,M12h+J6),Pi B 0.7 and 1.0?mol/L groups(M6h+P0.7,M6h+P1,M12h+P0.7,M12h+P1).Each group contain 6 subholes.The change of the cell morphological stucture observed under inverted phase contrast microscope;CCK-8 assay was applyed to detect the the activity of neurons;Hoechst33258 fluorescent staining was used to detection the cell apoptosis;Immunocytochemistry were used to measure the expressions of the proteins of Pin1,p-Bim EL and Bax in the neurons;Flow's cell analyzer was applyed to detect the mitochondrial membrane potential(??m)in neurons;the changes of ultrastructure were observed under transmission electron microscopy.Results(1)Compared to Control group,M6 h and M12 h group's cell refractivity decreased,protuberant shrined and neural network damaged,M12 h group even appear collapse,neuronal network seriously damaged,the neuronal activity were all significantly decreased(P<0.01),the cell apoptosis increased(P<0.01),the expression of Pin1 protein was rised(P<0.01),the expression of related mitochondrial apoptotic factors p-Bimel and Bax proteins were significantly increased(P<0.01),mitochondrial membrane potential were significantly decreased(P<0.05).M12 h group cell ultrastructure damage increased,mitochondria,endoplasmic reticulum swelling,vacuolar degeneration.(2)Using Juglone 6?mol/L and Pi B 0.7,1.0?mol/L immediately when 6h and 12 h,compared to M6 h,groups M6h+J6,M6h+P0.7,M6h+P1 the neuronal activity were all significantly decreased(P<0.01),the cell apoptosis obciously increased(P<0.01),Pin1 protein expression was markedly decreased(P<0.01),related mitochondrial apoptotic factors p-Bimel and Bax proteins expression were significantly increased(P<0.01),mitochondrial membrane potential was notly decreased(P<0.05).Compared to M12 h,groups M12h+J6,M12h+P0.7,M12h+P1 the neuronal activity were all significantly increased(P<0.01),the cell apoptosis dramatically decreased(P<0.01),Pin1 protein expression was markedly decreased(P<0.01),the expression of related mitochondrial apoptotic factors p-Bimel and Bax proteins were significantly decreased(P<0.01),the mitochondrial membrane potential were increased(P<0.05),ultrastructure damage alleviative.Conclusion(1)After OGD1h/R6 h and 12 h,with the extension of reoxygenation time,Pin1 protein expression was rised,cell damage was aggravated,the neuronal activity decreased,cell apoptosis increased,related mitochondrial apoptotic factors p-Bim EL and Bax proteins expression were increased,??m were decreased,showed that oxygen paradox-induced neuronal injury was related with the time of reoxygenation.(2)Restrain Pin1 by using Juglone 6?mol/L and Pi B 0.7,1?mol/L immediately when reoxygenation 6h,the neuronal activity were decreased,cell apoptosis increased,Pin1 protein expression was decrease,related mitochondrial apoptotic factors p-Bim EL and Bax proteins expression were significantly increased,??m were decreased,Prompting Pin1 may have interact with certain anti-damage factors and protect the neurons against apoptosis in OGD1h/R6 h,after Pin1 activity was inhibited,the interaction between Pin1 and anti-damage was decreased,lead to apoptosis.(3)Restrain Pin1 by using Juglone 6?mol/L and Pi B 0.7,1.0?mol/L immediately when reoxygenation 12 h,cell damage relieved,the neuronal activity were increased,cell apoptosis decreased,Pin1 protein expression was decrease,related mitochondrial apoptotic factors p-Bim EL and Bax proteins expression were significantly decreased,and the Pin1,p-Bim EL,Bax protein expression changes into positive correlation,??m were increased,ultrastructure damage alleviative.Promoting that Pin1 may stable by phosphorylation of p-Bim EL and start the mitochondrial apoptotic pathways at OGD1h/R12 h,accelerating apoptosis of neurons.After restrain activity of Pin1,its ability of stabilize phosphorylation of p-Bim EL was abated,cause the mitochondrial apoptosis pathway activation to receded,also reduce the apoptosis of neurons.Pin1 may regulate the stability of p-Bim EL in neuron mitochondrial apoptosis pathway,and participate in mediated apoptosis of neurons in neurons OGD1h/R12 h.