NAMPT对高脂饮食诱导小鼠脂肪肝的保护作用及机制研究
发布时间：2018-09-07 18:46
【摘要】：研究背景和研究目的:非酒精性脂肪性肝病(NAFLD)是指人体肝细胞内脂肪过度堆积导致的一种获得性代谢应激性肝损伤疾病,该疾病主要是由非酒精因素引起,与胰岛素抵抗以及遗传易感性密切相关。近些年来,由于我们的饮食结构和生活习惯的不断改变,NAFLD的病发率也越来越高,并随时间的变化而发展成肝硬化和肝癌的风险增加。尼克酰胺磷酸核糖转移酶(NAMPT)是烟酰胺腺嘌呤二核苷酸(NAD+)生物合成过程的关键酶,在哺乳动物各种器官的细胞代谢中起重要作用。已有研究报道NAD+含量下降与许多代谢性疾病有密切关系,而NAD+合成途径的关键酶NAMPT在NAFLD中对肝脏的具体作用及其机制仍然不明确。本研究旨在探索NAMPT在高脂饮食诱导NAFLD中的作用及其机制,以期为NAFLD的预防和治疗提供重要靶点和实验依据。实验方法:1.高脂饮食诱导脂肪肝模型的制备及其分析:先将实验动物分成四组:(1)正常饮食(ND)组;(2)ND+FK866(2 mg/kg/day,IP);(3)高脂饮食(HFD)组;(4)HFD+FK866(2 mg/kg/day,IP),腹腔给药NAMPT抑制剂FK866一周后,再继续分别用ND、HFD喂养12周,取其肝脏做成石蜡切片经HE染色观察脂滴的数量;并提取RNA和总蛋白用Q-PCR以及Western blot检测ND及HFD组小鼠肝脏组织中NAMPT、SREBP1及FASN的表达;同时检测HFD及HFD+FK866组小鼠肝脏组织中脂质合成相关基因SREBP1、FASN、ACC等表达情况。2.肝细胞脂肪累积模型的制备及其分析:1)HepG2细胞在给予油酸刺激下用20 n M FK866或联合NAD及NMN处理细胞24 h后,用油红染色检测细胞脂滴的堆积、甘油三酯试剂盒检测细胞内TG含量、并提取RNA和总蛋白用Q-PCR以及Western blot检测脂质合成相关基因的m RNA和蛋白的表达情况;2)在人源的Hep G2和鼠源的Hep1-6肝癌细胞中,用lipo3000转染NAMPT质粒(有两种给药方式:先转染NAMPT质粒再用油酸处理24h,第二种是先给油酸处理24h后再转染质粒NAMPT)。之后用油红染色检测细胞脂滴的堆积、甘油三酯试剂盒检测细胞内TG含量、并提取RNA和总蛋白用Q-PCR以及Western blot检测脂质合成相关基因的m RNA和蛋白的表达情况。3.机制分析:1)在动物水平上利用Western blot检测肝脏组织中Sirt1及AMPKα的蛋白水平;2)在细胞水平用Western blot检测给FK866后肝细胞胰岛素信号通路中Akt蛋白磷酸化水平;3)在细胞水平给予Sirt1的激动剂Res和Sirt1的特异性抑制剂EX-527处理细胞24h,检测脂质合成相关基因的蛋白表达水平。实验结果:1.NAMPT在高脂喂养野生型小鼠肝脏中的表达显著低于正常组,而脂质合成相关基因SREBP1及FASN的表达则显著高于正常组。HE染色显示,HFD组肝脏中脂滴数量明显多于ND组。2.NAMPT抑制剂FK866显著增加高脂饮食诱导的小鼠肝脏脂质堆积以及肝脏组织的脂质合成基因SREBP1、FASN、ACC和SCD1的表达。3.FK866显著增加正常或油酸刺激条件下的肝细胞脂质堆积,NAD或NMN则可改善这一作用。FK866还显著增加Hep G2细胞内TG含量、脂质合成基因SREBP1、FASN、ACC及SCD1的m RNA水平,同时增加FASN的蛋白表达水平,NAD或NMN则可部分逆转这些作用。4.Hep G2和Hep1-6细胞系过表达NAMPT可显著降低油酸诱导的脂质堆积和TG增加,同时降低脂质合成基因SREBP1、FASN、ACC的m RNA和蛋白水平。5.FK866可显著降低HFD喂养小鼠的肝脏组织Sirt1及AMPKα磷酸化水平,以及降低肝细胞胰岛素信号通路关键因子Akt的磷酸化。细胞实验显示,FK866联合Sirt1激动剂Res可明显抑制ACC表达以及油酸刺激条件下的FASN及ACC表达,而给予Sirt1特异性抑制剂EX527则呈现相反作用。NMN可以恢复由FK866增加的FASN及ACC表达。结论:1.NAMPT在高脂喂养小鼠肝脏及油酸处理的肝细胞中表达下降。2.抑制NAMPT在体内外均可增加肝脏脂质合成,NAD及NMN具有改善作用;而过表达NAMPT则改善油酸诱导的肝脂质合成。3.NAMPT通过激活Sirt1/SREBP1/AMPKα信号通路而抑制肝脏脂质积累。
[Abstract]:BACKGROUND AND OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is an acquired metabolic stress liver injury caused by excessive fat accumulation in human hepatocytes. It is mainly caused by non-alcoholic factors and is closely related to insulin resistance and genetic susceptibility. With the changing of living habits, the incidence of NAFLD is increasing, and the risk of developing cirrhosis and liver cancer increases with time. Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), plays an important role in the cellular metabolism of various mammalian organs. It has been reported that the decrease of NAD+ content is closely related to many metabolic diseases. However, the specific role of NAMPT, the key enzyme of NAD + synthesis pathway, in NAFLD, and its mechanism are still unclear. This study aims to explore the role and mechanism of NAMPT in high-fat diet-induced NAFLD, in order to provide an important target for the prevention and treatment of NAFLD. Methods: 1. Preparation and analysis of fatty liver model induced by high-fat diet: First, the experimental animals were divided into four groups: (1) normal diet (ND) group; (2) ND + FK866 (2 mg / kg / day, IP); (3) high-fat diet (HFD) group; (4) HFD + FK866 (2 mg / kg / day, IP), intraperitoneal administration of NAMPT inhibitor FK866 for one week, and then continued feeding with ND, HFD for 12 weeks, respectively. The number of lipid droplets was observed by HE staining, RNA and total protein were extracted and the expressions of NAMPT, SREBP1 and FASN were detected by Q-PCR and Western blot, and the expression of lipid synthesis-related genes SREBP1, FASN and ACC in liver tissues of HFD and HFD+FK866 mice were detected. Preparation and analysis of cell fat accumulation model: 1) HepG2 cells were stimulated by oleic acid and treated with 20 N M FK866 or combined with NAD and NMN for 24 hours. Cell lipid droplet accumulation was detected by oil red staining, intracellular TG content was detected by triglyceride kit, RNA and total protein were extracted and lipid synthesis related groups were detected by Q-PCR and Western blot. Because of the expression of M RNA and protein; 2) In human Hep G2 and mouse Hep 1-6 hepatoma cells, NAMPT plasmid was transfected with Lipo 3000 (two ways: first transfected with NAMPT plasmid and then treated with oleic acid for 24 hours; second, transfected with oleic acid for 24 hours and then transfected with NAMPT plasmid). Ester kit was used to detect intracellular TG content, RNA and total protein were extracted and the expression of M RNA and protein related to lipid synthesis was detected by Q-PCR and Western blot. The phosphorylation of Akt protein in the insulin signaling pathway of the posterior hepatocytes was detected after treatment with Sirt 1 agonist Res and Sirt 1 specific inhibitor EX-527 for 24 hours. HE staining showed that the number of lipid droplets in the liver of HFD group was significantly higher than that of ND group. 2. The expression of lipid synthesis genes SREBP1, FASN, ACC and SCD1 in the liver of mice induced by high-fat diet was significantly increased by the NAMPT inhibitor FK866. FK866 also significantly increased the content of TG in Hep G2 cells, the level of M RNA of lipid synthesis genes SREBP1, FASN, ACC and SCD1, and the level of FASN protein expression. NAD or NMN partially reversed these effects. 4. Hep G2 and Hep 1-6 cell lines overflow Expression of NAMPT significantly decreased oleic acid-induced lipid accumulation and TG increase, and decreased the levels of SREBP1, FASN, ACC m RNA and protein. 5. The results showed that FK866 combined with Sirt1 agonist Res could significantly inhibit the expression of ACC and FASN and ACC stimulated by oleic acid, but the expression of FASN and ACC increased by FK866 could be restored by NMN. Inhibiting NAMPT can increase liver lipid synthesis in vitro and in vivo, and NAD and NMN can improve it. Overexpression of NAMPT can improve oleic acid-induced liver lipid synthesis. 3. NAMPT inhibits liver lipid accumulation by activating Sirt1/SREBP1/AMPK alpha signaling pathway.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
本文编号：2229111
[Abstract]:BACKGROUND AND OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is an acquired metabolic stress liver injury caused by excessive fat accumulation in human hepatocytes. It is mainly caused by non-alcoholic factors and is closely related to insulin resistance and genetic susceptibility. With the changing of living habits, the incidence of NAFLD is increasing, and the risk of developing cirrhosis and liver cancer increases with time. Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), plays an important role in the cellular metabolism of various mammalian organs. It has been reported that the decrease of NAD+ content is closely related to many metabolic diseases. However, the specific role of NAMPT, the key enzyme of NAD + synthesis pathway, in NAFLD, and its mechanism are still unclear. This study aims to explore the role and mechanism of NAMPT in high-fat diet-induced NAFLD, in order to provide an important target for the prevention and treatment of NAFLD. Methods: 1. Preparation and analysis of fatty liver model induced by high-fat diet: First, the experimental animals were divided into four groups: (1) normal diet (ND) group; (2) ND + FK866 (2 mg / kg / day, IP); (3) high-fat diet (HFD) group; (4) HFD + FK866 (2 mg / kg / day, IP), intraperitoneal administration of NAMPT inhibitor FK866 for one week, and then continued feeding with ND, HFD for 12 weeks, respectively. The number of lipid droplets was observed by HE staining, RNA and total protein were extracted and the expressions of NAMPT, SREBP1 and FASN were detected by Q-PCR and Western blot, and the expression of lipid synthesis-related genes SREBP1, FASN and ACC in liver tissues of HFD and HFD+FK866 mice were detected. Preparation and analysis of cell fat accumulation model: 1) HepG2 cells were stimulated by oleic acid and treated with 20 N M FK866 or combined with NAD and NMN for 24 hours. Cell lipid droplet accumulation was detected by oil red staining, intracellular TG content was detected by triglyceride kit, RNA and total protein were extracted and lipid synthesis related groups were detected by Q-PCR and Western blot. Because of the expression of M RNA and protein; 2) In human Hep G2 and mouse Hep 1-6 hepatoma cells, NAMPT plasmid was transfected with Lipo 3000 (two ways: first transfected with NAMPT plasmid and then treated with oleic acid for 24 hours; second, transfected with oleic acid for 24 hours and then transfected with NAMPT plasmid). Ester kit was used to detect intracellular TG content, RNA and total protein were extracted and the expression of M RNA and protein related to lipid synthesis was detected by Q-PCR and Western blot. The phosphorylation of Akt protein in the insulin signaling pathway of the posterior hepatocytes was detected after treatment with Sirt 1 agonist Res and Sirt 1 specific inhibitor EX-527 for 24 hours. HE staining showed that the number of lipid droplets in the liver of HFD group was significantly higher than that of ND group. 2. The expression of lipid synthesis genes SREBP1, FASN, ACC and SCD1 in the liver of mice induced by high-fat diet was significantly increased by the NAMPT inhibitor FK866. FK866 also significantly increased the content of TG in Hep G2 cells, the level of M RNA of lipid synthesis genes SREBP1, FASN, ACC and SCD1, and the level of FASN protein expression. NAD or NMN partially reversed these effects. 4. Hep G2 and Hep 1-6 cell lines overflow Expression of NAMPT significantly decreased oleic acid-induced lipid accumulation and TG increase, and decreased the levels of SREBP1, FASN, ACC m RNA and protein. 5. The results showed that FK866 combined with Sirt1 agonist Res could significantly inhibit the expression of ACC and FASN and ACC stimulated by oleic acid, but the expression of FASN and ACC increased by FK866 could be restored by NMN. Inhibiting NAMPT can increase liver lipid synthesis in vitro and in vivo, and NAD and NMN can improve it. Overexpression of NAMPT can improve oleic acid-induced liver lipid synthesis. 3. NAMPT inhibits liver lipid accumulation by activating Sirt1/SREBP1/AMPK alpha signaling pathway.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
【参考文献】
相关期刊论文 前1条
1 Giovanni Tarantino;Carmine Finelli;;What about non-alcoholic fatty liver disease as a new criterion to define metabolic syndrome?[J];World Journal of Gastroenterology;2013年22期
，本文编号：2229111
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