高脂及炎症状态激活mTOR信号通路导致肝脏CD36翻译效率升高

发布时间：2018-08-13 12:30

【摘要】：目的：非酒精性脂肪肝病（non-alcoholic fatty liver disease，NAFLD）已经成为危害人类健康的主要代谢性疾病之一，但其发病机制尚不完全清楚。高脂及炎症状态均可以作为独立的危险因素导致NAFLD的发生发展。CD36属于B族清道夫受体，可以介导长链脂肪酸及氧化低密度脂蛋白的跨膜转运，其表达量与肝脏脂肪变性程度密切相关。哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）是一种高度保守的丝氨酸/苏氨酸激酶，它在调节细胞生长、增殖及蛋白质翻译中起着重要的作用。本课题旨在研究高脂及炎症状态是否可以通过激活mTOR信号通路导致CD36翻译效率升高，最终引起CD36蛋白表达量及肝细胞内脂质蓄积增加。方法：使用软脂酸、TNF-及IL-6处理人肝癌细胞株HepG2，高脂饲料喂养及酪蛋白皮下注射C57BL/6J小鼠的方法建立体外及体内高脂及炎症模型。HepG2细胞分为：对照组1（0.2%BSA）、软脂酸处理组（0.2%BSA+0.08mmol/L软脂酸）、对照组2（0.2%BSA+0.04mmol/L软脂酸）、TNF-处理组（0.2%BSA+0.04mmol/L软脂酸+25ng/mLTNF-）、IL-6处理组（0.2%BSA+0.04mmol/L软脂酸+20ng/mLIL-6），处理时间为24小时。雄性C57BL/6J小鼠分为：正常饲料组（正常饲料）、高脂饲料组（高脂饲料）、正常饲料+酪蛋白处理组（正常饲料+0.5mL10%酪蛋白皮下注射），，建模时间为14周。油红O染色观察肝细胞内脂质蓄积的情况。酶联免疫吸附试验及酶偶联比色法分别检测肝细胞内游离脂肪酸（FFA）及甘油三酯（TG）含量。Real-TimePCR检测肝细胞CD36mRNA表达。Western Blotting检测肝细胞CD36蛋白表达，mTOR及其下游翻译调控蛋白：核糖体蛋白p70S6激酶（p70ribosomal protein S6kinase，p70S6K）、eIF4E结合蛋白1（eukaryoticinitiation factor4E-binding protein1，4E-BP1）及真核翻译起始因子4E（eukaryotic initiation factor4E，eIF4E）的磷酸化水平。蛋白降解实验检测肝细胞CD36蛋白稳定性。多聚核糖体分析检测肝细胞CD36翻译效率。并应用mTOR特异性抑制剂雷帕霉素作用于高脂及炎症状态下的HepG2细胞（10ng/mL雷帕霉素）及C57BL/6J小鼠（2mg/kg体重的雷帕霉素皮下注射），观察其对上述检测指标的影响。结果：油红O染色、FFA及TG定量检测发现高脂及炎症状态能使HepG2细胞及C57BL/6J小鼠肝脏组织脂质蓄积增加（P0.05）。Western Blotting及Real-Time PCR检测发现高脂及炎症状态能使CD36蛋白表达量升高（P0.05），而CD36蛋白表达量却没有明显变化（P0.05）。蛋白降解实验发现高脂及炎症状态并没有改变HepG2细胞CD36蛋白稳定性（P0.05）。多聚核糖体分析发现高脂及炎症状态可以使CD36翻译效率明显升高。进一步行Western Blotting检测发现高脂及炎症状态可以升高mTOR及其下游翻译调控蛋白p70S6K、4EBP-1及eIF4E的磷酸化水平（P0.05）。加入雷帕霉素之后，相应的高脂及炎症处理组mTOR信号通路磷酸化水平降低（P0.05），导致CD36翻译效率及蛋白表达量减少（P0.05），最终引起HepG2细胞及C57BL/6J小鼠肝脏组织脂质蓄积减轻（P0.05）。结论：高脂及炎症状态可以激活HepG2细胞及C57BL/6J小鼠肝脏组织mTOR信号通路导致肝脏CD36翻译效率升高，最终引起CD36蛋白表达及脂质蓄积增加。
[Abstract]:Objective: Non-alcoholic fatty liver disease (NAFLD) has become one of the major metabolic diseases endangering human health, but its pathogenesis is not fully understood. High fat and inflammatory state can be used as independent risk factors leading to the occurrence and development of NAFLD. CD36 belongs to B scavenger receptor and can mediate the development of NAFLD. Long-chain fatty acids and oxidized low-density lipoproteins are transmembrane transporters whose expression is closely related to the degree of liver steatosis. Mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine kinase, which plays an important role in regulating cell growth, proliferation and protein translation. The purpose of this study was to investigate whether hyperlipidemia and inflammation could induce the increase of CD36 translation efficiency by activating the mTOR signaling pathway, and ultimately lead to the increase of CD36 protein expression and lipid accumulation in hepatocytes.
METHODS: Human hepatoma cell line HepG2 was treated with palmitic acid, TNF-and IL-6, and high-fat diet and subcutaneous injection of casein into C57BL/6J mice. HepG2 cells were divided into control group 1 (0.2% BSA), palmitic acid treatment group (0.2% BSA + 0.08mmol/L palmitic acid), control group 2 (0.2% BSA + 0.04 mmol/L palmitic acid). Acid, TNF-treated group (0.2% BSA + 0.04 mmol/L palmitic acid + 25 ng/mLTNF-), IL-6 treated group (0.2% BSA + 0.04 mmol/L palmitic acid + 20 ng/mLIL-6), treatment time was 24 hours. Male C57BL/6J mice were divided into normal diet group (normal diet), high-fat diet group (high-fat diet), normal diet + casein treated group (normal diet + 0.5 mL 10% casein subcutaneous injection). The content of free fatty acid (FFA) and triglyceride (TG) in hepatocytes were detected by enzyme-linked immunosorbent assay (ELISA) and enzyme-coupled colorimetry (ELISA). The expression of CD36 mRNA in hepatocytes was detected by Real-Time PCR. The expression of CD36 protein in hepatocytes was detected by Western Blotting. The expression of CD36 protein in hepatocytes was detected by mTOR and its subunits. The phosphorylation levels of p70S6 kinase (p70ribosomal protein S6kinase, p70S6K), eIF4E binding protein 1, 4E-BP1 and eukaryotic translation initiation factor 4E (eIF4E) in hepatocytes were determined by protein degradation assay. Sex. Polyribosome analysis was used to detect the translation efficiency of CD36 in hepatocytes. Rapamycin, a specific inhibitor of mTOR, was used to treat high-fat and inflammatory HepG2 cells (10ng/mL rapamycin) and C57BL/6J mice (2mg/kg body weight rapamycin subcutaneously) to observe the effects of rapamycin on the above indexes.
Results: Oil red O staining, FFA and TG quantitative detection showed that high fat and inflammation could increase the lipid accumulation of HepG2 cells and C57BL/6J mice liver tissue (P 0.05). Western Blotting and Real-Time PCR detection showed that high fat and inflammation could increase the expression of CD36 protein (P 0.05), but the expression of CD36 protein did not change significantly (P 0.05). Protein degradation assay showed that high fat and inflammation did not change the stability of CD36 protein in HepG2 cells (P 0.05). Polyribosome analysis showed that high fat and inflammation could significantly increase the translation efficiency of CD36. Phosphorylation levels of EBP-1 and eIF4E (P 0.05). When rapamycin was added, the phosphorylation levels of mTOR signaling pathway decreased (P 0.05) in the corresponding hyperlipidemia and inflammation treatment groups, resulting in the decrease of CD36 translation efficiency and protein expression (P 0.05), and ultimately the decrease of lipid accumulation in HepG2 cells and C57BL/6J mice liver tissue (P 0.05).
CONCLUSION: Hyperlipidemia and inflammation can activate the mTOR signaling pathway in HepG2 cells and C57BL/6J mice liver tissues, resulting in an increase in the efficiency of liver CD36 translation, and ultimately in the expression of CD36 protein and lipid accumulation.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.5

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