泽泻萜类组分调控自噬PI3K/Akt/mTOR通路致肾毒性作用机制研究

Autophagy is an important mechanism of the emergence and development of drug-induced renal injury. PI3K/AKT/mTOR is an important signaling pathway for autophagy regulation in drug-induced nephrotoxicity. This pathway may be a new target for interventing drug-induced nephrotoxicity. Numerous studies showed that the terpene component of Alisma plantago-aquatica Linn. (Zexie) had a potential renal toxicity. Our previous study confirmed this fact that Zexie indeed led to renal toxicity, which could cause renal tubular epithelial cell shedding and autophagy features. However, the underlying mechanism is not clear. Studies have shown that the terpene component of Zexie acts as autophagy inducer. In this study, "the nephrotoxicity of terpene component of Zexie is caused by inducing autophagy of renal cells " was taken as a scientific hypothesis. SD rats and HK-2 cell models were used to explore the nephrotoxicity of terpene component of Zexie, including doses-toxicity and time-toxicity relationship based on PI3K/Akt/mTOR pathway. Its autophagy features were explored with TEM, siRNA and other technologies, and the mechanism of PI3K/Akt/mTOR signaling pathway in Zexie-induced nephrotoxicity was revealed. The project studies innovatively the role and mechanism of Zexie-induced nephrotoxicity from regulating autophagy, provides new evidence for the scientific, rational use of Zexie, but also provide new strategies and targets for researches on drug-induced renal injury.

自噬是药物性肾损伤发生发展的重要机制之一。PI3K/AKT/mTOR是药物肾毒性自噬调控的重要信号通路，该通路有望成为干预药物肾毒性的新靶标。众多研究显示泽泻萜类组分具有潜在的肾毒性。我们前期研究证实泽泻确实能引起肾毒性作用，可引起肾小管上皮细胞脱落和自噬特征。但是其作用机制不明确。研究表明，泽泻萜类组分是新型的自噬诱导剂。本项目以“泽泻萜类组分肾毒性是其诱导肾细胞自噬引起的”为科学假说，基于PI3K/Akt/mTOR通路，以SD大鼠模型和HK-2细胞模型，探讨泽泻萜类组分肾毒性的"量-毒"、“时-毒”关系；借助透射电镜、siRNA等技术，阐明其诱导自噬特征，揭示PI3K/Akt/mTOR信号通路在泽泻肾毒性中的作用机制。本项目创新地从调控自噬的角度研究泽泻肾毒性的作用和机制，为科学、合理使用泽泻提供新的参考依据，也为药源性肾损伤研究提供新的策略和靶点。

本课题基于PI3K/Akt/mTOR通路，采用SD大鼠模型和HK-2细胞模型，借助透射电镜等技术，阐明泽泻萜类组分诱导自噬特征，揭示泽泻可能导致肾毒性的潜在机制。本课题考察泽泻慢性肾毒性作用，发现对雌性SD大鼠给予泽泻醇提物26周后，泽泻高、中剂量组大鼠肾脏细胞间质出现病理特征；高中剂量组中，肾损伤相关因子Ceruloplasmin、Clusterin、Kim-1、LCN2、Osteopontin和TIMP1蛋白表达及mRNA表达显著升高，HK-2细胞的Clustrein、Kim-1和TFF-3的mRNA水平显著升高。免疫荧光法检测结果显示泽泻萜类组分可引起HK-2细胞自噬的形成，可显著提高HK-2细胞LC3-II/LC3-I比值、Atg-5、Atg-12和Beclin-1表达。泽泻萜类组分可引起HK-2细胞凋亡。流式细胞术检测细胞凋亡率显示23-泽泻醇B、24-乙酰泽泻醇A和泽泻醇B给药组中HK-2细胞凋亡显著增加，并且该三种组分降低Bcl-2和Bcl-xl的 mRNA水平且提高Caspase-3 mRNA水平。PI3K/AKT/mTOR是自噬调控过程中的重要信号通路。结果显示，泽泻萜类组分给药组中HK-2细胞的PI3K、Akt和mTOR磷酸化水平都有降低，表明可通过抑制PI3K/Akt/mTOR通路，促进自噬的形成。以自噬抑制剂3-MA干预后，可显著地逆转升高的LC3-II/LC-3-I比值，表明自噬可被3-MA抑制；并且3-MA可逆转PI3K、Akt和mTOR蛋白磷酸化水平，从而调节萜类组分引起的HK-2细胞自噬。因此，推测泽泻萜类组分可能通过抑制PI3K/Akt/mTOR通路而激活HK-2细胞自噬，从而促进细胞凋亡和产生肾毒性。另外，在完成本课题原有工作内容的基础上，对泽泻萜类组分的组成结构特征进行了优化，限定了毒性成分的限度范围；评估该组分的成药性和“利水渗湿”的功效。本研究共发表标注本课题基金号学术论文3篇，中文核心论文1篇，SCI刊源文章2篇；在投SCI文章2篇。本研究为泽泻临床安全使用提供新的参考依据，也为药源性肾损伤研究提供新策略和新靶点。