基于自噬在M22诱导甲状腺细胞增殖中的作用探讨中药甲亢宁治疗Graves病的机制

Thyroid receptor antibody(TRAb) plays an important role in the pathogenesis of Graves Disease(GD), which triggers inappropriate activation of several signaling pathways including phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway and induces cell proliferation. But the relationship among those mechanisms remain unclear. Autophagy, a process of self-digestion in which damaged molecules or organelles are degraded and recycled, has been confirmed as an important player in cell death and survival. Furthermore, while the dysregulation of PI3K/Akt/mTOR was demonstrated to be correlated with TRAb-induced cell proliferation, this pathway is also reported to be closely associated with autophagy. Our precious research found that Jiakangning, a Chinese herbal compound, could reduce the expression of PI3K/Akt/mTOR pathway and inhibit the proliferation of thyroid cells in GD mouse models. Thus, we infer that autophagy is involved in the mechanism of TRAb-induced cell proliferation and the therapeutic mechanism of Jiakangning might be related with the ragulation of autophagy activitity. In this study, we intend to use M22, a simulating TRAb monoclonal antibody, to stimulate FRTL cells to establish GD cell model, and observe the change of autophagy activity during the process; use stimulators and inhibitors to manipulate the activity of autophagy and explore its effect on cell proliferation; use Jiakangning-contained serum to treat GD cells and explore its therapeutic mechanism based on autophagy activity. Through the exploration of the role of autophagy in M22-induced thyroid cell proliferation and the therapeutic mechanism of Jiakangning compound in the process, we hope to get a better understanding of the pathogenesis of GD, to find new targets for drug treatment, and to provide theoretical basis of traditional Chinese medicine treatment of GD.

在GD的发病中，自身免疫紊乱产生的TRAb与甲状腺细胞表面TSH受体结合，上调包括PI3K/Akt/mTOR在内的多条信号通路水平，刺激甲状腺上皮细胞增殖。然而其中间环节尚不清楚。自噬与细胞的生存和死亡密切相关，且研究证实细胞自噬活性主要受mTOR通路的调节。前期研究表明甲亢宁可下调该通路的表达，抑制GD小鼠模型甲状腺细胞的增殖。由此我们推测:自噬机制参与TRAb诱导的甲状腺细胞增殖；甲亢宁可能通过调节自噬活性发挥其抑制细胞增殖的作用。本次研究拟通过M22诱导FRTL细胞建立GD细胞模型，观察自噬活性的时序表达；通过分别抑制及诱导自噬功能，研究自噬在甲状腺细胞增殖中的作用；甲亢宁含药血清干预GD细胞，基于自噬活性的变化探索其抑制细胞增殖的机制。以此初步探索自噬在TRAb诱导甲状腺细胞增殖中的作用及甲亢宁的干预机制，为深入研究GD的发病机制、寻找新的药物干预靶点、揭示中药作用机理提供依据。

Graves病（Graves disease，GD）又称毒性弥漫性甲状腺肿，是一种伴甲状腺素合成增多的器官特异性自身免疫性疾病。TRAb通过结合TSHR，上调PI3K/Akt/mTOR通路诱导的甲状腺细胞增殖为GD发病的重要环节，课题组前期研究发现甲亢宁可下调该通路的表达，抑制GD细胞增殖，促进细胞凋亡。近期研究表明，mTOR为调节自噬的重要因子，PI3K/Akt/mTOR通路为mTOR调控的主要通路之一，因此mTOR介导的细胞自噬可能参与GD甲状腺细胞的增殖与凋亡。本次实验采用透射电镜、免疫荧光、流式细胞术、Western Blot、RT-PCR、siRNA等技术，从体内外两个方面探讨自噬在GD甲状腺细胞增殖、凋亡中的活性变化及作用，深度揭示甲亢宁通过调控自噬从而起到干预GD细胞增殖、促进GD细胞凋亡的作用机制。. 研究结果表明：细胞自噬参与GD细胞增殖和凋亡的病理过程，甲亢宁胶囊能有效降低GD模型小鼠血清甲状腺激素和TRAb水平，减轻甲状腺肿大，改善甲状腺上皮细胞结构异常等病理特征，其机制与调控mTOR通路，上调LC3、Beclin1、Atg5等蛋白和基因表达，增强自噬程度，抑制甲状腺细胞增殖、促进细胞凋亡有关。研究结果为了解GD发病的分子机制提供一个更深层次的认识，为中药甲亢宁治疗GD提供新的理论和实验依据。