Study Of Autophagy Level Of Astrocytes In EAE Mice And Effect Of Berberine On Autophagy Level Of Astrocytes In EAE Mice

Objectives: To explore the changes of autophagy on the astrocytes in the experimental autoimmune encephalomyelitis(EAE)mice and investigate the effects of berberine on the autophagy of astrocytes in vivo and in vitro.Methods: The EAE mouse model was built by immunizing C57BL/6 mice with myelin oligodendrocyte glycoprotein(MOG35-55).The mice were divided into 4 groups: group EAE,Complete Freund’s Adjuvant(CFA),EAE+Bererine(BBR)and EAE+rapamycin(RAPA),and their neurological function scores were observed respectively.On the 3rd,7th,15 th and 30 th days after EAE model was induced,the mice were perfused through hearts and then the brains and spinal cord were removed to make paraffin section.The hematoxylin-eosin staining and the luxol fast blue staining were used to observe the inflammatory infiltration and demyelination in the white matter of brain,and the immunofluorescence was applied to detect the expression of autophagy related proteins,Beclin1 and LC3 B,on the astrocytes of the mouse spinal cord.Astrocytes separated from the cerebral cortex of newly-born C57BL/6 mice were cultivated into stable astrocytes after two generations.Purification rate of astrocytes identified by GFAP antibody was > 95%.The cell samples were divided into 3 groups: Lipopolysaccharide(LPS),LPS+BBR,LPS+ RAPA.Quantitative analysis of cell sample proteins was applied by using BCA before the intervention and at 0.75 h,1.5h,3h,6h,and 12 h after the intervention.The expressions of autophagy related proteins,Beclin1 and LC3 B,were detected by Western Blot.Results:(1)Some EAE mice started to have symptoms on 8th days after the immunization.Most of the mice had symptoms from 13 th to15th days after the immunization and peaked from 15 th to 19 th days.Both BBR(P<0.01)and RAPA(P<0.01)groups had significantly lower score of neurological function and less severity of the disease compared to EAE group.(2)HE staining results showed that cerebral white matter of EAE mice had more inflammatory cell infiltration,which characterized as extensive lymphocyte and neutrophil infiltrations,when compared with CFA(P<0.01),BBR(P<0.01)and RAPA(P<0.01)groups.These infiltrated cells spread along the envelope and small blood vessels and extended outwards,which takes a typical’vascular cuff’ appearance.LFB staining results showed that the cerebral white matter of EAE mice had more serious demyelination compared to CFA(P<0.01),BBR(P<0.01)and RAPA(P<0.01)groups.In the area of inflammatory infiltration,there scattered demyelinating lesions in different size,which are characterized as loosened myelin sheath and lot of vacuoles.(3)Results from immunofluorescence imaging demonstrated that on 7th,15 th and 30 th days after immunization,the expression of Beclin1 in astrocytes of spinal cord in EAE group was significantly lower than that of BBR(P<0.05)and RAPA(P<0.05)groups.The expression of Beclin1 in CFA and EAE groups had no statistical significance(P>0.05).(4)Results from immunofluorescence imaging demonstrated that on 7th,15 th and 30 th days after immunization,the expression of LC3 B in astrocytes of spinal cord in EAE group was significantly lower than that of BBR(P<0.05)and RAPA(P<0.01)groups.The expression of LC3 B in CFA and EAE groups had no statistical significance(P>0.05).(5)WB results showed that the relative expression level of Beclin1 in LPS group had no significant change after LPS intervention(P>0.05).Whereas,in LPS+BBR group(P<0.01)and LPS+RAPA group(P<0.01),the relative expression level of Beclin1 increased significantly after the intervention.(6)WB results showed that the relative expression level of LC3-II/I in LPS group had no significant change after LPS intervention(P>0.05).On the contrary,in LPS+BBR group(P<0.01)and LPS+RAPA group(P<0.01),the relative expression level of LC3-II/I increased significantly after the intervention.Conclusions:(1)The expression level of autophagy related proteins,Beclin1 and LC3 B,in astrocytes of EAE mice is consistently low after immunization and the same phenomenon is observed in primary astrocytes induced by lipopolysaccharide in vitro.(2)Rapamycin and berberine reduce inflammatory infiltration in EAE mice and alleviate neural function defect and demyelination.(3)Rapamycin and berberine improve the expression level of autophagy related proteins Beclin1 and LC3B in astrocytes both in vivo and in vitro and induce the autophagy in astrocytes.This maybe one of the mechanisms of their improving the symptoms of EAE.