基于GSK-3β调控Nrf2通路的雷公藤甲素肾毒性机制研究

Triptolide(TP) has been shown to possess multiple biological activities. However, severe adverse effects, especially nephrotoxicity, limit its clinical use. The mechanism of TP-induced nephrotoxicity has not been fully elucidated. Our previous studies demonstrated that activation of Nrf2 played a protective role against TP-induced cytotoxicity in rat kidney cells through the counteraction of oxidative stress. However, prolonged treatment with TP led to Nrf2 inhibiton. We wonder the underlied mechanism against TP-induced Nrf2 inhibition. Recently, GSK-3β has been revealed to play a crucial role in controlling Nrf2 activity. Our preliminary results showed that TP increased the activity of GSK-3β in human proximal tubular epithelial cells; GSK-3β inhibitor could alleviate TP-induced Nrf2 inhibition and cytotoxicity. These results suggest that TP may induce nephrotoxicity through activating GSK-3β and inhibiting Nrf2 pathway. In the present study, techniques such as siRNA, co-IP and immunofluorescence will be employed to explore the role of GSK-3β in TP-induced Nrf2 regulation and to allow further insight into the mechanism of TP-induced renal injury. The current study will also provide direct implications for the development of novel therapeutic strategies against TP-induced nephrotoxicity.

雷公藤甲素(TP)药理作用广泛且明确，然而严重的毒副反应限制了其临床应用，其中肾毒性发生率高且后果严重。目前TP肾毒性的作用机制尚未完全明确。我们已经证明了在TP引起的肾小管上皮细胞损伤初期，ROS应激性激活Nrf2通路对抗氧化应激，然而随着给药时间延长，Nrf2通路被抑制。那么TP是如何抑制Nrf2通路的？研究表明GSK-3β在Nrf2通路的调控中发挥着重要作用。我们的预试验发现在肾小管上皮细胞中，TP能够激活GSK-3β；GSK-3β抑制剂使TP作用下的Nrf2通路抑制程度减弱，细胞存活增加。这提示TP可能通过激活GSK-3β、抑制Nrf2通路导致肾毒性。本项目拟从细胞及动物水平，采用siRNA、免疫共沉淀、激光共聚焦等方法，明确GSK-3β对Nrf2通路的调控在TP肾毒性中作用，以进一步阐明TP肾毒性机制，为其临床应用的减毒寻找新靶点，提供科学依据。

雷公藤甲素(triptolide, TP)是中药雷公藤的主要活性成分，具有免疫抑制、抗肿瘤、神经保护等多种药理作用，临床应用前景广泛。但明显的毒副作用限制了TP的临床应用，其中肾毒性发生率高且后果严重。目前TP导致肾毒性的分子作用机制尚未完全明确。本研究从细胞和整体动物水平，探讨GSK-3β对Nrf2的负性调控在TP所致肾毒性中的作用。我们的研究结果显示，TP可通过造成氧化应激损伤、诱导细胞凋亡而降低大鼠肾小管上皮细胞NRK-52E的存活率，造成BALB/c小鼠肾脏损伤。这可能与TP激活GSK-3β、抑制Nrf2相关，使用GSK-3β抑制剂氯化锂抑制GSK-3β活性在体内、外模型中均可减轻TP对Nrf2的抑制作用，缓解TP所致肾毒性。TP作用下GSK-3β对Nrf2的负性调控可能是通过Fyn途径降低Nrf2核内水平、促进其泛素化降解而实现的。这些研究结果提示，TP能够通过激活GSK-3β/Fyn信号通路，促进Nrf2泛素化降解而造成氧化应激损伤，导致肾毒性，抑制GSK-3β可缓解TP所致肾毒性。这为TP临床应用的减毒策略提供了依据。