基于细胞信号通路再平衡假说研究中药治疗糖尿病肾病的机制

Animal studies suggest that the inhibition of cell signaling pathways of PI3K/Akt/mTOR or TGF-β 1/Smad can improve pathologic lesions in diabetic kidney disease(DKD); nevertheless, the cell signaling pathways of PI3K/Akt/mTOR and TGF-β 1/Smad are essential in pathophysiological processes including the maintenance of cell survival and anti-inflammation. Furthermore, the specific blocking of these two cell signaling pathways can result in severe toxic and side-effects in DKD. Based on the Yin-Yang balance theory of traditional Chinese medicine, we put forward the following hypothesis: the homeostatic imbalance of multiple cell signaling pathways, and loss of auto-feedback of their downstream are the key-points of the occurrence and development of DKD; traditional Chinese medicine can modulate the homeostatic imbalance of cell signaling pathways via “complementing the deficiency and impairing the excess”, and achieve the re-balance of multiple cell signal pathways in DKD. The study is to investigate whether traditional Chinese medicine Huangqitujian compound can inhibit the expression or activation of the “excessive” cell signaling pathway factors, including NF-κB，Smad3, and PI3K/Akt/mTOR; in the meantime, enhance the expression of the “deficient” factors of cell signaling pathway, including Smad7 and PTEN; thus, Huangqitujian compound might re-balance the imbalance of cell signaling pathways and regain the normal cellular functions, so as to treat DKD.

动物实验研究结果提示抑制PI3K/Akt/mTOR或TGF-β1/Smad细胞信号通路可以改善糖尿病肾病（DKD）的病理损害，但这两条细胞信号通路在维持细胞的存活、抗炎症反应等病理生理过程中又必不可少，特异性阻断这两条信号通路的药物会产生严重的毒副作用。我们基于中医的阴阳平衡理论提出如下假说：各种信号通路之间的自我平衡失调，以及信号通路下游的自我反馈调控机制的丧失是导致DKD发生发展的关键，中药可通过“补其不足、泄其有余“使信号通路之间的平衡失调状态获得再平衡，从而达到治疗DKD的作用。本项目研究中药黄芪菟箭合剂是否可以抑制DKD时肾脏细胞内“亢盛”的信号通路因子(NF-κB，Smad3、 PI3K/Akt/mTOR)的表达或激活，同时提高“亏虚”的信号通路因子(Smad7、PTEN)的表达，从而使信号通路的平衡失调获得再平衡，恢复细胞正常功能达到治疗DKD的目的。

糖尿病肾病（Diabetic nephropathy, DN）是糖尿病严重的微血管并发症，已成为全球终末期肾病（end stage of renal disease, ESRD）的主要病因。DN的主要病理表现为系膜扩张，肾小球肥大，随后细胞外基质合成增加，最终导致肾小球硬化和间质纤维化，这与系膜细胞增殖有密切关系。既往本团队已发现黄芪菟箭合剂（Huang Qi Tu Jian Decoction, HQTJD）可降低STZ 诱导的糖尿病大鼠的尿白蛋白排泄率，本项目调整动物模型为db/db 小鼠自发糖肾模型进行在体试验，并使用低、中、高剂量中药干预，并分别干预6周和12周观察疗效。同时，通过体外培养系膜细胞，联合使用通路抑制剂，观察HQTJD含药血清对PI3K/Akt信号通路和TGF-β1/Smad 信号通路各因子的影响，以进一步确HQTJD的作用机制。结果发现，HQTJD在不升高db/db小鼠血糖的情况下，可明显改善肾功能、尿ACR，肾脏形态学变化，降低肾组织中基质相关蛋白、磷酸化AKT、TGF-β1的蛋白表达及smad2、smad3的mRNA表达，增加PTEN及smad7的蛋白表达，此效果在干预12周时更加明显，且以中剂量HQTJD效果更佳。此外，HQTJD含药血清可明显抑制高糖状态下系膜细胞增殖，改善基质相关蛋白的表达，增加PTEN和smad7的mRNA表达，降低AKT、smad2、smad3的mRNA表达，趋势同单独使用PI3K/Akt通路抑制剂以及TGF-β1/Smad通路抑制剂，在联合使用抑制剂后影响更加明显。结果提示HQTJD对DN的改善与其抑制高糖状态下系膜细胞的增殖和基质蛋白的表达有关，而PI3K/Akt途径和TGF-β1/Smad通路则起到主要调控作用。