丹参酮ⅡA对糖尿病肾病足细胞转分化及TGF-β1/Notch信号通路的调控机制

Podocyte injury plays an important role in the emergence of proteinuria in diabetic nephropathy (DN). High-glucose can cause podocyte phenotypic changes as epithelial-mesenchymal transition (EMT), which could cause the structure and function damage of podocytes and lead to the occurrence and development of proteinuria. The Notch pathway includes important molecules mediating diabetic kidney podocytes EMT, which can be activated by TGF-beta 1. Chinese medicine salvia miltiorrhiza has the effect of restoring qi to reinforce insufficiency and dispelling stasis to promote regeneration,which can therapy the pathogenesis of Qi and Yin deficiency and Stasis turbidity resistance in diabetic Nephropathy. Tanshinone ⅡA is the main effective component of salvia miltiorrhiza.Preliminary experimental results have showed that tanshinone ⅡA had obvious intervention effect on the expression of podocyte transdifferentiation related protein, and could affect the TGF-β1 expression level in renal cortex. And it has been confirmed that Tanshinone ⅡA could inhibit the Notch signaling pathways in cardiomyocyte. So we speculated that tanshinone ⅡA might intervene podocyte EMT through inhibiting the Notch pathway activated by TGF-beta1 in podocyte. No relevant research reports have been retrieved now.This topic was proposed to observe the impact of tanshinone IIA on diabetic kidney podocytes EMT and theTGF-beta 1/Notch signaling pathway, to discuss the mechanism of tanshinone IIA protecting diabetic glomerular podocytes. This study may give some academic evidence in looking for effective prevention and treatment drugs for diabetic nephropathy.

足细胞损伤在糖尿病肾病蛋白尿的发病中起重要作用。高糖可引起足细胞转分化，即上皮细胞-间充质细胞转化(EMT)，引起足细胞结构和功能破坏，导致蛋白尿发生发展。Notch通路是介导糖尿病肾病足细胞EMT的重要通路，可由TGF-β1激活。中药丹参功效益气补虚，祛瘀生新，能针对糖尿病肾病气阴两虚为本，瘀浊内阻为标的病机起治疗作用，其主要有效成分为丹参酮ⅡA。预实验结果表明丹参酮ⅡA对糖尿病肾病足细胞EMT相关蛋白表达有明显的干预作用，且能抑制肾皮质中TGF-β1 表达。丹参酮ⅡA已被证实能抑制心肌的Notch通路。我们推测丹参酮ⅡA能够干预足细胞EMT，机制可能与抑制TGF-β1激活的Notch通路有关。经文献检索，尚无相关研究报道。本课题拟通过观察丹参酮IIA对糖尿病肾病足细胞EMT及TGF-β1/Notch通路的影响，探讨其保护肾小球足细胞的机制，为寻找有效防治糖尿病肾病的药物提供实验依据。

糖尿病肾病（DN）是糖尿病最常见的慢性微血管并发症之一，最终将进展为终末期肾病。足细胞的结构和功能异常可导致DN蛋白尿的发生发展，而足细胞EMT可能是DN早期足细胞损伤的重要途径。本项目动物实验采用雄性db/db小鼠作为糖尿病肾病模型，以丹参酮IIA高中低剂量组干预，以厄贝沙坦作为阳性对照，并设立正常对照组。干预12周后，收集24小时尿液，取血及肾皮质组织。检测血糖、肾功能等生化指标，HE染色观察小鼠肾脏病理改变及透射电子显微镜下观察肾小球足细胞的超微结构。Western blot、免疫组化、RT-PCR等方法检测足细胞EMT相关蛋白的表达及小鼠肾皮质TGF-β1/Notch信号通路相关蛋白的水平。细胞实验采用条件永生系小鼠肾足细胞，采用高糖加小剂量棕榈酸钠诱导足细胞产生EMT，CCK8测定足细胞活性。给与不同剂量的丹参酮IIA干预，并与TGF-β1抑制剂、γ分泌酶抑制剂及Notch1激动剂相比较，以验证丹参酮IIA对足细胞EMT及相关TGF-β1/Notch信号通路的干预作用。研究结果发现，丹参酮ⅡA能改善db/db小鼠肾功能，减轻尿白蛋白/尿肌酐值，改善肾脏的病理改变及足细胞的损伤。进一步研究发现丹参酮ⅡA能改善糖尿病小鼠肾小球足细胞EMT，升高上皮细胞标志蛋白的表达，降低间充质细胞标志蛋白的水平，并对TGF-β1/Notch 信号通路的相关分子表达水平有抑制作用。体外研究表明，丹参酮ⅡA干预能改善高糖加小剂量棕榈酸钠诱导的小鼠肾足细胞EMT，能够抑制TGF-β1/Notch信号通路相关蛋白的表达。本项目通过体内和体外实验，观察了丹参酮ⅡA治疗糖尿病肾病的疗效，探讨了其保护肾小球足细胞的分子机制，初步验证了丹参酮ⅡA通过抑制 TGF-β1/Notch信号通路改善足细胞EMT进而减轻糖尿病肾损伤的假设。进一步探讨了中药丹参“活血祛瘀”的中医内涵，可为丹参酮IIA用于糖尿病肾病的防治及其进一步的机制研究提供实验依据。