3-巯基丙酮酸转硫酶在非酒精性脂肪性肝病中的作用及其分子机制研究

发布时间：2017-12-27 15:24

本文关键词：3-巯基丙酮酸转硫酶在非酒精性脂肪性肝病中的作用及其分子机制研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：背景和目的:肝脏中脂肪酸的过度沉积会通过脂毒性导致非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)的发生发展,但其中涉及的分子机制尚不明确。胱硫醚-β-合酶(cystathionine-β-synthase,CBS)和胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)是体内内源性硫化氢(hydrogensulfide,H2S)的主要产生酶,已被发现受到脂肪酸的调控并参与NAFLD发病机制。本研究旨在探究另一内源性H2S产生酶,3-巯基丙酮酸转移硫酶(3-mercaptopyruvate sulfurtransferase,MPST)在 NAFLD 发生发展中的作用及机制。材料和方法:采用游离脂肪酸(free fatty acid,FFA)(油酸:棕榈酸为2:1)混合培养液刺激人正常肝细胞L02细胞,及高脂饮食(high fat diet,HFD)喂养C57BL/6小鼠分别建立NAFLD细胞及动物模型,用Western Blot法检测MPST在NAFLD模型中的表达水平。采用免疫组化法检测MPST在NAFLD临床患者肝脏标本中的蛋白表达水平。采用重组腺病毒介导shRNA结合尾静脉高压注射技术对小鼠体内肝脏MPST的表达进行干预,并在借助转录激活样效应因子核酸酶(transcription activator-like effector nuclease,TALEN)技术构建的 C57BL/6 为背景的 MPST 基因敲除杂合子小鼠中进一步观察MPST对肝脏脂肪变性的影响作用。在FFA刺激的L02细胞中,分别运用siRNA和转染MPST过表达质粒以干预MPST表达,观察MPST对肝细胞脂肪变性的影响与作用。采用HE及油红O染色观察肝脏和肝细胞脂变程度,通过测定肝甘油三酯(TG)、总胆固醇(TC)含量及FFA水平,联系H2S水平变化,MDA及SOD活性等指标变化研究MPST对肝脏脂肪变性及氧化应激的影响。通过外源性H2S供体的应用,检测CSE及SREBP-1通路、JNK磷酸化水平变化,免疫共沉淀(Co-Imnunoprecipitation,CoIP)技术探究潜在的分子机制。结果:FFA的刺激诱导MPST在人脂变肝细胞中表达上调,并部分依赖于NF-1κB/p65。在HFD诱导的小鼠NAFLD模型及临床NAFLD患者肝脏中,MPST蛋白表达水平显著升高。应用MPST-shRNA重组腺病毒尾静脉高压注射介导小鼠肝脏MPST表达部分敲低,和使用MPST基因敲除杂合子小鼠介导遗传性的MPST表达部分缺失,均能显著改善HFD诱导的肝脏脂肪变性程度。和体内水平一致地,在L02细胞中抑制MPST表达后,FFA诱导的肝细胞脂变显著改善,而过表达MPST后则脂变加剧。有趣的是,部分敲低MPST会显著上调H2S水平,相反地,过表达MPST则显著下调H2S水平。CoIP实验揭示MPST可以通过直接蛋白-蛋白相互作用而负调控CSE——肝脏中H2S产生主要酶。且进一步的机制研究发现,H2S在MPST对脂肪肝的调节过程中起重要介导作用,与脂肪合成关键蛋白SREBP-1相关通路抑制,JNK磷酸化减弱及肝脏氧化应激改善有关。结论:FFA介导MPST表达显著上调,MPST通过CSE依赖途径调节H2S水平,在NAFLD发生发展中起到重要作用。这为基于MPST为潜在靶点的NAFLD新药研发提供了新的理论依据,部分抑制MPST有望成为治疗NAFLD新策略。
[Abstract]:Background and purpose: excessive deposition of fatty acids in the liver can lead to the occurrence and development of nonalcoholic fatty liver disease (NAFLD) through lipotoxicity. However, the molecular mechanism involved is not clear. Cystin cystathionine- -synthase (CBS) and cystathionine- -lyase -lyase (CSE) are the main enzymes producing endogenous hydrogen sulfide (HYDROGENSULFIDE, H2S). They have been found to be regulated by fatty acids and participate in the pathogenesis of NAFLD. The purpose of this study is to explore the role and mechanism of another endogenous H2S producing enzyme, 3- thiol pyruvate transferase (3-mercaptopyruvate sulfurtransferase (MPST)) in the occurrence and development of NAFLD. Materials and methods: using free fatty acids (free fatty, acid, FFA) (oleic acid palmitic acid 2:1) mixed medium stimulation of normal human liver cells L02 cells, and high fat diet (high fat, diet, HFD) NAFLD cells and animal models were established by feeding C57BL/6 mice, the expression level of Western detected by Blot MPST in the NAFLD model. The protein expression level of MPST in the liver specimens of patients with NAFLD was detected by immunohistochemistry. Were treated with recombinant adenovirus mediated shRNA combined with tail vein injection of mouse liver MPST expression, and with the help of transcription activator likeeffector nuclease (transcription activator-like effector nuclease TALEN) technology to build C57BL/6 for the background of MPST gene knockout further observe the effect of MPST on hepatic steatosis in heterozygous effect in mice. In the L02 cells stimulated by FFA, siRNA and MPST overexpression plasmids were used to interfere with the expression of MPST, and the effect of MPST on the fatty degeneration of hepatocytes was observed. HE and oil red O staining were used to observe the degree of liver and hepatocyte lipidosis. The effects of MPST on hepatic steatosis and oxidative stress were studied by measuring the contents of hepatic triglyceride (TG), total cholesterol (TC) and FFA level, and the changes of H2S level, MDA and SOD activity. Through the application of exogenous H2S donors, we detected the changes of CSE and SREBP-1 pathway and JNK phosphorylation level, and explored the potential molecular mechanism of Co-Imnunoprecipitation (CoIP) technology. Results: the expression of MPST was up-regulated in human lipoprotein hepatocytes by FFA stimulation, and partly dependent on NF-1 kappa B/p65. The expression of MPST protein was significantly increased in the HFD induced mouse NAFLD model and the liver of the clinical NAFLD patients. MPST-shRNA recombinant adenovirus mediated tail vein high pressure injection mediated partial knockdown of MPST expression in mice liver, and partial deletion of MPST expression mediated by MPST knockout heterozygous mice can significantly improve the degree of HFD induced hepatic steatosis. After the expression of MPST in L02 cells was consistent with the level of the body, the liver cell lipid induced by FFA significantly improved, and the fat increased after the overexpression of MPST. Interestingly, partial knockout MPST significantly up-regulated the H2S level, and on the contrary, overexpression of MPST significantly lowered the level of H2S. The CoIP experiment revealed that MPST can negatively regulate CSE by direct protein protein interaction - the main enzyme produced by H2S in the liver. Further mechanism studies showed that H2S plays an important mediating role in the regulation of MPST on fatty liver, and is related to inhibition of SREBP-1 related pathway, JNK phosphorylation and liver oxidative stress. Conclusion: FFA mediated MPST expression is significantly up-regulated, and MPST regulates the level of H2S through CSE dependent pathway, which plays an important role in the development of NAFLD. This provides a new theoretical basis for the research and development of new NAFLD drugs based on MPST as a potential target, and partial inhibition of MPST is expected to be a new strategy for the treatment of NAFLD.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.5

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