The Protective Effects On Rapamycin Through Mediating MTOR Signaling Pathways To Regulate The Autophagy Of Spinal Cord Neurons In EAE Mice

Objective: To investigate whether the protective effects on rapamycin could mediate the mTOR signaling pathways to regulate the levels of autophagy in spinal cord neurons of EAE mice. Methods: 45 healthy female C57BL/6 mice were randomly divided into three groups: normal control group?EAE model group and rapamycin treatment group, 15 mice in each group, and then the groups of mice were divided into three subgroups randomly,5 mice in each subgroup. EAE model group and rapamycin treatment group were injected with MOG35-55 and complete freund's adjuvant (CFA) to make the EAE model, nonnal control group was given the same dose of saline and CFA. Meanwhile intraperitoneal injecting the rapamycin (1kg/mg) to the NeuN, namely LC3 mainly accumulated in the spinal cord neurons.? The expression of autophagy marker LC3 proteins in the spinal cord neurons with western blot: at the peak stage of incidence, the expression of LC3 proteins were significantly reduced in the EAE groups, and the LC3-II/LC3-I levels also were decreased (P<0.01), while the use of rapamycin intervention, the expression of LC3 were increased, at the same time, the LC3-?/LC3-? levels were risen in contrast to EAE groups (P<0.05),between with the EAE groups and normal control groups. ?The expression of related proteins in mTOR autophagy pathways: the phosphorylation levels of mTOR proteins in EAE groups and Rap treatment groups were higher than normal control groups, and in contrast to the EAE groups, the phosphorylation levels of mTOR proteins had higher expression in Rap groups (P<0.01) , for P70s6k proteins, had roughly the same phosphorylation levels of mTOR proteins. ?The contents of ROS in the spinal cord neurons at different groups: the contents of ROS changes in EAE groups and Rap treatment groups were higher than the normal control groups (P<0.05),and comparing with EAE groups, the contents of ROS in Rap groups were significantly decreased (P<0.05), the difference were statistically significant. ?Correlational analysis: reactive oxygen stress levels were negatively correlated with the onset of the incubation period?the number of spinal cord neurons with nissl marked, and were positively correlated with progression period?nerve dysfunction scores (P<0.05); Autophagy marker LC3 protein levels were negatively correlated with the contents of ROS?the expression of phosphorylated mTOR and P70s6 protein kinases (P<0.05).Conclusion: 1. Using the C57BL/6 mice immunized with MOG35-55 to preparate the EAE models, by observing the mice of different groups, in EAE model groups, the incubation periods shortened ?progression extended and the nerve dysfunction scores obviously increased, the results of HE staining showed that a large number of inflammatory cells infiltrated in spinal cord neurons of EAE groups at the peak stage, typical "sleeve" changes, white matter demyelination around blood vessels,above changes illustrated that the EAE animal models had established. 2. Defection of autophagy existed in spinal cord neurons of EAE groups at the peak stage, and could further increased the levels of ROS, which accumulated abundantly in spinal cord neurons to cause its seriously damage. 3. Rapamycin could extend the onset of incubation period?shorten the progressive period?decrease the nerve dysfunction scores and add the number of spinal cord neurons which marked with nissl staining, prompting that rapamycin can protect the spinal cord neurons of EAE mice. 4. The protective effects of rapamycin might through inhibiting the mTOR signaling pathways to activate the autophagy activity,negatively regulated the contents of ROS, and thus relieved the oxidative stress injuries to play a important roles in spinal cord nerve protection.