基于TNF-α介导的sTNFR1/PI3K/Akt炎症通路探究雷公藤改善糖尿病肾病足细胞损伤的分子机制

It is a difficult point to treat proteinuria in diabetic nephropathy（DN）. The extracts from Tripterygium wilfordii Hook. f.（TWHF）including multiglycoside of Tripterygium wilfordii Hook. f.（GTW）and triptolide（TP），the multi-fractions Traditional Chinese Medicine（TCM）which have a great significance for delaying the development of DN，have been applied extensively for the treatment of proteinuria in the patients with DN in China. However，the correlative molecular mechanism is fully unclear. It is known that，the activated inflammatory signaling pathway in kidney leads to proteinuria and podocyte lesion including nephrin down-regulation and cell apoptosis in the progressive state of DN. Our previous study demonstrated that TP could improve podocyte dysfunction by regulating the downstream signaling target in inflammatory pathway in vitro. It is supposed that，tumor necrosis factor（TNF）-α mediated soluble tumor necrosis factor receptor 1（sTNFR1）/phosphatidylinositol-3-kinase（PI3K）/serine-threonine kinase（Akt）inflammatory pathway（sTNFR1/PI3K/Akt pathway）is not only the classical regulative signaling channel but also the potential novel therapeutic target in the treatment of proteinuria with TWHF under inflammation in DN. Among which，sTNFR1，p85-PI3K，and 473pAkt，as three key signaling molecules，are involved respectively. In this report，using the animal DN models and cellular models under high-glucose condition，we firstly purpose to clarify whether TNF-α mediated sTNFR1/PI3K/Akt pathway could be related to podocyte damage，and if so，we secondly aim to demonstrate the effectiveness and mechanism of GTW or TP ameliorating podocyte injury via regulating TNF-α mediated sTNFR1/PI3K/Akt pathway in vivo and in vitro. Finally，we plan to establish in vitro the protective action of TP on podocyte lesion through interfering with the expression of sTNFR1，as an important upstream target in TNF-α mediated sTNFR1/PI3K/Akt pathway，in transgenic cellular model. In sum，for the sake of the translational application of TWHF in the clinical practice in TCM，we in the present study aim at revealing the deep rule of the treatment with TWHF in proteinuria on the basis of the new theory of podocyte injury under inflammation in DN.

蛋白尿是糖尿病肾病（DN）治疗的难点。雷公藤提取物雷公藤多苷（GTW）和雷公藤甲素（TP）是临床上治疗DN蛋白尿的组分中药，但机制不明。DN进展过程中肾组织炎症通路激活可导致足细胞损伤（nephrin表达下调、细胞凋亡）而出现蛋白尿。前期研究表明，TP通过调控炎症通路关键信号而改善足细胞功能。假设肿瘤坏死因子（TNF）-α介导的sTNFR1/PI3K/Akt通路既是DN炎症状态下足细胞损伤的经典信号调控途径，也是雷公藤治疗DN蛋白尿的新靶点。我们借助动物和细胞模型，在DN 或体外高糖状态下，首先阐明TNF-α介导的炎症通路对足细胞损伤的作用；其次阐明GTW或TP调控该炎症通路而改善足细胞损伤的效果和机制；最后建立基因工程细胞模型，阐明TP调控该炎症通路关键上游信号而改善足细胞损伤的作用。本项目基于对DN足细胞炎症性损伤的新认识，揭示雷公藤治疗DN蛋白尿的新靶点，促进其在中医临床的转化应用。

雷公藤多苷（GTW）和雷公藤甲素（TP）是临床上治疗糖尿病肾病（DN）蛋白尿的组分中药，但机制不明。假设：在DN 或体外高糖状态下，肿瘤坏死因子（TNF）-α所介导的sTNFR1/PI3K/Akt/mTORC1炎症通路既是足细胞损伤的上游信号调控途径，也是雷公藤治疗DN蛋白尿的新靶点。针对①TNF-α介导的炎症通路在体内对DN足细胞损伤的作用以及GTW的影响；②TNF-α介导的炎症通路在体外对高糖诱导的足细胞损伤的作用以及TP的影响；③调控炎症通路的关键上游信号——sTNFR1而改善高糖诱导的转基因足细胞损伤的作用以及TP的影响，首先，借助改良型DN模型鼠，我们发现，足突融合、podocin和CD2AP蛋白表达下调以及nephrin和neph1蛋白结合障碍是DN足细胞损伤的三种病理特征；GTW、TNF-α抑制剂——沙利度胺（THA）以及mTOR抑制剂——雷帕霉素（RAP）既能改善足细胞损伤，又能减轻相关的硬化性肾小球病变；与THA、RAP不同，GTW既能上调podocin和CD2AP的蛋白表达水平，又能上调nephrin和neph1蛋白结合水平；GTW、THA以及RAP均能针对不同靶点而调控炎症通路的活性，从而，改善DN足细胞损伤。其次，借助足细胞模型，我们发现，高糖与TNF-α共培养在体外能激活炎症通路和Wnt3α/β-catenin通路而诱导足细胞发生上皮-间充质转分化（EMT）；低剂量TP和THA在体外能明显抑制炎症通路和Wnt3α/β-catenin通路活性而改善高糖诱导的足细胞EMT。最后，借助转基因足细胞模型，我们发现，TP调控炎症通路和NF-κB/NLRP3/IL-1β通路的关键上游信号——sTNFR1而改善高糖诱导的足细胞损伤。本项目基于对DN足细胞炎症性损伤上游信号调控途径的新认识，揭示了GTW或TP治疗DN蛋白尿的新靶点，促进其在DN治疗领域的转化应用。