益气解毒复方对实验性自身免疫性重症肌无力大鼠胸腺中Aire及ROR-γt/Foxp3相关炎症因子表达的影响

Effects of Yiqi Jiedu Compound on the Expression of Aire and ROR-γt/Foxp3-Related Inflammatory Factors in the Thymus of Experimental Autoimmune Myasthenia Gravis Rats

目的研究益气解毒复方对实验性自身免疫性重症肌无力(Experimental autoimmune myasthenia gravis,EAMG)大鼠胸腺中自身免疫调节因子(Aire)及转录因子维甲酸相关核孤儿受体(ROR-γt)/叉头翼状螺旋转录因子3(Foxp3)相关炎症因子表达的影响。方法通过免疫诱导注射鼠源性AchR-α亚基97-116肽段复制EAMG模型,分为正常组、模型组、阳性对照组(强的松)及益气解毒复方高、中、低剂量组,每组10只。观察大鼠行为学及Lennon评分,检测血清转化生长因子-β1(TGF-β1)、白细胞介素-6(IL-6)水平,Western blot检测EAMG大鼠胸腺组织中Aire和Foxp3的表达,RT-PCR检测各组大鼠胸腺组织中RORγt的表达情况。结果与治疗前相比,各药物组Lennon分级评分显著降低;与强的松组相比,益气解毒复方各剂量组Lennon评分无明显差异(P>0.05)。RT-PCR结果,模型组胸腺内RORγt mRNA相对表达量明显高于正常组(P<0.05),各药物组胸腺中RORγt mRNA相对表达量明显低于模型组(P<0.05)。Western blot结果,与正常组比较,模型组Aire和Foxp3蛋白的表达量均显著降低(P<0.05);与模型组比较,各药物组大鼠胸腺中Aire和Foxp3蛋白的表达量均显著升高(P<0.01)。ELISA结果,模型组大鼠血清IL-6含量明显高于正常组(P<0.01),而TGF-β1含量明显低于正常组(P<0.01);给药后,各药物组IL-6含量明显降低,TGF-β1含量明显升高(P<0.01)。结论益气解毒复方能改善EAMG大鼠肌无力症状,其作用机制可能通过上调Aire蛋白表达,调节免疫炎症反应,影响Th17/Treg分化,从而调控ROR-γt/Foxp3免疫平衡发挥调节自身免疫反应的作用,可能是其治疗MG的作用机制之一。

Objective To study the effect of Yiqi Jiedu compound on autoimmune regulator(Aire)and transcription factor retinoic acid-related orphan receptor(ROR-γt)/Effects of forkhead wing helix transcription factor 3(Foxp3)-related inflammatory factor expression.Methods The EAMG model was replicated by immunization-induced injection of mouse-derived AchR-αsubunit 97-116 peptide,including normal group,model group,positive control group(prednisone),and Yiqi Jiedu compound high-dose,medium-dose and low-dose groups.Group of 10,the behaviors and Lennon scores of the rats in each group were observed.ELISA method was used to detect serum transforming growth factor-β1(TGF-β1)and interleukin-6(IL-6)levels.Western blot was used to detect the thymus tissue of EAMG rats.The expression of Aire and Foxp3,and the expression of RORγt in the thymus tissue of rats in each group was detected by RT-PCR.Results Compared with before treatment,the Lennon grading score of each drug group was significantly decreased;compared with the prednisone group,there was no significant difference in the Lennon score of each dose group of Yiqi Jiedu compound(P>0.05).RT-PCR detection results showed that compared with the normal group,the relative expression of RORγt mRNA in the thymus of the model group was significantly increased(P<0.01).Western blot detection results showed that the expressions of Aire and Foxp3 in the model group were significantly lower than those in the normal group(P<0.05);The levels of Aire and Foxp3 proteins in thymus of the rats in each drug group were significantly higher than those of model group(P<0.01).ELISA results showed that serum IL-6 content in model group was significantly higher than that in normal group(P<0.01),while TGF-β1 content was significantly lower than that in normal group(P<0.01).After intragastric administration,the content of IL-6 in all drug groups was significantly decreased,and the content of TGF-β1 was significantly increased(P<0.01).Conclusion Yiqi Jiedu compound can improve the symptoms of muscle weakness in EAMG rats,and its mechanism may be through up-regulating the expression of Aire protein,regulating immune inflammatory response,affecting the differentiation of Th17/Treg,thereby regulating the immune balance of ROR-γt/Foxp3.The role of autoimmune response may be one of the mechanisms of its treatment of MG.