天阳丸通过抑制钙敏感受体(CaSR)介导的mTORC2-Akt2-mTORC1信号环路调控糖尿病肾病足细胞自噬的分子机制

Diabetic nephropathy (DN) has become the leading cause of end-stage renal failure. Recent studies have shown that decreased autophagy levels and increased mTOR activity are one of the key mechanisms of podocyte injury induced by high glucose. Clinical application of rapamycin to inhibit mTORC1 can prevent renal injury progression in a short period of time, but long-term inhibition of mTORC1 may aggravate podocyte injury, which may be related to feedback activation of the mTORC2-Akt2-mTORC1 signaling loop. Intracellular calcium [Ca2+]i, as the important second messenger of organism, is essential for maintaining autophagy and homeostasis in podocytes. Previous studies in the research group have confirmed that calcium-sensitive receptor (CaSR) antagonists can inhibit Akt2 phosphorylation and improve insulin resistance in diabetic rats. Tianyang wan which consists of Morinda officinalis How 20g, Cistanches Herba 15g, CynomorII Herba 15g, Rehmanniae Radix Praeparata 15g, Corni Fructus 15g, and Dioscoreae Rhizoma 15g is the experience of Professor Li ji in clinical treatment of kidney yang deficiency type diabetic nephropathy and related disease syndrome. Pre-experimental results showed that Tianyang wan can improve renal damage significantly and inhibit the expression of CaSR in podocytes of DN rats. Based on this, this subject uses CaSR as the entry point to further explore the mechanism of Tianyang wan in regulating autophagy of DN podocytes.

糖尿病肾病(DN)已成为终末肾衰的首要原因，最新研究表明，足细胞自噬水平降低及mTOR活性升高是高糖引起足细胞损伤的关键机制之一。临床应用雷帕霉素抑制mTORC1可短期防止肾损伤进展，但长期抑制mTORC1反而会加剧足细胞损伤，其原因可能与反馈性激活mTORC2-Akt2-mTORC1信号环路有关。细胞内钙[Ca2+]i作为机体重要的第二信使，对维持足细胞自噬及内环境稳态至关重要。课题组前期研究已证实，钙敏感受体(CaSR)拮抗剂可抑制Akt2磷酸化、改善糖尿病大鼠胰岛素抵抗。天阳丸由巴戟天20g肉苁蓉15g锁阳15g熟地黄15g山萸肉15g山药15g配伍组成，是李冀教授临床治疗肾阳虚型糖尿病肾病及相关病证的经验方，疗效确切。预实验结果发现，天阳丸可显著改善DN大鼠肾损伤、抑制足细胞CaSR表达。据此本课题以CaSR为切入点，深入探讨天阳丸调控DN足细胞自噬的作用机制。

最新研究表明，足细胞自噬水平降低及mTOR活性升高是高糖引起足细胞损伤的关键机制。本项目研究通过钙敏感受体(CaSR)抑制剂、雷帕霉素干预高糖培养的足细胞，检测PI3K/Akt/mTOR信号途径关键蛋白的表达水平及自噬水平，进而验证天阳丸改善糖尿病肾病足细胞损伤的分子机制。体内及体外研究结果表明：天阳丸能够显著改善DKD小鼠尿蛋白、肾小球滤过率及自噬蛋白表达水平，减轻肾组织病理损伤；天阳丸含药血清可通过CaSR调节高糖诱导MPC5足细胞的自噬水平，其分子途径确与mTOR通路相关；药学实验部分通过HPLC法建立天阳丸特征图谱，确定了天阳丸水煎液14种化学成分，并已明确其药材归属。初步明确了天阳丸水煎液制备条件及工艺流程，完成天阳丸特征图谱的建立，以及相似度、稳定性、精密度及重复性考察。本项目研究内容及结果对中医药临床治疗糖尿病及糖尿病肾病等代谢性疾病提供有力的理论指导和实验依据，复方天阳丸具有申请国家专利及开发成临床有效制剂的潜力。