基于HIF-1α及其下游通路探讨肾衰II号方保护肾小管上皮细胞功能的作用机制研究

Basing on the preliminary work of our research group, we concentrate on renal tubular interstitial fibrosis (TIF), which is characterized by chronic ischemia and hypoxia. We find “Meliorated Renal Failure Decoction”, having an ideal effect in clinical studies, can downregulate the hypoxia inducible factor-1α (HIF-1α) and finally improve TIF. Combining with the new knowledge that HIF-1α plays an important role in the incidence and development of TIF, we raised the hypothesis that Meliorated Renal Failure Decoction may inhibit partial epithelial-to-mesenchymal transition and improve cell cycle arrest by regulating the HIF-1αand its downstream signaling pathways so as to protect the function of tubular epithelial cells (TECs). So we aim at the relationship between hypoxia and TECs’ function. We planned to use the 5/6th kidney (ablation/infarction) model in rats and the hypoxic injury model in human renal proximal tubular cells (HK2) with siRNA to Twist or p53 gene as the research objects, and to explore the new mechanism how the Meliorated Renal Failure Decoction regulates HIF-1α and its downstream signaling pathways (HIF-1α/Twist, p53/p21) to anti TIF from the whole, the cellular and the molecular levels by renal oxygen consumption test, pathological examination and so on. It will provide scientific bases of our Meliorated Renal Failure Decoction, which contains not only invigorating qi and nourishing blood but also removing blood stasis and purging turbidity, for using in clinical treatment widely and further development of new drugs for TIF.

本课题组前期针对肾间质纤维化（TIF）慢性缺血缺氧的特征，经临床研究已发现疗效显著的肾衰II号方，能下调缺氧诱导因子-1α（HIF-1α），改善TIF。鉴于该方作用及其近年来对HIF-1α在TIF发生发展中作用的新认识，本课题提出“肾衰II号方通过调节HIF-1α相关信号通路，抑制肾小管上皮细胞(TECs)不完全EMT，改善上皮细胞周期阻滞，从而保护其功能”的科学假说。拟从缺氧与TECs功能间关系切入，采用5/6(A/I)慢性肾衰模型大鼠和HK2细胞缺氧模型（siRNA沉默Twist或p53基因）为研究对象，从体内与体外两个层面，通过氧代谢、细胞与分子水平、病理等手段，探讨该方调节HIF-1α及其下游HIF-1α/Twist和p53/p21信号通路抗TIF作用的新机制，为诠释临床广泛应用具有益气养血、化瘀泄浊的肾衰II号方防治TIF的理论及今后研发新药提供坚实基础。

本课题基于肾小管上皮细胞(TECs)持续损伤以上调Twist及p53基因表达，发生上皮间充质不完全转分化(不完全EMT)及G2/M期阻滞为最显著病理变化的认识，在前期研究发现肾衰II号方改善肾内氧耗，下调 HIF-1α蛋白表达，延缓肾功能衰竭的基础上，从缺氧与TECs损伤关系切入，分别采用5/6(A/I)肾切除慢性肾功能衰竭大鼠模型及低氧诱导HK2转分化模型，从整体、细胞、分子水平，进一步剖析肾衰II号方对以慢性缺血缺氧为特征的肾间质纤维化(TIF)中TECs损伤关键事件（不完全EMT、G2/M期阻滞、肾小管转运功能）的作用。. 研究进展顺利，体内研究重要结果及关键数据显示在大鼠5/6(A/I)慢性肾衰模型中，肾衰II号方及其拆方（益气活血方）可明显降低肾内氧耗，减轻p53/p21活化导致的G2/M期阻滞。同时，肾衰II号方及其拆方（补肾泄浊方）减少下游Twist表达，增加上皮表型标志物ZO-1、E-Cadherin及肾小管转运功能蛋白表达，减轻TECs不完全EMT，改善TECs转运功能不全。体外研究显示肾衰II号方有效组分IEFT下调低氧诱导HK2细胞模型中Twist及p53基因转录表达，增加E-Cadherin蛋白表达，降低G2/M期细胞比例，减少Fibronectin等细胞外基质成分分泌。采用siRNA沉默Twist或p53干预低氧诱导HK2细胞验证IEFT作用机制确与HIF-1α下游HIF-1α/Twist及p53/p21信号通路相关。. 研究结果表明，具有益气养血、化瘀泄浊的肾衰II号方调节HIF-1α下游HIF-1α/Twist及p53/p21信号通路，减轻TECs不完全EMT，改善G2/M期阻滞，保护TECs转运功能，延缓TIF进展。复方及其有效组分研究部分结果均已申请国家发明专利，为诠释临床广泛应用的肾衰II号方防治TIF的理论及今后研发新药提供坚实基础。