Effect Of Autophag On Mitochondrial Apoptosis And Inflammation In Experimental Autoimmune Encephalomyelitis Mice

BACKGROUND AND OBJECTIVEMultiple sclerosis (MS)is a central nervous system (CNS) autoimmune disease, It mainly manifested in the following aspects: demyelination in white matter, leukocyte infiltration and blood brain barrier injuries. It is still unclear that how multiple sclerosis happens specificly. It has been found recently through autopsy and a series of imaging modality that oligodendrocytes apoptosis was the earliest pathology in the early lesions, followed by microglia gathering and activating while the morphological of myelin was normal and without any inflammation cells. The infiltrating of monocytes, macrophages and lymphocytes and demyelination were found after a few weeks or months. The researchers describe the early pathological change of new lesion in MS as "preactive lesion". The apoptosis of oligodendrocyte and the activate of microglia are the characteristic changes of pathological. Experimental autoimmune encephalomyelitis C57/BL mice is a perfect model of MS. Our early findings shows that the exhaustion and damage of mitochondria energy is the initial reason of oligodendrocyte apoptosis, which might be the main reason of preactive lesion formation, result in typical pathological change happens.we also found mitochondria damage and neuron apoptosis through out the EAE, and is consistent with the acuty of disease. But the reason why mitochondria damage happens is still unknown. Cell can start autophagy to reset the damaged mitochondria to protect cells from necrosis and apoptosis. Researchers have proved that autophagy has dual function to T cells, called presurvive and predeath, and they are important mediater of autophagy and inflammation. In this study, we explore the autophagy in EAE C57BL/6 mice at different time points post-immunization,and demonstrate its effect on the formation of preactive lesions and mitochondrial apoptosis pathway pattern in EAE mice.we aim to provide a new method for multiple sclerosis’clinical treatment.METHODSPart 1.96 female C57BL/6 mice were divided into the normal control group, EAE group, EAE+Rapmycin group and EAE+PBS group. EAE+Rapmycin group intramuscular injection Rapmycin(1mg/kg/d) every day post immunization. Western blot was performed to observe the expression of LC3B and cyt-c in each group on 3d,7d,16d and 30d post immunization. Double-label immunofluorescence was performed to explore the expression of caspase-3 in oligodendrocytes.Part 2.Western blot was performed to observe the change of L C3B expression in CNS at 3,7,16,30d after the tweenty C47BL/6 rats model was succesfully conducted. Other sixty rats was devided into normal control group,EAE group,EAE+3-Methyladenine group(3-MA). EAE+3-MA group intramuscular injection 3-MA(100nmol/uL,2uL/d) p ost immunization. Immunohistochemistry was performed to explore th e activation of microglia post immunization. Western blot was perfor med to observe the expression of IL-1β at acut phase(16d), chronic p hase(30d) post immunization.HE staining performed to observe the inf lammatory infiltration of CNS.RESULTS1.Western blot showed the expression of LC3-II/LC3-I of EAE group decreased at 7 days post immunization((P<0.05), and stayed in a lower level contrasted to the normal group((P<0.05). The expression of beclinl was consistent with the expression of LC3B. The expression of cyt-c was elevated at 7 days and reached to the highest level at 16 days post immunization((P<0.05). Double-label immunofluorescence showed there are significant caspase-3 expression in oligodendrocytes at 7 days post immunization(P<0.05), which were reduced in EAE+Rap group(P<0.05).2. Immunohistochemistry showed that there are significant activated microglia at 7 days post immunization(P<0.05), which is more severe in the EAE+3-MA group compared to the EAE group(P<0.05). Western blot showed a high level expression of IL-1β at 16 days and 30 days post immunization in EAE group, which were higher in EAE+3-MA group at the same time point(P<0.05).HE staining showed the inflammatory infiltration in the 3-MA group was elevated contrasted to the EAE group at the same time point(P<0.05).CONCLUSIONS1. The autophagy level decreased 7 days post immunization in EAE group, which prompt that the reduction of autophagy level can be the significant reason of EAE pathological procedure.2. Rapmycin can enhance the autophagy level of EAE, decrease the apoptosis of oligodendrocyte at the early stage post immunization and suppress the release of cyt-c. This probably because Rap suppress mitochondria damage to restrain the cell apoptosis mediated by it.3. Autophagy inhibitor 3-MA can provoke the activation and gathering of microglia at early stage post immunization, increase expression of IL-lb and inflammatory infiltration at both acut phase and chronic phase,then promote the inflammation.4. Autophagy can inhibit the formation of preactive lesions and mitochondrial apoptosis, and also have the anti-inflamatory effect in EAE. The results will guide us to find effective treatment methods for the MS patients.