泻火消瘿方通过mTOR信号通路调控Tfh/Tfr细胞平衡治疗Graves病的机制研究

Graves Disease (GD) is a common clinical refractory autoimmune disease. How to reduce the TRAb level is the most difficult problem. Xiehuo Xiaoying Formula, a kind of hospital preparations, has shown a significant effect in the treatment of GD. Our previous study shows that this formula can effectively reduce TRAb levels, but the exact mechanism remains unclear. It has been proven that the imbalance of Tfh/Tfr contribute to the high level of TRAb leading to the onset of GD. Also, the inhibition of mTOR signaling pathway may recover the balance of Tfh/Tfr and may promote the relief of GD. The GD patients has shown an obviously imbalance of Tfh/Tfr and Xiehuo Xiaoying Formula can suppress the expression of mTOR in CD4+T cells of thyroid tissue according to our previous study. Thus, We put forward the hypothesis: Xiehuo Xiaoying Formula could treat Graves Disease by regulating the balance of Tfh/Tfr via the mTOR signaling pathway. In this project, we would firstly sort T cells by MACS technology and establish Graves Disease mouse model as research subjects. Then, detect the changes of inflammatory cytokines, immune cells, mTOR pathway proteins and downstream factors. Finally, clarify the treatment mechanism of Xiehuo Xiaoying Formula on Graves Disease from holistic, cellular and molecular levels. It may provide new evidence on promoting the formula.

Graves病（GD）是临床难治的自身免疫性甲状腺病，降低自身抗体TRAb水平是其临床治疗难点。泻火消瘿方是我院疗效显著的临床经验协定方。前期研究显示，该方可显著降低TRAb水平，但其机制不明。业已证实TRAb的产生主要源于Tfh/Tfr细胞失衡，抑制mTOR信号通路可恢复Tfh/Tfr细胞平衡，有望促进GD缓解。我们前期亦发现GD患者存在Tfh/Tfr细胞失衡，且预实验证实泻火消瘿方能下调GD小鼠甲状腺组织CD4+T细胞mTOR的表达。故我们提出假说：泻火消瘿方通过mTOR信号通路调控Tfh/Tfr细胞平衡治疗GD。本研究以GD模型小鼠和磁珠分选的小鼠CD4+T细胞为研究对象，通过检测炎性因子、免疫细胞、mTOR信号通路蛋白及下游因子表达的变化，从整体、细胞、分子水平阐明泻火消瘿方治疗GD的机理，为该方的进一步推广应用提供依据。

Graves病（GD）是临床难治的自身免疫性甲状腺病，免疫细胞功能异常引起TRAb升高在GD致病中发挥重要作用，降低自身抗体TRAb水平是其临床治疗难点。泻火消瘿方（XHXY）是我院疗效显著的临床经验协定方。前期研究显示，该方可显著降低TRAb水平，但其机制不明。本课题围绕GD发生的关键环节—TRAb开展研究，探讨泻火消瘿方抑制TRAb的效应及其机制。本研究以GD模型小鼠和磁珠分选的小鼠CD4+T细胞为研究对象，研究发现：（1）XHXY改善了GD小鼠甲状腺功能亢进。给药4周后，与模型组相比，XHXY组显著降低了小鼠的甲状腺激素及TRAb水平，减轻GD模型小鼠甲状腺滤泡上皮细胞病理损伤；（2）XHXY治疗纠正了Tfh/Tfr细胞失衡，增加Treg水平，改善了小鼠免疫异常。与模型组相比，XHXY治疗有效抑制了Tfh细胞产生，促进了Tfr细胞增殖，Tfh/Tfr比值降低；XHXY治疗后Tfh相关细胞因子IL-4、IL-21及特异性转录因子Bcl-6降低，Tfr相关细胞因子IL-10、TGF-β水平升高，Treg比例及FOXP3表达增加。（3）XHXY治疗激活GD小鼠脾脏keap1/Nrf2信号通路，显著减轻GD小鼠氧化应激反应，调节免疫反应。XHXY治疗能够上调Nrf2、HO-1，抑制Keap1蛋白表达，有效降低GD小鼠NOX2、4-HNE、8-OHdG水平，改善氧化应激反应。本研究阐释了泻火消瘿方治疗GD免疫失常的机制，为临床推广应用泻火消瘿方治疗GD，改善免疫失常提供了现代科学依据，具有重要意义。