| Objective:To investigate the immunological mechanism of Jianpi Yiqi Busui Prescription in the treatment of experimental autoimmune myasthenia gravis model rats.Methods:1.The experimental autoimmune myasthenia gravis animal model was actively immunized by injection of immunogen.The clinical symptoms,body weight changes and the peripheral blood AchR-ab of the rat model were used to evaluate the Jianpi Yiqi Busui Method in the treatment of experimental autoimmune myasthenia gravis;2.ELISA was used to detect the levels of serum IFN-?,IL-4 and IL-10 in rats.3.Fluorescence activated cell sorter(FACS)was used to detect number of subgroups changes CD4+T cells(Treg cell,Th1,Th2 and Th17 cell)in spleen mononuclear cells of EAMG;4.Real-time qPCR was used to detect the expression of cytokines IL-17,TGF-?,IL-6,IFN-?and IL-21 in spleen tissues of healthy EAMG groups involved in regulating the balance of Th17/Treg cell transformation.Results:1.Model establishment:The third immunization was performed 45 days after the first immunization.The clinical symptoms of EAMG were further aggravated,and there was shortness of breath.The tremor was further severe,and the neck could not be lifted.The head was close to the table,and the EAMG score was higher than that of the CFA group.The difference was statistically significant(P<0.001),the hair color was dry,the voice was low,the incisors grew too long,the upper jaw was touched,and the symptoms were further reduced.After 25 days,the rats in the EAMG group and the CFA group were treated with Anti ratAchR-ab in the peripheral serum.There was no statistical difference in secretion levels(P=0.08>0.05).After the third booster immunization,the level of Anti ratAchR-ab secretion in peripheral blood of EAMG group was significantly higher than that of CFA group,and there was significant difference between them(p<0.0001).2.Weight and clinical symptoms of EAMG:The body weight of the EAMG group did not increase with the progress of the experiment,and there was always a significant difference in body weight between the CFA group and the rats in the CFA group(****p<0.0001);Comparing the rats in the high,middle and low dose groups of Chinese medicine,the clinical symptoms of the rats in each group gradually eased with the experiment.The weight gain of the rats in each dose group was statistically different from that of the EAMG group(from the 20th day of administration)(**p<0.01),the above results indicate that Jianpi Yiqi Busui Prescription can improve the weight and clinical symptoms of EAMG.3.The level of Anti ratAchR-ab antibody in EAMG peripheral serum:In the EAMG group,the level of Anti ratAchR-ab in peripheral blood of rats was always higher than that in CFA group.Compared with EAMG,the levels of AntiratAchR-ab in peripheral blood of rats in each dose group of Chinese medicine were significantly lower than those in low dose group.The level of Anti ratAchR-ab in peripheral blood of rats in high dose group of Chinese medicine decreased significantly,with significant difference(**p=0.22<0.01),the level of AntiratAchR-ab in EAMG peripheral serum was significantly lower than that in the prednisone group,and there was no significant difference(p=0.22>0.05),indicating that prednisone and The levels of Anti ratAchR-ab in EAMG peripheral blood were decreased in all doses of traditional Chinese medicine.The lower dose of Chinese medicine in the high dose group,the level of AntiratAchR-ab in peripheral blood decreased more significantly,and the high dose was equivalent to prednisone;4.Cytokine levels:Compared with CFA group,the levels of pro-inflammatory cytokine IFN-?in peripheral blood of EAMG group increased significantly,anti-inflammatory cytokines IL-4 and IL-10 decreased significantly,and there were significant differences between groups(***p<0.001);the levels of pro-inflammatory cytokine IFN-?decreased significantly after anti-inflammatory cytokines IL-4 and IL-10(**p<0.01).5.CD4~+T cell differentiation subgroup:Compared with CFA group,spleen tissue mononuclear cells CD4IL4~+T cells(Th2 cells)and CD4~+CD25~+(regulatory T cells)were significantly decreased in EAMG group,and there was significant difference between groups.Difference(***p<0.001),the number of CD4~+T cells,CD4~+IL-17~+T cells(Th17cells)and CD4~+IFN-?~+T cells(Th1 cells)increased significantly,and the comparison between groups was significant.Difference(***p<0.001);Compared with the model group,Th2 cells and regulatory T cells were significantly up-regulated after high-and medium-dose intervention of Jianpi Yiqi Busui Prescription,and the number of CD4~+T cells,Th17 cells and Th1 cells was significantly reduced.There was a significant difference between the groups(***p<0.001),and it was equivalent to the effect of the prednisone group.In the low dose group of Jianpi Yiqi Busui Prescription,the regulatory T cells were up-regulated,CD4~+T cells,Th17 cells,and Th1 cells.The number was significantly reduced,and there was a significant difference between the groups(***p<0.001),but the number of Th2 cells was not significantly different from the model group(p>0.05);6.Gene expression of cytokines IL-17,TGF-?,IL-6,IFN-?,IL-21:Compared with CFA group,the expression levels of IL-17,IFN-?,IL-21,IL-6 mRNA in EAMG group were significantly increased.There was a significant difference(***p<0.001),and the expression levels of TGF-?were significantly decreased,with significant difference(***p<0.001).Compared with the model group,Jianpi Yiqi Busui Prescription were high and medium After dose intervention,the expression levels of IL-17,IFN-?,IL-21 and IL-6 mRNA were significantly decreased,and there was significant difference between the groups(**p<0.01).The expression level of TGF-?mRNA was significantly increased,which was significant.The difference(**p<0.001);the expression levels of IL-17 and IL-21 mRNA in the low dose group of Jianpi Yiqi Busui Prescription were significantly decreased,and the expression of TGF-?mRNA was increased.There was a statistically significant difference between the groups(*p<0.05).However,there was no significant difference between IL-6and IFN-?mRNA expression levels and EAMG group(p>0.05).Conclusion:1.Jianpi Yiqi Busui Prescription reduced the level of anti-chR-ab in peripheral blood and improves the clinical symptoms of experimental autoimmune myasthenia gravis model rats;2.Jianpi Yiqi Busui Prescription can inhibit the production of pro-inflammatory cytokines IL-17 and IFN-?and gene level,up-regulating the expression of anti-inflammatory cytokine IL-4,promoting the differentiation and inhibition of CD4~+T cells Th2 cells.Differentiation of CD4~+T cells into Th17 cells and Th1 cells improves Th1/Th2/Th17 cell immune imbalance3.Jianpi Yiqi Busui Prescription can inhibit the expression of cytokines IL-6 and IL-21,up-regulate the production of anti-inflammatory cytokine IL-10 and TGF-?gene expression,inhibit differentiation of CD4~+T cells into Th17 cells,promote the transformation of Th17cells into Treg cells,thereby inhibit the development of EAMG. |
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