从HIF-1/BNIP3/Beclin-1通路调控的线粒体自噬研究三七总皂苷保护顺铂肾损害的分子机制

Drug-induced renal damage is common in clinical, and there have not been effective drug to protect kidney presently. Cisplatin is one of the common drugs which cause renal damage, it can induce kidney ischemia, hypoxia and oxidative damage. Kidney cells can resist hypoxia and oxidative damage through mitophagy mediated by hypoxia-inducible factor 1(HIF-1). Panax Notoginsenosides (PNS) can protect against cisplatin-induced renal damage and not inhibit cisplatin’s antitumor effect. We found that PNS could protect against cisplatin-induced nephrotoxicity, reduced the mitochondria damage and increasing mitochondrial autophagy in rat renal tubular epithelial cells, elevated the protein expression of LC3, HIF-1α, BNIP3, Beclin-1 and Atg5 in renal tissue, these results suggest that PNS can enhance mitophagy mediated by HIF-1αand protect against cisplatin-induced nephrotoxicity. This project intends to establish rat model and cellular model of cisplatin-induced nephrotoxicity, intervening with PNS and HIF-1α inhibitor, then determine the mitophagy activity, mitochondrial function and morphology, reactive oxygen species content, detect the protein expression of LC3, HIF-1α, BNIP3 and Beclin-1, detect the expression of HIF-1αmRNA in renal tubular epithelial cell at different time points. Furthermore, we will clarify the molecular mechanisms of PNS protection against cisplatin-induced nephrotoxicity through the mitophagy regulated by HIF-1/BNIP3/Beclin-1 pathway.

药源性肾损害临床常见，目前缺乏保护肾脏的有效药物。顺铂是引起肾损害的常见药物，可致肾缺血缺氧和氧化损伤。肾细胞可通过低氧诱导因子1(HIF-1)介导线粒体自噬抵御缺氧和氧化损伤。三七总皂苷（PNS）可保护顺铂肾损害且不抑制顺铂抗肿瘤效应。本课题组发现PNS 可保护顺铂肾损害，减轻肾小管上皮细胞线粒体损伤和增多线粒体自噬体，升高肾LC3Ⅱ、HIF-1α、BNIP3、Beclin-1和Atg5蛋白表达；提示PNS可增强HIF-1α介导的线粒体自噬而保护顺铂肾损害。本项目拟建立体内、体外顺铂肾损害模型，以PNS、HIF-1α抑制剂干预，了解不同时间点肾小管上皮细胞的线粒体自噬活性、线粒体功能与形态、ROS含量等，以及LC3、HIF-1α、BNIP3、Beclin-1蛋白表达、mRNA表达等，从HIF-1/BNIP3/Beclin-1通路调控的线粒体自噬探讨PNS保护顺铂肾损害的作用机制。

顺铂可引起肾组织缺血缺氧和氧化损伤，而缺血缺氧可诱导产生HIF-1α。本课题组前期研究发现三七总皂苷（PNS）可保护顺铂损伤的肾功能和减轻肾小管上皮细胞线粒体损伤、增多自噬。但HIF-1α和自噬的关系及其在PNS保护顺铂肾损伤中的作用尚不清楚。本项目建立体内、外模型，以PNS、HIF-1α抑制剂2ME2（体外采用效果更好的siRNA-HIF-1α技术）干预，从 HIF-1/ BNIP3/Beclin-1通路调控的线粒体自噬研究 PNS 保护顺铂肾损害的分子机制。结果如下：.1、顺铂显著升高大鼠血液BUN、Cr和尿NAG水平，病理学显示肾组织损害明显；PNS显著改善大鼠肾功能，减轻肾组织病理损伤，提示顺铂肾损害模型制作成功及PNS有保护作用。.2、顺铂诱导HK-2细胞和大鼠肾组织HIF-1α蛋白和mRNA表达升高，抑制HIF-1α加重顺铂肾损伤；PNS可升高HIF-1α蛋白和mRNA表达。提示PNS通过升高HIF-1α而保护肾脏。.3、体内外实验发现顺铂显著损伤肾细胞线粒体，减少ATP、MMP和MPTP开放增加，抑制HIF-1α加重线粒体功能障碍；PNS改善线粒体功能障碍。提示PNS通过升高HIF-1α而保护肾细胞线粒体。.4、顺铂诱导线粒体自噬小体产生，升高LC3-Ⅱ/LC3-Ⅰ比例；抑制HIF-1α使线粒体自噬小体减少；PNS增加线粒体自噬小体和升高LC3-Ⅱ/LC3-Ⅰ比例。提示PNS可通过升高HIF-1α而增强线粒体自噬。另外，顺铂升高肾细胞的线粒体蛋白BNIP3、Beclin-1表达，抑制HIF-1α使BNIP3、Beclin-1蛋白表达下降，PNS可升高以上蛋白表达。提示PNS增强HIF-1α/BNIP3/Beclin-1而提高线粒体自噬。.5、顺铂显著升高肾组织、细胞的ROS、MDA，降低SOD；抑制HIF-1α进一步升高ROS、MDA，以及降低SOD；PNS改善顺铂诱导的ROS、MDA、SOD变化；提示PNS 升高HIF-1α而减少顺铂诱导肾氧化损伤。.以上结果说明，PNS通过HIF-1/BNIP3/Beclin-1增强线粒体自噬和减轻氧化损伤，从而改善顺铂诱导的肾损害。