Study On The Immunoregulatory Effect Of Buyang Huanwu Decoction On Mice With Experimental Autoimmune Encephalomyelitis

ObjectiveA chronic-progressive EAE（experimental autoimmune encephalomyelitis,EAE）C57BL/6 mice model was established by treatment of myelin oligodendrocyte glycoprotein35-55（MOG35-55）.Our aim was to observe the clinic assessment levels,the abilities of repairment of myelin sheaths after intervented was done by Buyang Huanwu decoction（BYHWD）in early stage of the disease.For further steps we have explored the efficacy of immunomodulatory and inflammatory responses in peripheral imuune organs and central nervous system（CNS）.We tried to explore the expression and activation of ROCK/TLR-4/NF-κB,to pay more attention to the multi-targets for the neuroprotection and neuroregeneration of MS/EAE,in order to reveal the mechanisms of the inflamatory signaling paythways of cellular and molecular of this disease.These studies will expand the therapeutic strategies of Yiqihuoxue Method for treatment of MS/EAE.MethodsFemale C57BL/6 mice were immunized subcutaneously with MOG_35-55.They were divided randomly into saline group as control,BYHWD group as treatment,16 mice in each group.Since the 3rd day post-immunization（p.i.）,original BYHWD was given by mouth to each mouse in treatment group（50g/kg）in everyday,and saline in the same way was given to each mouse in control group.We have weighed the mice every day since first day immunization,we also have done the clinical score evaluation and analysis for the mice according to the international criteria of EAE.On the day.17 p.i and 28 p.i.which are the peak period of the disease,we extracted the spinal cords and brains from the mice for HE,myelin,and CD4⁺T cell staining.The spleen mononuclear cells were obtained to analyze the subtypes of CD4⁺T cells by flow cytometry.We extracted total proteins from spinal cords,brains and spleens for the measurement of ROCKII.Meanwhile TLR4,Myd88,NF-κB and cox-2 in spinal cords were detected.The data was analysed by Graph Pad Prism 5 software.Results1.EAE model in C57BL/6 mice were established successfully by MOG_35-555-55 peptides induced.EAE mice fell sick on the 10th day p.i,.Saline group mice were characterized by fatigue,decreased activity,hair no luster,spontaneous perspiration,the tail gradually mop,bilateral hind limbs weakness and even paralysis,also showed symptoms with dark purple tongue and gatism.BYHWD mice have good activity,no dark purple tongue,no gatism,only part of the mice with tail weakness.Clinical score and body weight have beed monitored from day 0 to 28 p.i.Compared with the control group,BYHWD treatment delayed clinical onset appearing（mean onset time of saline group was 10.46±2.18,BYHWD treatment group was 16.6±2.32,p<0.01）,decreased the symptoms of EAE,reduce the clinical score（the highest average score of saline group was 3.62±0.46 points,the highest average clinical score in BYHWD group was 1.19±0.8,p<0.01）.For mice body weight loss,control group is more obvious（15.67±0.35,p.i.28）than BYHWD group.（19.42±2.22,p.i.28）（p<0.001）.2.In the HE staining,there were much more inflammatory cells in the saline group than treatment group.The wounds were mainly located at white matter of the anterior of the spinal cord.The saline group of inflammatory cell infiltration for white matter ratio（9.95±5.36）%,BYHWD group is（2.25±1.29）%,the degree of inflammatory infiltration in BYHWD group was significantly less than the control group（p<0.01）.Spinal cord myelin staining in control group had obvious demyelination,myelin is not continuous,saline group amyelination ratio throughout the white matter area was（27.69±8.44）%,BYHWD group was（8.28±3.02）%.BYHWD group reduced the myelin sheath compared with the control group（p<0.001）,there are statistically significant in two groups（p<0.001）.3.In our study,we explored whether administration of BYHWD could modulate systemic immune responses in EAE mice.Flow cytometry was used to observe the effects of BYHWD on the changes of T lymphocyte subsets,such as CD4⁺CD25⁺,CD4⁺IL-10⁺,CD4⁺IFN-γ⁺and CD4⁺TGF-β⁺in spleen mononuclear cells.The results showed that BYHWD can significantly increase the expression of CD4⁺CD25⁺（control group is 2.4%,BYHWD group is 5.47%,p<0.001）,CD4⁺IL-10⁺（control group is 9.25%,BYHWD group is 14.30%,p<0.001）,CD4⁺TGF-β⁺（control group is 6.37%,BYHWD group is 18.24%,p<0.001）,CD4⁺IFN-γ⁺（control group is 6.34%,BYHWD group is 14.22%,p<0.001）compared with saline group.4.Cell types of CNS inflammatory infiltration were determined by immunostaing.We found that there were more numbers of CD4⁺T cells in the spinal cord and brain of control mice,especially the CD4⁺T cells infiltration is more obvious in the brain.The numbers of CD4⁺T cells was significantly reduced in the treatment mice with BYHWD（p<0.01）.5.In this study,we measured ROCKII,TLR4,NF-κB and cox-2 expression by western blotting.The expression of ROCKII was remarkably inhibited in spinal cord,brain and spleen of EAE mice treated with BYHWD（p<0.01）.Also,BYHWD reduce protein expression of TLR4,Myd88,p-NF-κB/p65,cox-2 in the spinal cord（p<0.01）.ConclusionBYHWD shows the good potential treatment in EAE mice,especially in the earlier stage.（1）BYHWD can suppress the reaction of inflammatory and the demyelination of EAE by preventing the migration of inflammatory cells or CD4⁺T cells into the CNS（2）BYHWD can reduce the inflammatory pathological damage of EAE by inhibiting the expression of ROCK/TLR4/NF-κB signaling pathway;（3）BYHWD promotes the anti-inflammatory effect of EAE by increasing the proportion of regulatory T cells and promoting the release of immunosuppressive factors in Th2 cells.