薯蓣皂苷对顺铂诱导肾损伤的保护作用及调控miR-34a/Sirt1信号通路的分子机制研究

Cisplatin-induced kidney injury is one of major factors limiting its clinical application. Exploration of effective drugs and elucidation of the molecular mechanisms is one major project in life science. Dioscin, one classical natural product, has been widely studied by our research group, has the new biological activities in protecting liver, kidney and brain injury, and against metabolic diseases. In this study, we investigated the effect of dioscin on cisplatin-induced kidney injury. The preliminary results indicated that dioscin could significantly improve cisplatin-induced NRK-52e and HK-2 cells kidney injury, as well as rats and mice kidney injury. Furthermore, we found that dioscin down- regulated the expression levels of inflammatory factors, miR-34a, NF-κB, and up-regulated the expression levels of Sirt1 and Nrf2. Therefore, in this project, the in vivo kidney injury models caused by cisplatin in Wistar rats and C57BL/6J mice, and the in vitro models on rat primary renal tubular epithelial cells, NRK-52e and HK-2 cells will be used to evaluate the protective effects of the compound against the injury. Furthermore, the technologies of western blot, gene overexpression, siRNA and inhibitor intervention will be applied to investigate the possible mechanisms of dioscin on adjusting miR-34a/Sirt1 signaling pathway. This project will provide useful data for further studying dioscin and development of innovative drugs with independent property rights.

顺铂诱导肾损伤是限制其临床应用的主要因素之一，探寻有效的保护药物并阐明分子机制是现代生命科学领域的热点研究课题之一。薯蓣皂苷是一经典天然产物，申请者所在课题组对其进行了大量研究，发现了该天然产物保护肝肾脑器官损伤和抗代谢性疾病等新的生物学活性。在抗顺铂诱导的肾损伤研究中，初步试验结果显示薯蓣皂苷能显著改善顺铂诱导NRK-52e和HK-2细胞损伤以及大小鼠肾损伤，显著下调肾组织中炎症因子、miR-34a和NF-κB表达，上调Sirt1和Nrf2表达。因此，本项目拟在前期研究基础上，采用顺铂诱导大小鼠肾损伤模型，以及大鼠原代肾小管上皮细胞、NRK-52e和HK-2细胞损伤模型来全面评价薯蓣对顺铂肾损伤的保护作用，采用Western blot、全基因表达和siRNA干扰等技术探讨薯蓣皂苷调控miR-34a/Sirt1信号通路的分子机制。本项目将为薯蓣皂苷的深入研究和开发以及创新药物研制提供资料。

目的：本课题探讨薯蓣皂苷对顺铂(CDDP)诱导肾毒性的保护作用，并探讨可能的分子机制。.方法：NRK-52E和HK-2细胞经薯蓣皂苷预处理后，再用9 μg/mL CDDP处理24 h建立细胞损伤模型，MTT法检测细胞活力。体内实验中，48只Wistar雄性大鼠随机分成空白组、单纯给药组(Dio 40 mg/kg)、模型组(CDDP)、高剂量组(CDDP + Dio 40 mg/kg)、中剂量组(CDDP + Dio 20 mg/kg)、低剂量组(CDDP + Dio 10 mg/kg)，连续口服给药12天；48只C57BL/6J雄性小鼠随机分成空白组、单纯给药组(Dio 60 mg/kg)、模型组(CDDP)、高剂量组(CDDP + Dio 60 mg/kg)、中剂量组(CDDP + Dio 30 mg/kg)、低剂量组(CDDP + Dio 15 mg/kg)，连续口服给药10天；给药7天后一次性腹腔注射CDDP(大鼠7 mg/kg；小鼠25 mg/kg)，末次给药4 h后处死实验动物；考察体重变化，检测BUN、Cr水平和组织MDA、SOD等水平，观察肾脏组织病理结构变化，并考察薯蓣皂苷的分子作用机制。.结果：薯蓣皂苷显著改善CDDP诱导NRK-52E和HK-2细胞损伤，降低ROS水平；体内研究表明，与模型组相比，薯蓣皂苷显著下调血清BUN、Cr、MDA水平，升高SOD、GSH和GSH-Px水平，显著改善肾组织细胞刷状缘脱落、线粒体肿胀，减轻肾损伤。分子机制研究表明，与模型组相比，薯蓣皂苷显著下调miR-34a水平，激活Sirt1，促进Nrf2胞核转移，上调HO-1、GST、GCLC和GCLM抗氧化蛋白的表达水平，抑制NF-κB核易位，下调AP-1、COX-2和HMGB-1等蛋白表达水平；双荧光素酶报告基因实验表明薯蓣皂苷对Sirt1的调控至少是通过miR-34a实现的，薯蓣皂苷能抵抗miR-34a过表达引起的Sirt1通路抑制，基因转染和抑制剂实验证实薯蓣皂苷是介导miR-34a/Sirt1信号通路改善炎症和氧化应激，分子对接技术初步预测Keap1和NF-κB p65蛋白可能是薯蓣皂苷抗CDDP肾毒性的药物作用靶点。.结论：薯蓣皂苷介导miR-34a/Sirt1信号通路调控氧化应激和炎症抗CDDP肾毒性，薯蓣皂苷在抗顺铂与阿霉素肾毒性方面具有较大研究和开发价值。