基于内质网钙ATP酶信号研究黄芪皂苷甲改善糖尿病肾病的机制

Edoplasmic reticulum (ER) stress and podocyte apoptosis are the research focus on diabetci nephropath recently. Sarcpasmic reiculm Ca2+-ATPase (SERCA) plays a vital role in ER stress. We have demonstrated that Astragaloside IV (AS-IV), which is a major component isolated from Radix Astragali that has a protective effect on kidney, could not only improve renal tubulointerstitial fibrosis, but also inhibit high glucose or free fatty acid induced down-regulation of SERCA, and ER stress induced podocyte apoptosis. Thus, we hypothesized that podocyte apoptosis was mastered by SERCA signaling in diabetic nephropathy, which can be regulated by treatment with AS-IV. On this hypothesis, the present project intends to investigate the effect of AS-IV on high glucose and palmitic acid induced SERCA expression and activity, as well as apoptosis mediated by ER stress and mitochondrial dysfunction by using calcium fluorescent probe, siRNA, immunofluorescence technique. Moreover, the influence of AS-IV on SERCA-ER stress mediated apoptosis, and the downstream signaling molecules such as CHOP, JNK, Caspase-12, and mitochondrial dependent apoptosis pathways will be investigated by means of immunohistochemistry, RT-PCR, Western blot in type I and II diabetic mice models. This project will provide the experimental and theoretical evidences and new treatment strategies for the clinical application of AS-IV in therapy of diabetic nephropathy.

内质网（ER）应激与足细胞凋亡在糖尿病肾病中的作用是目前研究的热点，研究发现ER钙ATP酶（SERCA）及其亚型的改变在ER应激过程中起着关键作用。我们前期工作发现糖尿病肾病动物模型SERCA表达异常；黄芪皂苷甲（AS-IV）能明显抑制高糖/棕榈酸引起的足细胞损伤，并上调SERCA的表达。提示AS-IV可能通过影响SERCA及亚型的表达与活性，缓解ER应激，抑制肾小球足细胞凋亡，改善糖尿病肾病发病进程。基于该假说，我们应用钙荧光探针、siRNA、免疫荧光标记等技术研究AS-IV对高糖和棕榈酸引起足细胞SERCA及亚型影响，继而介导ER应激和线粒体功能紊乱导致细胞凋亡的作用；应用western blot、荧光定量PCR技术等观察AS-IV对I、II型糖尿病肾病动物模型肾小球损伤的影响，阐明AS-IV通过调控SERCA信号改善糖尿病肾病的作用机制，为临床治疗提供新的措施和策略。

糖尿病肾病是糖尿病中最普遍的微血管并发症，也是世界范围内引起终末期肾病的首要病因。近年来，持续的证据表明足细胞凋亡在糖尿病肾病中起到重要作用。并且内质网应激与足细胞凋亡密切相关，而ER钙ATP酶（SERCA）及其亚型的改变在ER应激过程中起着关键作用。然而，潜在的机制尚不明确。基于前期研究，我们工作发现糖尿病肾病动物模型SERCA表达异常；黄芪皂苷甲（AS-IV）能明显抑制高糖/棕榈酸引起的足细胞损伤，并上调SERCA的表达。提示AS-IV可能通过影响SERCA及亚型的表达与活性，缓解ER应激，抑制肾小球足细胞凋亡，改善糖尿病肾病发病进程。基于该假说，我们的研究结果显示：1）糖尿病肾病（DN）进程中涉及内质网应激通路的激活，缓解内质网应激改善DN；2）黄芪皂苷甲可通过上调SERCA2b亚基，抑制高糖引起的内质网应激诱导的足细胞凋亡，调节自噬，缓解线粒体功能紊乱，改善I型和II型糖尿病引起的糖尿病肾病小鼠足细胞损伤、肾功能异常、肾小球硬化等病理进程；3）足细胞离体实验证实棕榈酸引起的钙离子紊乱诱导的足细胞凋亡是由线粒体钙单向转运体（MCU）介导的；4）在此基础上进一步研究发现黄芪皂苷甲抑制棕榈酸诱导的足细胞凋亡与调节胞内钙离子紊乱相关。通过此项研究，阐明AS-IV通过调控SERCA信号改善糖尿病肾病的作用机制，为临床治疗提供新的措施和策略。