基于调控PI3K/Akt通路的大黄附子汤保护肾小管上皮细胞凋亡的研究

Dahuangfuzi Decoction (DFD) is one of the representatives in Synopsis of Golden Chamber. In recent years, several Chinese scholars used this decoction to treat chronic renal insufficiency, uremia, diabetic nephropathy, etc., and achieved some effects. Our previous one in vivo study demonstrated that, DFD can ameliorate apoptosis of renal tubular epithelial cells in adenine-induced renal damage rats through regulating JNK/Bcl-2 signaling pathway. This research aims to measure different syndromes by different herbs and study the material base of kidney yang-deficiency, in order to prove that DFD is a warm and purgative method to treat chronic kidney disease (CKD). In addition, we will investigate the action of PI3K/Akt signaling pathway in apoptosis of renal tubular epithelial cells by the in vitro study of the intervention of Akt plasmid and Akt inhibitor in renal tubular epithelial cells, as well as in vivo study of tail vein injection of Akt plasmid and Akt-siRNA plasmid in kidney injury rats. Moreover, with the treatment of DFD in vivo and vitro studies, we will do research on the effect of DFD in renal tubular epithelial cells via regulating PI3K/Akt signaling pathway. The purpose of this research is to further explore the mechanism of DFD in treating CKD and supply the scientific bases for effectively preventing and treating CKD.

大黄附子汤是《金匮要略》中温下的代表方，近年一些国内学者用该方加减治疗慢性肾功能不全、尿毒症、糖尿病肾病等，取得一定疗效。我们的前期体内研究发现，大黄附子汤能通过调控JNK/Bcl-2信号通路缓解腺嘌呤诱导的肾损伤大鼠模型的肾小管上皮细胞凋亡。本研究拟通过"以药测证"和对肾阳虚证物质基础的研究，证实大黄附子汤为治疗慢性肾脏病的温下方；通过Akt质粒和Akt抑制剂对肾小管上皮细胞干预的体外研究、Akt质粒和Akt-siRNA质粒对肾损伤大鼠尾静脉注射的体内研究，观察PI3K/Akt信号通路在肾小管上皮细胞凋亡中的作用；以及大黄附子汤干预后的体内、外研究，观察大黄附子汤通过调控PI3K/Akt信号通路对肾小管上皮细胞凋亡的影响。进一步阐明大黄附子汤治疗慢性肾脏病的作用机理，为慢性肾脏病的有效防治提供科学依据。

大黄附子汤是临床上常用于治疗糖尿病肾病，高血压肾病等慢性肾脏病的经典方剂。既往也有研究提示大黄附子汤及其主要成分大黄可以通过调节细胞凋亡，从而改善肾脏病的进展。但是对于此方剂的具体有效成分以及作用机理尚未明确。我们在体内研究中发现，大黄附子汤可以改善模型鼠的一般状态、生理生化指标，减轻肾组织的病理损伤，调节TGF-β1/JNK信号通路减少腺嘌呤诱导的大鼠肾脏组织中Bax和Cleaved caspase-3等凋亡相关蛋白的表达，增加Bcl-2抗凋亡蛋白的表达，还能减少模型鼠肾脏自噬标志蛋白LC3以及纤维化指标蛋白Collagen I、Fibronectin蛋白表达。在后期的深入研究中，我们建立了HBSS诱导的NRK-52e细胞自噬模型，发现大黄酸可以抑制HBSS诱导的LC3的表达，机制探讨中证实大黄酸可以抑制HBSS诱导的AMPK、p38、JNK蛋白的磷酸化，增加mTOR、p70S6K等蛋白的表达，单独作用于NRK-52e并不会产生影响以上蛋白的表达。以上结果提示大黄酸是通过调控AMPK-mTOR-p70S6K信号通路调节自噬，改善HBSS诱导的肾小管细胞损伤，且其作用机理还与调控MAPK信号通路相关。MAPK信号通路与AMPK, mTOR的关系尚不明确，在未来的实验研究中，我们将进一步进行探讨。大黄附子汤在治疗慢性肾脏病的作用机理与调控肾脏细胞的凋亡、自噬均相关，而且其君药大黄的主要有效成分大黄酸可以通过调节自噬发挥其肾脏的保护作用。