Hsp20-AMPK-mTOR轴通过调控细胞自噬影响非酒精性脂肪性肝病的作用和机制研究

Autophagy represents a pivotal mechanism involved in non-alcoholic fatty liver disease (NAFLD). while regulation of autophagy using genetic or pharmacological approaches results in effective handling of NAFLD. We recently found that the globle deletion of Hsp20 markedly reduces hepatosteatosis. However, whether Hsp20 regulates NAFLD in a cell-autonomous manner and the underlying mechanism has never been reported worldwide. Our preliminary data demonstrate that Hsp20 knockdown inhibits mTOR via AMPK activation and concomitantly increases autophagy flux. Based on the above findings, we hypothesize that Hsp20-AMPK-mTOR axis acts as a regulator of autophagy in hepatocyte, thereby participating in the development of NAFLD. Herein our proposal aims to elucidate the function of Hsp20 in NAFLD and lipotoxicity-induced hepatocyte injury using hepatocyte-specific Hsp20 gene knockout and transgenic mice models as well as in vitro experiment. Furthermore, to get insight into the mechanism underlying the regulation by Hsp20 in AMPK-mTOR pathway via LC-MS/MS、Co-IP and RNAi techniques. Which will provide theory evidence for searching for the novel therapeutic target of NAFLD.

自噬在非酒精性脂肪性肝病(NAFLD)发展中发挥关键调控作用，通过调节细胞自噬能够有效应对NAFLD。申请者新近研究首次发现热休克蛋白20（Hsp20)基因全身性敲除小鼠NAFLD的发展较对照小鼠显著减轻。然而，Hsp20对NAFLD的调控是否呈肝细胞自主性及其分子机制尚不明确。我们前期研究发现在棕榈酸刺激下，与对照细胞相比，Hsp20基因敲低肝细胞中磷酸化AMPK表达水平增加明显，mTOR活性因此降低，自噬显著增强。基于上述发现，我们假设：Hsp20-AMPK-mTOR轴通过调控肝细胞自噬影响NAFLD的发展。为此本项目拟通过肝细胞特异性的Hsp20敲除及转基因动物模型结合体外实验来系统地阐明Hsp20在NAFLD中的作用；并采用LC-MS/MS、Co-IP及RNAi技术明确Hsp20影响AMPK-mTOR自噬调节通路的分子机制，从而为寻找NAFLD新的防治靶点提供理论依据。

在心脏组织中，HSP20是新型自噬调节因子，然而，其对肝脏组织及自噬的影响尚不清楚。本研究中，我们观察到HSP20在小鼠和NAFLD患者的肝脏组织中表达增加。肝脏特异性敲低Hsp20，可减轻肥胖小鼠肝脏脂肪变性和胰岛素抵抗，而上调Hsp20可促进脂质沉积和肝细胞死亡。在机制上，LC-MS/MS显示HSP20与磷酸化的ERK2相互作用，并阻止其被磷酸酶双特异性磷酸酶6 (DUSP6)去磷酸化，导致ERK2介导的自噬抑制，从而导致饱和脂肪酸(SFA)触发的肝细胞死亡加重。重要的是，这种不良的细胞影响可以通过使用ERK抑制剂来抑制。我们的数据揭示了SFA治疗如何导致肝细胞ERK2磷酸化的框架，为治疗肥胖提供了一种可能的策略。

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