NAMPT對(duì)高脂飲食誘導(dǎo)小鼠脂肪肝的保護(hù)作用及機(jī)制研究
發(fā)布時(shí)間：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)是指人體肝細(xì)胞內(nèi)脂肪過(guò)度堆積導(dǎo)致的一種獲得性代謝應(yīng)激性肝損傷疾病,該疾病主要是由非酒精因素引起,與胰島素抵抗以及遺傳易感性密切相關(guān)。近些年來(lái),由于我們的飲食結(jié)構(gòu)和生活習(xí)慣的不斷改變,NAFLD的病發(fā)率也越來(lái)越高,并隨時(shí)間的變化而發(fā)展成肝硬化和肝癌的風(fēng)險(xiǎn)增加。尼克酰胺磷酸核糖轉(zhuǎn)移酶(NAMPT)是煙酰胺腺嘌呤二核苷酸(NAD+)生物合成過(guò)程的關(guān)鍵酶,在哺乳動(dòng)物各種器官的細(xì)胞代謝中起重要作用。已有研究報(bào)道NAD+含量下降與許多代謝性疾病有密切關(guān)系,而NAD+合成途徑的關(guān)鍵酶NAMPT在NAFLD中對(duì)肝臟的具體作用及其機(jī)制仍然不明確。本研究旨在探索NAMPT在高脂飲食誘導(dǎo)NAFLD中的作用及其機(jī)制,以期為NAFLD的預(yù)防和治療提供重要靶點(diǎn)和實(shí)驗(yàn)依據(jù)。實(shí)驗(yàn)方法:1.高脂飲食誘導(dǎo)脂肪肝模型的制備及其分析:先將實(shí)驗(yàn)動(dòng)物分成四組:(1)正常飲食(ND)組;(2)ND+FK866(2 mg/kg/day,IP);(3)高脂飲食(HFD)組;(4)HFD+FK866(2 mg/kg/day,IP),腹腔給藥NAMPT抑制劑FK866一周后,再繼續(xù)分別用ND、HFD喂養(yǎng)12周,取其肝臟做成石蠟切片經(jīng)HE染色觀察脂滴的數(shù)量;并提取RNA和總蛋白用Q-PCR以及Western blot檢測(cè)ND及HFD組小鼠肝臟組織中NAMPT、SREBP1及FASN的表達(dá);同時(shí)檢測(cè)HFD及HFD+FK866組小鼠肝臟組織中脂質(zhì)合成相關(guān)基因SREBP1、FASN、ACC等表達(dá)情況。2.肝細(xì)胞脂肪累積模型的制備及其分析:1)HepG2細(xì)胞在給予油酸刺激下用20 n M FK866或聯(lián)合NAD及NMN處理細(xì)胞24 h后,用油紅染色檢測(cè)細(xì)胞脂滴的堆積、甘油三酯試劑盒檢測(cè)細(xì)胞內(nèi)TG含量、并提取RNA和總蛋白用Q-PCR以及Western blot檢測(cè)脂質(zhì)合成相關(guān)基因的m RNA和蛋白的表達(dá)情況;2)在人源的Hep G2和鼠源的Hep1-6肝癌細(xì)胞中,用lipo3000轉(zhuǎn)染NAMPT質(zhì)粒(有兩種給藥方式:先轉(zhuǎn)染NAMPT質(zhì)粒再用油酸處理24h,第二種是先給油酸處理24h后再轉(zhuǎn)染質(zhì)粒NAMPT)。之后用油紅染色檢測(cè)細(xì)胞脂滴的堆積、甘油三酯試劑盒檢測(cè)細(xì)胞內(nèi)TG含量、并提取RNA和總蛋白用Q-PCR以及Western blot檢測(cè)脂質(zhì)合成相關(guān)基因的m RNA和蛋白的表達(dá)情況。3.機(jī)制分析:1)在動(dòng)物水平上利用Western blot檢測(cè)肝臟組織中Sirt1及AMPKα的蛋白水平;2)在細(xì)胞水平用Western blot檢測(cè)給FK866后肝細(xì)胞胰島素信號(hào)通路中Akt蛋白磷酸化水平;3)在細(xì)胞水平給予Sirt1的激動(dòng)劑Res和Sirt1的特異性抑制劑EX-527處理細(xì)胞24h,檢測(cè)脂質(zhì)合成相關(guān)基因的蛋白表達(dá)水平。實(shí)驗(yàn)結(jié)果:1.NAMPT在高脂喂養(yǎng)野生型小鼠肝臟中的表達(dá)顯著低于正常組,而脂質(zhì)合成相關(guān)基因SREBP1及FASN的表達(dá)則顯著高于正常組。HE染色顯示,HFD組肝臟中脂滴數(shù)量明顯多于ND組。2.NAMPT抑制劑FK866顯著增加高脂飲食誘導(dǎo)的小鼠肝臟脂質(zhì)堆積以及肝臟組織的脂質(zhì)合成基因SREBP1、FASN、ACC和SCD1的表達(dá)。3.FK866顯著增加正�；蛴退岽碳l件下的肝細(xì)胞脂質(zhì)堆積,NAD或NMN則可改善這一作用。FK866還顯著增加Hep G2細(xì)胞內(nèi)TG含量、脂質(zhì)合成基因SREBP1、FASN、ACC及SCD1的m RNA水平,同時(shí)增加FASN的蛋白表達(dá)水平,NAD或NMN則可部分逆轉(zhuǎn)這些作用。4.Hep G2和Hep1-6細(xì)胞系過(guò)表達(dá)NAMPT可顯著降低油酸誘導(dǎo)的脂質(zhì)堆積和TG增加,同時(shí)降低脂質(zhì)合成基因SREBP1、FASN、ACC的m RNA和蛋白水平。5.FK866可顯著降低HFD喂養(yǎng)小鼠的肝臟組織Sirt1及AMPKα磷酸化水平,以及降低肝細(xì)胞胰島素信號(hào)通路關(guān)鍵因子Akt的磷酸化。細(xì)胞實(shí)驗(yàn)顯示,FK866聯(lián)合Sirt1激動(dòng)劑Res可明顯抑制ACC表達(dá)以及油酸刺激條件下的FASN及ACC表達(dá),而給予Sirt1特異性抑制劑EX527則呈現(xiàn)相反作用。NMN可以恢復(fù)由FK866增加的FASN及ACC表達(dá)。結(jié)論:1.NAMPT在高脂喂養(yǎng)小鼠肝臟及油酸處理的肝細(xì)胞中表達(dá)下降。2.抑制NAMPT在體內(nèi)外均可增加肝臟脂質(zhì)合成,NAD及NMN具有改善作用;而過(guò)表達(dá)NAMPT則改善油酸誘導(dǎo)的肝脂質(zhì)合成。3.NAMPT通過(guò)激活Sirt1/SREBP1/AMPKα信號(hào)通路而抑制肝臟脂質(zhì)積累。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575
本文編號(hào)：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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
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
，本文編號(hào)：2229111
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