Study On Mechanism Of B Cell-mediated Immunity In Experimental Autoimmune Myasthenia Gravis Rats Treated With Jianpi Yiqi Busui Method

Objective: To explore the immune mechanism of Jianpi Yiqi Busui method(qishendihuang granule)on B cells of experimental autoimmune myasthenia gravis(EAMG)rats.Methods: 1.Eight Lewis female rats were random Ly selected as adjuvant control group(CFA group),and the remaining rats were immunized with Rα97-116 peptide.Forty experimental autoimmune myasthenia gravis(EAMG)rats were random Ly divided into model group(Model group),low-,medium-,and high-dose qishendihuang granules groups(QSDH-L,QSDH-M,QSDH-H groups)and prednisone group(Prednisone group)and 8 rats in each group.The body weight and clinical symptoms of rats were observed,and the content of ACh R-Ab was detected by ELISA to evaluate the effect of Jianpi Yiqi Busui method on symptoms and antibodies of experimental autoimmune myasthenia gravis rats.2.The protein expressions of CD19 and CD27,and the protein expressions of BTK,Lyn,Syk,PLC-γ 2,p-btk,p-lyn,p-syk and p-plc-γ2 were detected by Western blot.3.Immunohistochemistry was used to detect the expression of CD19,CXCL13,B220 and CD44 in spleen and thymus.4.The m RNA expressions of BAFF,CXCL13 and CXCR5 were detected by RT-PCR.Results: 1.Before administration,compared with adjuvant control group,weight loss(P < 0.001),aggravation of clinical symptoms(P < 0.001),and increase of ACh R-Ab content(P < 0.001)were significant differences.2.After the treatment,compared with the model group,the weight gain of qishendihuang granules in low,medium and high dose groups(P < 0.05,P < 0.01,P < 0.001),the clinical symptoms were reduced(P < 0.01,P < 0.001),and the content of ACh R-Ab decreased(P < 0.05,P < 0.01,P < 0.001),which had significant difference.3.Western blot showed that the expression of CD19 and CD27 in model group increased(P < 0.001),Btk,Syk,plc-γ2 protein expression increased(P < 0.01,P < 0.001), and decreased expression of Lyn protein(P < 0.01),Compared with the model group,the expression of CD19 in the low,medium and high dose groups and the positive drug groups was decreased(P < 0.05,P < 0.001),CD27 protein expression decreased(P < 0.01,P < 0.001),Btk,Syk,plc-γ2 protein expression decreased(P < 0.05,P < 0.01,P < 0.001),and the expression of Lyn protein increased(P < 0.05,P < 0.01),The expression of Syk and plc-γ2 phosphorylation was decreased(P < 0.05,P < 0.01,P < 0.001),and the expression of lynphosphorylation increased(P < 0.05,P < 0.01),which had significant difference,and there was no significant difference in the expression of Btk phosphorylation among the groups.4.The results of immunohistochemistry showed that the expression of CD19,CXCL13,B220 and CD44 in model group was increased compared with the adjuvant control group in spleen and thymus tissues of rats.Compared with model group,CD19,CXCL13,B220 and CD44 in qishendihuang granule low,medium and high dose groups were decreased.In spleen tissue,CD19 qishendihuang granule high dose group,CXCL13 qishendihuang granule high dose group,B220 qishendihuang granule high dose group and CD44 qishendihuang granule medium and high dose group were the most significantly lower expression,close to the positive drug group;in thymus tissue,CD19 qishendihuang granules in medium and high dose group,CXCL13 qishendihuang granule high dose group,B220 qishendihuang in medium and high dose group and CD44 qishendihuang granule in medium and high dose group were the most significantly lower expression,which were close to the positive drug group.5.RT-PCR results showed that compared with adjuvant control group,BAFF,CXCL13 and CXCR5 m RNA gene expression level in model group increased(P < 0.05,P < 0.01).Compared with model group,BAFF m RNA expression level of qishendihuang granules in medium,high dose and positive drug group decreased The m RNA expression level was decreased(P < 0.05),and there was statistical difference.Conclusion: 1.The rat model of EAMG can be successfμLly established by R α 97-116 peptide modeling method,which provides a certain guarantee for the model construction of EAMG research.2.Jianpi Yiqi Busui method(qishendihuang granule)can increase the weight of EAMG rats,effectively improve the clinical symptoms,reduce the content of ACh R-Ab,and has the effect of treating myasthenia gravis.3.Jianpi Yiqi Busui method(qishendihuang granule)can regulate the protein expression of Btk,Syk,PLC-γ2 and Lyn and phosphorylation expression of Syk,PLC-γ2 and Lyn,reduce the signal transduction of BCR,inhibit the activation mechanism of B cells,regulate the immune system,and play a therapeutic role in MG.4.Jianpi Yiqi Busui method(qishendihuang granule)can reduce the expression of CD19,CD27,B220,CD44,BAFF,CXCL13 and CXCR5 in EAMG rats,reduce the differentiation and proliferation of B cells,inhibit the production of ACh R-Ab by B cells,reduce the damage to ACh R,correct the immune response,and has therapeutic effect on MG.