黄葵胶囊调控PI3K/Akt/mTOR信号通路活性而保护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）. Huangkui capsule（HKC），a Chinese patent medicine，has been applied extensively for the treatment of proteinuria in the patients with early DN in China，however，its therapeutic mechanism is fully unclear. It is known that，in the process of early DN，the activation of phosphatidylinositol-3-kinase（PI3K）/ serine-threonine kinase（Akt）/ mammalian target of rapamycin（mTOR）signaling pathway in the kidney leads to structural and functional podocyte lesion，which could result in proteinuria. Our previous study demonstrated that HKC in vitro could improve podocyte dysfunction by regulating the downstream signaling target in PI3K/Akt/mTOR pathway. It is supposed that，PI3K/Akt/mTOR pathway is not only the egulative signaling channel in DN but also the novel therapeutic target in the treatment of proteinuria with HKC. In this report，using the animal DN models and cellular models under high-glucose condition，we firstly purpose to clarify whether PI3K/Akt/mTOR pathway could be related to podocyte damage，and if so，we secondly aim to demonstrate the effectiveness and mechanism of HKC ameliorating podocyte injury via regulating PI3K/Akt/mTOR pathway in vivo and in vitro. Finally，we plan to establish in vitro the action of HKC on podocyte lesion through interfering with the downstream signaling target in PI3K/Akt/mTOR pathway in transgenic cellular model. In sum，for the sake of the second developmental clinical application of HKC，an classical anti-nephritic Chinese patent medicine，in traditional Chinese medicine（TCM），we in the present study aim at revealing a new therapeutic target of the treatment with HKC in proteinuria on the basis of the novel regulatory mechanism of podocyte injury in DN.

蛋白尿是糖尿病肾病（DN）治疗的难点。黄葵胶囊（HKC）是临床上治疗早期DN患者蛋白尿的有效中成药制剂，但机制不明。DN早期发病过程中肾组织PI3K/Akt/mTOR信号通路激活可导致足细胞结构和功能异常而出现蛋白尿。前期研究表明，HKC在体外通过调控PI3K/Akt/mTOR通路关键下游信号分子而改善足细胞功能。假设PI3K/Akt/mTOR通路既是DN足细胞损伤的信号调控途径，也是HKC治疗DN蛋白尿的新靶点。笔者借助动物和细胞模型，在DN 或体外高糖环境下，首先阐明PI3K/Akt/mTOR通路激活对足细胞损伤的作用；其次阐明HKC调控该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HKC调控该通路关键下游信号分子而改善足细胞损伤的作用。本项目基于对DN足细胞损伤分子调控机制的新认识，揭示HKC治疗DN蛋白尿的新靶点，促进经典的抗肾炎中成药在中医临床的二次开发应用。

黄蜀葵花中成药制剂——黄葵胶囊（HKC）是临床上广泛应用于治疗早期糖尿病肾病（DN）患者蛋白尿的有效药物，但机制不明。假设在DN 或体外高糖状态下PI3K/Akt/mTOR信号通路既是DN足细胞损伤的信号调控途径，也是HKC及其有效成分金丝桃苷（HPY）治疗DN蛋白尿的新靶点。针对①PI3K/Akt/mTOR信号通路激活在体内对DN足细胞损伤的作用以及HKC的影响；②PI3K/Akt/mTOR信号通路激活在体外对高糖诱导的足细胞损伤的作用以及HYP的影响；③调控PI3K/Akt/mTOR信号通路的关键下游信号p-70S6K而改善高糖诱导的转基因足细胞损伤的作用以及HYP的影响，首先，借助改良型DN模型鼠，我们发现，PI3K/Akt/mTOR信号通路激活会诱导足细胞损伤，其病理特征是“nephrin、neph1蛋白表达下调”和“足突融合”；HKC在体内能改善足细胞损伤，其功效不同于mTOR抑制剂——雷帕霉素（RAP）；HKC保护足细胞损伤的作用是通过调控PI3K/Akt/mTOR信号通路活性来实现的，这一机制与RAP类似。其次，借助足细胞模型，我们发现，高剂量D-葡萄糖（高糖、HG）在体外能激活PI3K/Akt/mTOR信号通路而诱导足细胞损伤和上皮-间充质转分化（EMT）； HYP在适当的剂量范围内能抑制PI3K/Akt/mTOR信号通路激活而改善足细胞损伤和EMT，这可能是HYP保护DN足细胞损伤的关键分子机制之一。最后，借助转基因足细胞模型，我们发现，HYP调控PI3K/Akt/mTOR信号通路和NLRP3/Caspase-1/IL-1β信号通路，靶向干预下游信号p-70S6K，改善HG诱导的足细胞损伤。本项目基于对DN足细胞损伤信号调控途径的新认识，揭示“清利法”代表性中成药制剂HKC及其有效成分HPY治疗DN蛋白尿的新靶点，促进其在DN治疗领域的转化应用。