GSK-3β磷酸化失活及其调控在金丝桃苷抗氧化性肝损伤中的作用研究

xidative stress-induced liver damage plays a vital role in the pathogenesis of various liver diseases. The adjuvant treatment on liver diseases provided by traditional Chinese medicine mostly for this point. Flavonoids have been reported to elicit liver protective effect against oxidative stress through activation of Nrf2 signaling pathways and involving a number of ubiquitous intracellular kinases, including the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/Akt (PI3K/Akt). Such kinases may act in isolation or in concert to facilitate Nrf2 nuclear translocation. Recent research has shown that, GSK-3β can make the serine phosphorylation of Nrf2, and then inhibit the upregulation of cytoprotective gene. Therefore GSK-3β might integrate these kinases pathways at the level of Nrf2 in the protective effect of flavonoids. Our preliminary study on hyperoside(Hyp), a naturally occurring quercetin-3-galactoside have found that it attenuated hydrogen peroxide-induced L02 cell damage via p38 and ERK-dependent Nrf2 signaling pathway. However, the role of PI3K/Akt and GSK-3β in hyperoside-mediated upregalation of antioxidant enzymes remains to be elucidated. The present study was designed to investigate whether GSK-3β is a common target for above kinase in Hyp-induced Nrf2 signaling pathway. The results will be helpful for full understanding the pharmacological effects and mechanisms of flavonoids and can also make use of traditional Chinese medicine to develop new strategies on the treatment of oxidative stress-induced liver injury.

在各种肝脏疾病的发病机制中，由氧化应激所致的肝细胞氧化-还原稳态失衡是关键环节，传统中医药对肝病的辅助治疗大多针对该环节。文献报道植物黄酮可通过激活MAPK或(和)PI3K/Akt等多条激酶途径，诱导Nrf2介导的HO-1等细胞保护基因表达上调，恢复肝细胞氧化-还原稳态。新近研究发现，GSK-3β可使Nrf2结构中的丝氨酸磷酸化，进而抑制Nrf2对细胞保护基因的转录激活作用。因此，GSK-3β磷酸化失活可能是植物黄酮激活激酶途径与诱导Nrf2的中间环节。我们前期对槲皮素半乳糖苷Hyp的研究观察到明显肝保护效应，且与激活ERK和p38途径，促进Nrf2核转位有关，但是否涉及PI3K/Akt和GSK-3β尚未明确。本项目拟以Hyp为工具药、HO-1为代表酶，基于GSK-3β磷酸化失活及其调控，探讨植物黄酮的肝保护效应与机制，可为充分利用传统中医药宝库对抗氧化性肝损伤开拓新策略。

氧化应激参与酒精性肝病、非酒精性脂肪肝、病毒性肝炎、缺血-再灌注肝损伤、药物性肝病、肝癌等多种肝脏疾病的病理生理过程。新近研究发现，GSK-3β/Nrf2通路参与氧化性肝损伤修复过程中细胞保护基因的转录调节，其中GSK-3β可能以多条激酶途径的共同下游靶分子的角色参与Nrf2的负调控。因此，抑制GSK-3β，诱导Nrf2对细胞保护基因的转录调控有望成为氧化性肝损伤防治的新靶点。课题组前期研究发现，植物黄酮金丝桃苷(Hyperoside, Hyp)可通过激活ERK和p38途径，促进Nrf2核转位，上调细胞保护蛋白的水平与活性，保护氧化损伤的肝细胞，且单独给予ERK和p38的特异性抑制剂对Hyp诱导Nrf2核转位的抑制作用较二者合用更强，提示可能还有PKC、PI3K/Akt等其他信号途径的参与。因此我们推测：Hyp可能通过激活ERK、p38、PI3K/Akt等激酶途径，抑制其下游共同靶分子GSK-3β，诱导Nrf2核聚集，上调细胞保护蛋白的水平与活性，增强细胞内源性的抗氧化能力，从而发挥肝细胞保护效应。.本项目围绕这个核心科学假说开展研究，取得如下发现：1. Hyp在mRNA和蛋白水平上调Nrf2表达；2. Hyp对G3K-3β蛋白和mRNA水平无明显调节作用；3. Hyp可通过诱导GSK-3β(Ser9)磷酸化失活，促进Nrf2核转位，进而在mRNA和蛋白水平上调细胞保护蛋白表达，发挥肝保护效应；4. Hyp可同时通过Keap1依赖途径和GSK‐3β依赖途径激活Nrf2-ARE信号通路，两条途径相互独立，且GSK-3β依赖途径较Keap1依赖途径对Hyp诱导Nrf2-ARE信号通路具有更重要的作用；5. ERK、p38和PI3K/Akt途径均参与Hyp诱导Nrf2入核，但GSK‐3β仅为PI3K/Akt途径的下游靶分子；6. PI3K/Akt-GSK‐3β途径较ERK和p38途径对Hyp诱导Nrf2-ARE信号通路的贡献更大。.本项目首先阐明了GSK-3β磷酸化失活在Hyp诱导Nrf2-ARE通路，保护氧化损伤肝细胞中的重要作用，并揭示了Nrf2的胞浆抑制分子Keap1在Hyp诱导Nrf2-ARE途径中的作用及其与GSK‐3β依赖途径的关系，为氧化性肝损伤的防治提供了潜在干预靶点。相关成果发表SCI论文一篇(PLoS One, 2015,10(12): e0145