磷酸化Hsp27与STAT3结合促进自噬抑制肝细胞脂质变性的分子机制研究
发布时间：2018-07-23 12:55
【摘要】：非酒精性脂肪肝病是指以无过量饮酒史,以肝细胞脂质变性为特征的临床综合征,是常见的慢性肝病,也是导致终末期肝病的危险因素。目前,尚无有效防治药物。自噬是真核细胞中普遍存在的溶酶体降解程序。研究表明,自噬能够促进脂质代谢,缓解肝脏脂质变性。热休克蛋白27(heat shock protein 27, Hsp27)是重要的分子伴侣,能够发生磷酸化修饰,而磷酸化导致Hsp27聚合度和功能的改变。研究表明,Hsp27参与自噬调控。但是,Hsp27,尤其是磷酸化Hsp27参与自噬调控的具体机制尚不明确,有待进一步研究。在本论文中,我们研究了磷酸化Hsp27在激活自噬以及抑制肝细胞脂质变性中的作用。我们发现Hsp27与STAT3(信号转导与转录激活因子3)结合,促进自噬及脂质代谢。首先,通过高脂喂养C57/BL6小鼠,建立小鼠肝脏脂质变性模型,我们发现高脂喂养上调小鼠肝脏组织中Hsp25(人Hsp27在啮齿类中的同源蛋白)的磷酸化和自噬水平。同样,在体外培养的人肝脏L02细胞中,软脂酸促进Hsp27磷酸化和自噬激活。在软脂酸处理前,利用RNA干扰下调Hsp27的表达,或者利用Hsp27磷酸化抑制剂KRIBB3处理,或者过表达非磷酸化突变的Hsp27-3A,则抑制软脂酸激活的自噬,并加剧脂质变性。若过表达野生型的Hsp27-WT或者持续磷酸化突变的Hsp27-3D,则提高L02细胞的自噬水平,并缓解脂质变性。利用羟氯喹抑制自噬体的降解,则阻断磷酸化的Hsp27对脂质变性的缓解作用。在高脂喂养的小鼠模型中,利用KRIBB3抑制Hsp25磷酸化,则抑制自噬激活,并加剧肝脏脂质变性。我们进一步研究了Hsp27磷酸化激活自噬的分子机制。通过免疫共沉淀实验,我们发现Hsp27能与STAT3形成复合物,更为重要的是,软脂酸处理后,Hsp27与STAT3形成的复合物增加,与此同时,STAT3与PKR的复合物明显减少。如果利用RNA干扰下调Hsp27的表达,则抑制STAT3从PKR解离。如果利用KRIBB3抑制Hsp27磷酸化,则抑制Hsp27/STAT3复合体形成,并抑制STAT3从PKR解离。此外,过表达野生型的Hsp27-WT和持续磷酸化突变的Hsp27-3D,则促进STAT3从PKR解离。过表达非磷酸化突变的Hsp27-3A,则抑制STAT3从PKR的解离。与野生型的Hsp27-WT相比,持续磷酸化突变的Hsp27-3D与STAT3结合的能力更强。综上所述,本论文的研究发现,磷酸化Hsp27与STAT3结合,干扰了STAT3与PKR复合物的形成,削弱STAT3对PKR的抑制作用,促进PKR磷酸化eIF2a,激活自噬,从而缓解脂质变性。本论文的研究结果明确了磷酸化Hsp27在自噬活化中的重要作用,深入揭示了磷酸化Hsp27的功能。同时,揭示了磷酸化Hsp27活化自噬,促进脂质分解的作用,为临床采用促进脂质代谢防治脂质变性提供理论基础。
[Abstract]:Non-alcoholic fatty liver disease is a clinical syndrome characterized by liver cell lipids degeneration without excessive drinking history. It is a common chronic liver disease and a risk factor leading to end-stage liver disease. At present, there is no effective control drug. Autophagy is a common lysosomal degradation process in eukaryotic cells. Studies have shown that autophagy can promote lipid metabolism and alleviate liver lipid degeneration. Heat shock protein 27 (Hsp27) is an important molecular chaperone capable of phosphorylation modification, while phosphorylation changes the degree of polymerization and function of Hsp27. The results showed that Hsp27 was involved in autophagy regulation. However, the mechanism of Hsp27, especially phosphorylated Hsp27, involved in autophagy regulation is not clear, which needs further study. In this study, we investigated the role of phosphorylated Hsp27 in activating autophagy and inhibiting hepatocyte lipid degeneration. We found that Hsp27 binds to STAT3 (signal transduction and transcription activator 3) and promotes autophagy and lipid metabolism. Firstly, the lipids denaturation model was established in C57/BL6 mice by high-fat feeding. We found that high-fat feeding up-regulated the phosphorylation and autophagy level of Hsp25 (homologous protein of human Hsp27 in rodents) in mice liver. Similarly, in cultured human liver L02 cells, palmitate promotes Hsp27 phosphorylation and autophagy activation. Before palmitic acid treatment, Hsp27 expression was down-regulated by RNA interference, or treated with Hsp27 phosphorylation inhibitor KRIBB3 or overexpression of non-phosphorylated mutant Hsp27-3A, which inhibited palmitic acid-activated autophagy and aggravated lipid denaturation. Overexpression of wild-type Hsp27-WT or continuous phosphorylation mutation Hsp27-3D increased autophagy level of L02 cells and alleviated lipid denaturation. Hydroxychloroquine was used to inhibit the degradation of autophagy and to block the effect of phosphorylated Hsp27 on lipid denaturation. In hyperlipidemic mice, KRIBB3 inhibited Hsp25 phosphorylation, inhibited autophagy activation and aggravated liver lipid degeneration. We further studied the molecular mechanism of Hsp27 phosphorylation activated autophagy. By immunoprecipitation, we found that Hsp27 can form complex with STAT3, and more importantly, the complex of Hsp27 and STAT3 increased after palmitate treatment, while the complex of statin and PKR decreased significantly. If the expression of Hsp27 was down-regulated by RNA interference, the dissociation of STAT3 from PKR was inhibited. If KRIBB3 was used to inhibit the phosphorylation of Hsp27, the formation of Hsp27/STAT3 complex and the dissociation of STAT3 from PKR were inhibited. In addition, overexpression of wild-type Hsp27-WT and persistent phosphorylation mutation Hsp27-3D promoted the dissociation of STAT3 from PKR. Overexpression of non-phosphorylated Hsp27-3 A inhibits the dissociation of STAT3 from PKR. Compared with wild type Hsp27-WT, Hsp27-3D with persistent phosphorylation mutation has stronger ability to bind to STAT3. In conclusion, we found that the combination of phosphorylated Hsp27 and STAT3 interferes with the formation of STAT3 / PKR complex, weakens the inhibitory effect of STAT3 on PKR, promotes PKR phosphorylation of eIF2a, activates autophagy, and alleviates lipid denaturation. In this paper, the important role of phosphorylated Hsp27 in autophagy activation was clarified, and the function of phosphorylated Hsp27 was revealed. At the same time, it revealed the role of phosphorylated Hsp27 activated autophagy and promoted lipid decomposition, which provided a theoretical basis for the prevention and treatment of lipid denaturation by promoting lipid metabolism.
【学位授予单位】：南京师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575
本文编号：2139479
[Abstract]:Non-alcoholic fatty liver disease is a clinical syndrome characterized by liver cell lipids degeneration without excessive drinking history. It is a common chronic liver disease and a risk factor leading to end-stage liver disease. At present, there is no effective control drug. Autophagy is a common lysosomal degradation process in eukaryotic cells. Studies have shown that autophagy can promote lipid metabolism and alleviate liver lipid degeneration. Heat shock protein 27 (Hsp27) is an important molecular chaperone capable of phosphorylation modification, while phosphorylation changes the degree of polymerization and function of Hsp27. The results showed that Hsp27 was involved in autophagy regulation. However, the mechanism of Hsp27, especially phosphorylated Hsp27, involved in autophagy regulation is not clear, which needs further study. In this study, we investigated the role of phosphorylated Hsp27 in activating autophagy and inhibiting hepatocyte lipid degeneration. We found that Hsp27 binds to STAT3 (signal transduction and transcription activator 3) and promotes autophagy and lipid metabolism. Firstly, the lipids denaturation model was established in C57/BL6 mice by high-fat feeding. We found that high-fat feeding up-regulated the phosphorylation and autophagy level of Hsp25 (homologous protein of human Hsp27 in rodents) in mice liver. Similarly, in cultured human liver L02 cells, palmitate promotes Hsp27 phosphorylation and autophagy activation. Before palmitic acid treatment, Hsp27 expression was down-regulated by RNA interference, or treated with Hsp27 phosphorylation inhibitor KRIBB3 or overexpression of non-phosphorylated mutant Hsp27-3A, which inhibited palmitic acid-activated autophagy and aggravated lipid denaturation. Overexpression of wild-type Hsp27-WT or continuous phosphorylation mutation Hsp27-3D increased autophagy level of L02 cells and alleviated lipid denaturation. Hydroxychloroquine was used to inhibit the degradation of autophagy and to block the effect of phosphorylated Hsp27 on lipid denaturation. In hyperlipidemic mice, KRIBB3 inhibited Hsp25 phosphorylation, inhibited autophagy activation and aggravated liver lipid degeneration. We further studied the molecular mechanism of Hsp27 phosphorylation activated autophagy. By immunoprecipitation, we found that Hsp27 can form complex with STAT3, and more importantly, the complex of Hsp27 and STAT3 increased after palmitate treatment, while the complex of statin and PKR decreased significantly. If the expression of Hsp27 was down-regulated by RNA interference, the dissociation of STAT3 from PKR was inhibited. If KRIBB3 was used to inhibit the phosphorylation of Hsp27, the formation of Hsp27/STAT3 complex and the dissociation of STAT3 from PKR were inhibited. In addition, overexpression of wild-type Hsp27-WT and persistent phosphorylation mutation Hsp27-3D promoted the dissociation of STAT3 from PKR. Overexpression of non-phosphorylated Hsp27-3 A inhibits the dissociation of STAT3 from PKR. Compared with wild type Hsp27-WT, Hsp27-3D with persistent phosphorylation mutation has stronger ability to bind to STAT3. In conclusion, we found that the combination of phosphorylated Hsp27 and STAT3 interferes with the formation of STAT3 / PKR complex, weakens the inhibitory effect of STAT3 on PKR, promotes PKR phosphorylation of eIF2a, activates autophagy, and alleviates lipid denaturation. In this paper, the important role of phosphorylated Hsp27 in autophagy activation was clarified, and the function of phosphorylated Hsp27 was revealed. At the same time, it revealed the role of phosphorylated Hsp27 activated autophagy and promoted lipid decomposition, which provided a theoretical basis for the prevention and treatment of lipid denaturation by promoting lipid metabolism.
【学位授予单位】：南京师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575
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1 沈磊;磷酸化Hsp27与STAT3结合促进自噬抑制肝细胞脂质变性的分子机制研究[D];南京师范大学;2016年
，本文编号：2139479
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