基于宏基因组学探讨健脾益气法调节重症肌无力肠道微生态-免疫机制研究

Myasthenia gravis (MG) is a rare and stubborn disease with the characteristics of severe symptoms, high mortality rate and high recurrence rate. Once suffer from this kind of disease, will lose labor force, affected the patient's quality of life badly. Strengthening spleen and supporting Qi fang which guided by traditional Chinese medicine theory has obtained good curative effect in MG, Experience useful by professor Dengtietao，but its mechanism have not yet been confirmed. Recent researches show that Treg cells and cytokines play an important role on the pathogenesis of myasthenia gravis and progression of disease by activating the specific anti-acetylcholine-receptor. Meanwhile, the intestinal bacteria and metabolism is crucial for the immunologic balance of intestinal mucosa in human body. Therefore, we speculate the strengthening spleen and supporting Qi fang may through the ways above to play its therapeutic effect. We intends to establish a rat model of autoimmune acquired myasthenia gravis(EAMG) as study object. First we detect Tregs and cytokine immune expression levels. Meanwhile we analysis gut microbiota and explore the key genes that lead to immune imbalance of myasthenia gravis by using high-throughput gene sequencing technologies.The above pathways were further clinically verified，To explore the mechanism of the method of strengthening spleen and supporting Qi on gut microbes-immunity intervention in MG on the level of genes and cells, which might provide important scientific basis for prevention and treatment of myasthenia gravis with traditional Chinese medicine.

重症肌无力(MG)具有病情重、死亡率高、易复发的特点，是世界性难治疾病之一。国医大师邓铁涛教授验方“强肌健力方”治疗MG具有良好的疗效，但其作用机理尚不明确。近年研究发现AChR特异性Treg细胞及其细胞因子介导的免疫机制通过激活自身抗体在MG的发病及进展中发挥重要作用，而肠道菌群结构及靶向调控对体内免疫平衡起决定性作用。因此，我们推测MG发病可能与上述通路相关，菌群靶向调控的免疫调节作用是健脾益气法治疗MG的关键机制。本研究以自身免疫性获得性重症肌无力大鼠模型(EAMG)为对象，进行“强肌健力方”干预，应用宏基因测序方法对EAMG肠道菌群进行分析并筛选关键基因，检测特异菌群导向的Treg细胞及其细胞因子的表达水平，并进一步对上述通路进行临床验证，从基因-细胞水平阐释健脾益气法对EAMG肠道微生态-免疫调节的作用机制，对揭示中医药治疗MG的机制具有重要意义。

背景：重症肌无力是一种神经免疫罕见病，国医大师邓铁涛教授认为，脾胃虚损是其主要病机，健脾补气法治疗具有良好的临床疗效，但其作用机理尚不明确。MG患者的肠道微生物组成与健康人存在显著差异，患者的肠道关键微生物特征在实验性自身免疫性重症肌无力EAMG小鼠动物模型中保持不变。免疫细胞如Treg细胞及其细胞因子介导的免疫机制通过激活自身抗体在MG的发病及进展中发挥重要作用，而肠道菌群结构改变及代谢调控对体内免疫平衡起决定性作用。.主要研究内容：本研究采用主动免疫法建立实验性自身免疫性小鼠模型，对模型进行体重、抗体滴度评估，造模成功后给予健脾补气方（强肌健力胶囊）干预，进行粪便16sRNA基因测序，生物信息学分析对比组间的菌群差异。流式细胞技术检测其中各组T淋巴细胞的比例，并筛选出强肌健力胶囊靶向干预的短链脂肪酸。体内实验验证观察强肌健力方靶向的短链脂肪酸MG患者Treg细胞分化的影响。基于菌群宏基因测序技术，应用健脾补气方主药成分黄芪甲苷（AS-IV）干预治疗EAMG进行干预研究，宏基因测序技术分析肠道菌群的差异及基因功能特征，对差异菌群进行诊断模型预测。从肠道菌群-免疫调节角度探讨中医药治疗MG的作用机制。.重要结果：EAMG模型符合脾胃虚损的表现，与健康小鼠对比存在肠道菌群差异。强肌健力胶囊通过肠道菌群改善重症肌无力的症状，其中阿克曼氏菌属可能是重要的靶点。肠道菌群代谢产物短链脂肪酸中的丁酸可以通过激活MG体内Treg细胞的自噬进而促进MG的Treg细胞的分化和抑制功能。强肌健力胶囊可通过提高机体促丁酸生成菌的相对丰度进而提升丁酸的含量来发挥治疗MG的作用。AS-IV干预EAMG小鼠可改变肠道菌群的多样性，并显著恢复体内T淋巴细胞的比例失衡，细菌磷酸转移酶系统和双组分系统可能是其主要的调控通路，预测模型显示标志性菌株可有效区别健康小鼠及EAMG小鼠。.科学意义：健脾补气法治疗MG的作用机制之一是通过调节体内肠道微生物群，改善体内免疫失衡反应、纠正免疫紊乱而起到改善肌无力症状的疗效。该研究结果提示，菌群-免疫调节在MG的发病机制、药物作用机制、靶向菌群新药研究及粪菌移植中有广阔前景，