Autophagy Protects Against PI3K/AKT/mTOR-mediated Apoptosis After The Mechanical Injury Of Spinal Cord Neurons

Objective:To investigate the relationship between PI3K/AKT/mTOR mediated autophagy and mitochondrial apoptosis after spinal cord injury Methods:(1)To culture primary cultured spinal cord neurons,we used pregnant 16 days SD rats.Then the model of spinal cord injury was established in seventh days by Que Haiping method;(2)The cell growth state of the neurons was observed by microscope,and the purity of neurons was identified by using neuron specific antibody TUBB3 and NSE;(3)Wecollected cytoplasmic proteins at different time points after injury(1h,4h,8h,24h).Analysis of autophagy(LC3)and apoptosis related proteins(cleaved caspase-3,Bax,Bcl-2,cytochrome c)by western blot;(4)We used to detect the apoptosis of neurons in different groups(control,SCI,SCI+LY294002,SCI+BAF)by TUNEL method;(5)JC-1 kit was used to detect the level of mitochondrial membrane potential;(6)AO staining was used to detect the expression of autophagy lysosome after mechanical injury;Results:(1)We observedthe neuronsat seventh days by ordinary inverted microscope.It can be seen that the neuronal cell body is small and the synapse formation.The results of immunofluorescence showed that most of the neurons could be labeled with high purity.(2)Apoptosis inducing injury: JC-1 fluorescence staining showed that the neurons in 8hours after injury and mitochondrial membrane potential were significantly decreased(p<0.05).The mitochondrial apoptosis protein Bax,cytochrome c,cleaved caspase-3 was significantly increased at 8hours after injury(p<0.05);The anti-apoptosis protein Bcl-2 was expressed peakedat 1 hours after injury,then began to decrease(p<0.05);(3)After injury induced autophagy: The results of western blot showed that the level of LC3 was increased at 4 hours and reached the highest level at 24 hours after the injury of neurons(p<0.05).The application of Bafilomycin A1 could reduce the occurrence of autophagy;(4)AO staining showed that the control group in the cytoplasm of weak red fluorescence,with the time of injury increased at 4hours after injury,increased red fluorescence in the cytoplasm(p<0.05),24 hours in the cytoplasm which the red fluorescence was the strongest,there were dense hyperchromatic green fluorescent yellow or yellow green pieces.(5)PI3K/Akt/mTOR signaling pathway: Thewestern blot results showed that the in phosphate-Akt level was significantly increasedat 1hours after injuryand reached the peak level of 4 hours(p<0.05).Then its downstream phosphorylation level of mTOR increased significantly after 1hours and reached the peak at 4 hours(p<0.05);We used the PI3 K inhibitor LY294002 after injuryat 24 hours and then downstream phosphate-Akt,phosphate-mTOR level the inhibited autophagy level have increased significantly(p<0.05);(6)The results of TUNEL showed that the level of apoptosis after LY294002 was significantly lower than that in the simple injury group(p<0.05).Conclusion:(1)The autophagy and apoptosis of neurons were induced by mechanical injury,and the level of autophagy occurred earlier than that of apoptosis;(2)Spinal cord neurons after mechanical injury mediated autophagy through PI3K/Akt/mTOR signaling pathway,inhibiting the phosphorylation of the signal pathway can enhance the level of autophagy and reduce apoptosis;...