| OBJECTIVE:To observe the medullary thymic epithelial cells(mTECs)and regulatory T-cell(Treg)related factors in EAMG rats by intragastric intervention of Yi-qi-Jie-du prescription and prednisone,so as to elucidate the immunoregulatory mechanism of thymus in myasthenia gravis(MG)and the intervention target of Yi-qi-Jie-du prescription,which provides theoretical support and experimental basis for the treatment of MG and the development of Yi-qi-Jie-du prescription.METHODS:(1)Sixty healthy female Lewis rats were selected,6 were used as adjuvant controls,and 54 were subcutaneously injected with immune emulsion.The success of the animal model of experimental autoimmune myasthenia gravis(EAMG)was comprehensively evaluated in combination with Lennon score,weight change of rats,low frequency repetitive electrical stimulation results and serum AchR-Ab content changes.Antidote Compound and prednisone were administered intragastrically.And Yi-qi-Jie-du prescription and prednisone were given intragastric administration to rats after successful model establishment.(2)The expressions of Foxp3 and CTLA-4 in thymus tissues Characteristic markers of Treg were detected by Rt-qPCR,Western-Blot and immunohistochemistry methods respectively;The expressions of Aire in thymus tissues Characteristic markers of mTECs were detected by Rt-qPCR,Western-Blot.(3)The expressions of Foxp3,CTLA-4 and Treg cell-related cytokines IL-4,IL-10 and TGF-β1 in peripheral blood were detected by ELISA and flow cytometry.RESULT:(1)Animal model evaluation:43 of the 54 rats in the immune emulsion group were successfully established.The success rate of EAMG rat model was 79.63%.(2)The results of Rt-q PCR and Western-Blot showed that the expression of Aire protein and gene in thymus tissue of model group was lower than that of adjuvant control group(P<0.01).And the results of immunohistochemistry,Rt-q PCR and Western-Blot showed that the expression of Foxp3 protein and gene in thymus tissue of model group was significantly lower than that of adjuvant control group(P<0.0001,P<0.01,P<0.0001).The results of immunohistochemistry and Rt-q PCR indicated that the expression of CTLA-4 protein and gene in thymus tissue of model group was significantly lower than that of adjuvant control group(P<0.05,P<0.01)(3)The results of IL-10 and TGF-β1 detected by ELISA indicating that the level of IL-10 in model group was significantly higher than that in adjuvant control group(P<0.0001),and the level of TGF-β1 was significantly lower than that in adjuvant control group(P<0.01).Flow cytometry was used to investigate the expression of Treg cell characteristic surface factors Foxp3,CTLA-4 and Treg-related cytokines IL-4 and IL-10 in peripheral blood.The results showed that the percentage of CD4+CD25+Foxp3+Treg cells and CTLA-4+T cells in the model group was significantly lower than that in the adjuvant control group(P<0.01,P<0.0001),and the percentage of IL-10+T cells and IL-4+ T cells in the model group was significantly higher than that in the adjuvant control group(P<0.05,P<0.0001)(4)After treated with Yi-qi-Jie-du prescription,the gene and protein expression levels of Aire,Foxp3 and CTLA-4 were improved than those of model group(P<0.01,P<0.05).The results of ELISA indicating that the content of IL-10 decreased significantly after treatment with Yi-qi-Jie-du prescription(P<0.0001),while the content of TGF-β1 was no significant difference(P>0.05).Flow cytometry results showed that the percentage of CD4+CD25+Foxp3+Treg cells and CTLA-4+T cells had no significant difference compared with the adjuvant control group(P>0.05)Compared to the model group,the expression of the percentage of IL-10+T cells and IL-4+T cells were decreased significantly(P<0.0001).After comprehensive evaluated the results,the middle dose group of Yi-qi-Jie-du prescription had the best therapeutic effect.CONCLUSION:(1)EAMG rat model can be successfully reproduced by subcutaneous injection of immune emulsion.(2)Treg cell dysfunction may be associated with mTECs functional abnormality and Aire expression decline,which may be the cause of autoimmune diseases such as MG,and the specific mechanism needs further study.(3)The changes of characteristic markers and cytokines of Treg cells in peripheral blood of model group were consistent with those in thymus,it may be suggested that the treatment of MG could begin with thymus,or may interfere with peripheral immunity to achieve therapeutic effect.(4)Yi-qi-Jie-du prescription can interfere with the expression of mTECs cell-specific factor Aire in the thymus of EAMG rats,and can simultaneously interpret the expression of Treg cell-related factors in thymus and peripheral blood.After treatment,the symptoms,body weight and Lennon score of the rats were improved,suggesting that the target of Yi-qi-Jie-du prescription in treating MG may be to promote the release and regulation of immunoregulatory factors such as TGF-β1 and Aire.The function of Treg and mTECs,but its specific mechanism needs further experimental proof. |
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