mTOR调控巨噬细胞极化参与肝脏缺血再灌注损伤的机制

Liver ischemia and reperfusion injury (IRI) remains one of the major causes of graft malfunction post liver transplantation. During IRI, innate immunity plays significant roles in regulating liver inflammation. As the primary participant of innate immune response, liver macrophages, also known as Kupffer cells, polarized to proinflammatory M1 and alternative M2 status upon IR. However, the regulation of macrophage polarization remains to be elucidated. Our previous study showed that mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and complex 2 (mTORC2) had different impact on local inflammation during mouse liver IRI. In addition, the regulation of mTOR in macrophage polarization has been increasingly reported recently. Thus, we assume that mTOR regulates liver inflammation induced by IR through adjusting the polarization of macrophages. To prove this hypothesis，we plan to inhibit mTORC2 in macrophages through selectively knocking out Rictor gene in mouse myeloid cells and then evaluate the liver inflammatory responses and the polarization of macrophages, and finally to conclude the regulatory mechanisms of mTOR on macrophage polarization. These findings may develop novel strategies for treating IRI after liver transplantation.

肝脏缺血再灌注损伤（IRI）是肝脏移植术后移植物功能障碍的主要原因之一，固有免疫反应在这一病理过程中发挥重要调节作用。巨噬细胞（Kupffer细胞）作为固有免疫反应的主要参与者，在肝脏IRI中的调节作用仍未完全阐明。我们的前期研究结果表明，在小鼠肝脏IRI模型中，哺乳动物雷帕霉素受体复合物1（mTORC1）和复合物2（mTORC2）显示出不同的炎症调节作用。此外，诸多体内外实验显示mTOR对巨噬细胞极化亦具有调节作用。由此我们推测，mTOR通过调控巨噬细胞的极化方向，即具有促炎作用的经典活化M1型和具有抗炎作用的选择性活化M2型巨噬细胞，从而调节肝脏IR损伤过程中的炎症反应。我们拟采用小鼠70%肝脏热缺血再灌注损伤模型，通过髓系敲除Rictor基因抑制mTORC2的活性，观察其对巨噬细胞极化的调节和对炎症反应的影响，并进一步阐明其调节机制，从而为临床改善肝脏IR损伤寻找新的治疗靶点。

目的：肝脏缺血再灌注损伤是临床过程中常见的一种病理生理过程，常常会导致严重的后果。自噬是由溶酶体介导的细胞自我吞噬的现象，其在缺血再灌注损伤中发挥重要作用。Rictor是mTORC2的特异性结合蛋白，在肝脏缺血再灌注损伤发生发展中的作用及其机制尚不明确。本研究旨在探讨Rictor蛋白通过调节自噬在肝脏缺血再灌注损伤中的机制研究。方法：体内实验建立小鼠肝脏缺血再灌注损伤模型，体外实验建立小鼠原代肝细胞缺氧/复氧损伤模型；分别采用ELISA、qRT-PCR、Western blot、免疫组织化学等方法检测小鼠肝脏损伤程度以及免疫细胞的浸润程度和炎症因子的释放水平；采用细胞毒性实验检测缺氧/复氧过程中肝脏细胞损伤程度；分别采用Western Blot、透射电镜和免疫荧光等方法检测肝脏缺血再灌注和缺氧/复氧损伤过程中自噬的变化；采用Western Blot检测自噬相关信号通路、Rictor/mTORC2信号通路的变化。结果：Rictor蛋白的表达量随着再灌注时间的增加而逐渐增加，在小鼠体内敲低Rictor蛋白表达，再灌注之后小鼠血清转氨酶水平明显增加，肝脏组织损伤区域明显增多，炎症细胞浸润明显增加，活性氧ROS水平也明显增加。Rictor 蛋白缺失在缺血再灌注损伤中降低了细胞自噬水平并且激活了MAPK信号通路。结论：Rictor蛋白的缺失通过减轻自噬并且激活MAPK信号通路而加重小鼠肝脏缺血再灌注损伤。