硫辛酸活化SIRT1分子改善肝脏脂代谢的机制研究

发布时间：2018-04-30 05:31

本文选题：硫辛酸 + FoxO1　；参考：《山东大学》2014年博士论文

【摘要】：因能量过剩造成的肥胖及由肥胖引起的非酒精性脂肪肝(non-alcoholic fatty liver disease, NAFLD)日益成为全世界公众最关注的健康问题。NAFLD的主要发病机制至今尚未完全明确,目前许多理论都停留在假说阶段,其中以“二次打击假说”最具有说服力。该学说认为胰岛素抵抗(insulin resistance,IR)导致肝脏脂肪沉积成为NAFLD发病过程中的第一次打击,而在肝脏脂肪沉积基础上所发生的氧化应激和脂质过氧化损伤则形成第二次打击,第二次打击增加了肝细胞对凋亡和坏死的易感性,进一步促进肝纤维化和肝硬化的发生和发展,并最终导致NAFLD的发生。由此可见,肝脏脂质代谢稳态的变化是形成脂肪肝的基础。在内外环境因素作用下肝细胞对脂肪酸的摄取、合成多于氧化及分泌,可促使脂肪酸在肝内积聚。现阶段对NAFLD的临床治疗,主要是通过合理控制体重、给予胰岛素增敏剂、降血脂药物、抗氧化剂、肝脏保护剂等等。但是,但大样本的荟萃分析发现,上述大部分药物均不能有效缓解肝组织学炎症病变程度。 SIR (silence information regulator)基因家族是一种保守的NAD+(烟酰腺嘌呤二核苷酸)依赖的组蛋白/非组蛋白去乙酰基酶,该家族成员基因结构高度保守。沉默信息调节蛋白2(silencing information regulator2, SIRT2)为染色体的异源结构组分,具有独特的生物学性质和功能,因其具有将机体能量代谢、氧化状况与基因表达调控相耦联的重要作用而成为调控寿命基因研究的热点。沉默信息调节因子1(silencing information regulator1, SIRT1)是在哺乳动物细胞中发现的与SIRT2同源性最高的同系物。研究发现SIRT1不仅参与了细胞的能量代谢,而且还能通过各种途径改善肝脏的胰岛素敏感性、调节脂质代谢、减轻炎性反应和氧化应激损伤。因此,其已成为NAFLD治疗的新靶点及寻找新一代NAFLD防治新药的重要途径。 AMP-activated protein kinase (AMPK)是一种重要的能量代谢调节因子,主要生物学效应是通过感受胞浆内AMP/ATP比值的变化,或者受其上游的激酶LKB1、CaMKK (Ca2+/calmodulin-dependent protein kinase kinase)或TAK1(transforming growth factor-β-activated kinase-1)调节激活,进而影响细胞物质代谢的多个环节以维持细胞能量供求平衡。文献报告以白藜芦醇为代表的多酚类化合物能够增强SIRT1去乙酰基酶的活性,促进其下游LKB1和AMPK-p的表达与活化并通过SIRT1/LKB1/AMPK通路进行脂质代谢调节。活化后的SIRT1/LKB1/AMPK通路主要是利用去乙酰化或磷酸化作用修饰转录因子p53,FoxO, NF-kB, PGC-α以及SREBP-1,加速脂肪酸氧化、抑制脂肪酸的从头合成过程从而在肝脏脂代谢过程中发挥核心调节作用。 nuclear factor E2-related factor2(Nrf2)在阻止肝脏氧化应激及调节肝脏代谢、脂肪肝方面有极大的保护作用。研究表明,SIRT1与AMPK及Nrf2可能存在着内在联系,从而在调节能量代谢过程尤其是脂代谢过程中起着重要的调控作用。硫辛酸(Alpha-lipoic acid, ALA)是一种内源性的强抗氧化剂,以辅酶的形式参与机体能量代谢。本研究通过体内、体外两部分实验验证ALA对肝脏内SIRT1表达及活性的直接调节作用；通过SIRT1的siRNA及化学激动剂、抑制剂实验,证实ALA活化SIRT1/AMPK、SIRT1/Nrf2通路调节肝脏脂质代谢,改善NAFLD。希望通过上述研究明确ALA对肝脏脂代谢调节的基本机制,为拓展和深化ALA的临床用药提供理论基础。研究方法：本研究首先采用不同剂量ALA处理人肝癌细胞株HepG2细胞,通过NAD+/NADH比值测定及SIRT1去乙酰化酶活性测定初步判断ALA对SIRT1分子表达及活性调节的可能机制。ALA处理HepG2细胞后应用免疫共沉淀方法明确SIRT1与LKB1分子的相互作用。运用蛋白免疫印迹实验证实ALA对AMPK、ACC的磷酸化表达调节作用。利用SIRT1化学抑制剂(NA),AMPK化学抑制剂(CC)以及AMPK化学激动剂(AICAR)孵育实验,验证SIRT1对AMPK、ACC的直接调节作用以及对肝细胞内甘油三酯合成、储存的影响。运用RNAi技术确定SIRT1对肝细胞内甘油三酯合成、分解途径限速酶FAS、ATGL表达的直接调节作用。 ALA处理HepG2细胞,同时分别给予NA、CC、AICAR孵育,运用免疫荧光技术检测FoxO1、SREBP-1的表达及核迁移情况。ALA处理被SIRT1化学抑制剂NA孵育的HepG2细胞,运用蛋白免疫印迹法检测SIRT1、核内Nrf2、抗氧化酶SOD、Catalase、HO-1的表达变化。进而运用RNAi技术抑制SIRT1的细胞内表达,再次检测上述蛋白质的表达变化。运用高脂饮食持续喂养C57BL/6J小鼠建立NAFLD模型,检测小鼠体重、血糖、血脂水平变化。通过摄食量、体重以及肝脏内甘油三酯含量测定,显示各组小鼠肝脏脂代谢基本状况。利用组织学手段确定肝脏组织内脂滴堆积情况。运用蛋白免疫印迹方法检测SIRT1/AMPK下游脂代谢相关信号分子的表达变化。运用超氧化阴离子荧光免疫探针DHE进行各组小鼠肝组织内活性氧含量测定。研究结果： 1.ALA对HepG2细胞内SIRT1活性有显著激活作用,这种作用有明显的剂量效应关系,且与NAD+/NADH比值增加有相关性。免疫共沉淀实验证实,ALA对AMPK的活化作用依赖SIRT1/LKB1的互作效应。化学激动剂、抑制剂实验以及SIRT1RNAi实验证实,ALA对肝细胞内脂代谢的调节效应依赖SIRT1/LKB1/AMPK信号通路。 2.SIRT1、AMPK的化学激动剂、抑制剂孵育HepG2细胞并辅以ALA处理,运用免疫荧光实验证实ALA减弱了胞浆内p-FoxO1表达,增强了甘油三酯脂肪酶ATGL的表达。同样的处理条件显著增强了胞浆内p-SREBP-1的表达,抑制了脂肪酸合成酶FAS的表达。 3.SIRT1化学抑制剂NA孵育HepG2细胞辅以ALA处理,蛋白免疫印迹实验显示SIRT1、核内Nrf2、以及抗氧化酶SOD、Catalase、HO-1的表达显著下降,SIRT1siRNA实验结果与之吻合。ALA长期处理对于高脂持续喂养诱导的NAFLD小鼠脂肪重量有显著降低作用,同时对于体重和摄食量也体现明显抑制效应(P0.05)。各组小鼠肝脏重量没有明显差异,但是ALA处理组的肝内甘油三酯含量较高脂模型组明显降低(P0.05),且油红O染色显示,该组小鼠肝细胞内脂滴积存现象显著改善。ALA处理组血糖、血脂指标都显著优于高脂诱导组(P0.05)。高脂喂养并辅以ALA处理的C57BL/6J小鼠,肝脏组织内ROS含量少于高脂组小鼠。蛋白免疫印迹实验显示,ALA处理明显增加小鼠肝脏内SIRT1、p-AMPK、p-ACC、 p-SREBP-1、ATGL的表达,并伴随p-FoxO1、FAS的表达下调。研究结论： 1.ALA对肝细胞内脂代谢的调节作用通过SIRT1/LKB1/AMPK通路的活化加以实现,其下游靶分子可为FoxO1/ATGL以及SREBP-1/FAS。 2.ALA可通过增强SIRT1与Nrf2的相互作用,活化Nrf2/ARE通路,调节肝脏内氧化还原体系平衡,进而改善肝细胞内脂代谢。
[Abstract]:Obesity caused by excess energy and non-alcoholic fatty liver disease (NAFLD), which are caused by obesity, are increasingly becoming the major health problems of the public all over the world. The main pathogenesis of.NAFLD is not completely clear. Many theories are currently in the hypothesis stage, among which the "two attack hypothesis" is the most important. It is persuasive. It is believed that insulin resistance (insulin resistance, IR) causes liver fat deposition to be the first attack in the pathogenesis of NAFLD, while oxidative stress and lipid peroxidation damage on the basis of liver fat deposition have formed second strikes, and second strikes increase the susceptibility to apoptosis and necrosis of liver cells. Sensibilities, further promote the occurrence and development of liver fibrosis and cirrhosis, and eventually lead to the occurrence of NAFLD. This shows that the changes in the homeostasis of lipid metabolism are the basis for the formation of fatty liver. In the presence of internal and external environmental factors, the uptake of fatty acids in liver cells is more than oxygenation and secretion, which can induce fatty acids to accumulate in the liver. The clinical treatment of NAFLD is mainly through reasonable weight control, insulin sensitizer, blood lipid drug, anti oxidant, liver protective agent and so on. However, large sample meta-analysis shows that most of these drugs can not effectively alleviate the degree of liver histologic inflammation.
The SIR (silence information regulator) gene family is a conservative NAD+ (nicotinyl adenine dinucleotide) dependent histone / non histone deacetylase. The gene structure of the family members is highly conserved. The silent information regulating protein 2 (silencing information regulator2, SIRT2) is a heterologous structural component of the chromosome, which is unique. Its biological properties and functions have become the hot spots in the study of the regulation of life genes because of their important role in coupling the energy metabolism of the body and the regulation of gene expression and regulation. The silent information regulator 1 (silencing information regulator1, SIRT1) is the most homologous to the homology of SIRT2 found in mammalian cells. It has been found that SIRT1 not only participates in the energy metabolism of cells, but also improves the insulin sensitivity of the liver, regulates lipid metabolism, reduces inflammatory response and oxidative stress damage. Therefore, it has become a new target for the treatment of NAFLD and an important way to find new generation of NAFLD to prevent and cure new drugs.
AMP-activated protein kinase (AMPK) is an important regulator of energy metabolism, and its main biological effect is regulated by the changes in the AMP/ATP ratio within the cytoplasm, or by the upstream kinase LKB1, CaMKK (Ca2+/calmodulin-dependent protein kinase kinase) or TAK1. In the literature report, the polyphenols represented by resveratrol can enhance the activity of SIRT1 deacetylase, promote the expression and activation of the downstream LKB1 and AMPK-p, and regulate the lipid metabolism through the SIRT1/LKB1/ AMPK pathway. The activated SIRT The 1/LKB1/AMPK pathway mainly modifies the transcription factor p53, FoxO, NF-kB, PGC- alpha and SREBP-1 by deacetylation or phosphorylation. It accelerates fatty acid oxidation and inhibits the initio synthesis of fatty acids and plays a core regulatory role in liver lipid metabolism.
Nuclear factor E2-related FACTOR2 (Nrf2) has a great protective effect on preventing liver oxidative stress and regulating liver metabolism and fatty liver. The study shows that SIRT1 may have an intrinsic relationship with AMPK and Nrf2, which plays an important role in regulating energy metabolism, especially in lipid metabolism. Acid, ALA) is a kind of endogenous strong antioxidant that participates in energy metabolism in the form of coenzyme. In this study, two experiments were conducted to verify the direct regulation of ALA on the expression and activity of SIRT1 in the liver. Through SIRT1 siRNA and chemical agonists, inhibitor experiments confirmed ALA activation SIRT1/AMPK, SIRT1/Nrf2 pathway modulation. The lipid metabolism of the liver and the improvement of NAFLD. hope to clarify the basic mechanism of ALA regulation on liver lipid metabolism through these studies, and provide a theoretical basis for expanding and deepening the clinical use of ALA.
Research methods:
This study first used different doses of ALA to treat human hepatocellular carcinoma cell line HepG2 cells, the determination of NAD+/NADH ratio and the activity of SIRT1 deacetylase were used to determine the possible mechanism of ALA on the expression and activity of SIRT1 molecules..ALA treated HepG2 cells after.ALA treatment, and the interaction between SIRT1 and LKB1 molecules was confirmed by immunoprecipitation. The effect of ALA on the phosphorylation of AMPK and ACC was confirmed by white Western blot. Using SIRT1 chemical inhibitor (NA), AMPK chemical inhibitor (CC) and AMPK chemical agonist (AICAR) incubation experiment, the effect of SIRT1 on AMPK, the direct regulation of ACC and the effects on the synthesis and storage of triglyceride in the liver cells were verified. It directly regulates triglyceride synthesis and expression of FAS and ATGL in hepatocyte.
ALA treated HepG2 cells, incubated with NA, CC, AICAR, and detected FoxO1, SREBP-1 expression and nuclear migration by immunofluorescence..ALA treated HepG2 cells incubated by SIRT1 chemical inhibitor NA. The expression of SIRT1 in cell was inhibited and the expression changes of the above protein were detected again. The NAFLD model was established by feeding C57BL/6J mice continuously with high fat diet, and the changes of body weight, blood sugar and blood lipid were detected in mice. The basic status of liver lipid metabolism in each group was revealed by the intake of food, weight and the content of triglyceride in the liver. The accumulation of lipid droplets in the liver tissues was determined by histological method. The expression of lipid metabolism related signal molecules in the lower SIRT1/AMPK was detected by the method of protein immunoblotting. The content of active oxygen in the liver tissues of each group was measured by the superoxide anion immunofluorescence probe DHE.
The results of the study:
The activation of SIRT1 in HepG2 cells was significantly activated by 1.ALA, which had a significant dose effect relationship and was correlated with the increase in the ratio of NAD+/NADH. The immunoprecipitation experiment confirmed that the activation of ALA on AMPK depended on the interaction effect of SIRT1/LKB1. Chemical agonists, inhibitory agents and SIRT1RNAi experiments confirmed that ALA was in the liver cells. The regulatory effect of lipid metabolism depends on the SIRT1/LKB1/AMPK signaling pathway.
2.SIRT1, AMPK chemical agonist, the inhibitor incubated HepG2 cells and treated with ALA. The immunofluorescence test showed that ALA weakened the p-FoxO1 expression in the cytoplasm and enhanced the expression of triglyceride lipase ATGL. The same treatment conditions significantly enhanced the expression of p-SREBP-1 in the cytoplasm and inhibited the expression of the fatty acid synthase FAS.
3.SIRT1 chemical inhibitor NA incubated HepG2 cells and treated with ALA. The protein immunoblotting experiment showed that the expression of Nrf2 in the nucleus and the expression of SOD, Catalase and HO-1 decreased significantly in the nucleus, and the SIRT1siRNA experimental results coincided with the.ALA long-term treatment for the fat weight of NAFLD mice induced by high fat continuous feeding, and at the same time, The weight and feeding amount also showed obvious inhibitory effect (P0.05). There was no significant difference in liver weight in each group, but the content of triglyceride in the liver of the ALA treatment group was significantly lower (P0.05), and the accumulation of lipid droplets in the liver cells of the group showed that the lipid droplet accumulation in the liver cells of the group significantly improved the blood sugar of the.ALA treatment group, and the blood lipid indexes were all significant. It was better than high fat induction group (P0.05). The ROS content in the liver tissue was less than that of the high fat group of C57BL/6J mice treated with high fat feeding and supplemented with ALA. The protein immunoblotting experiment showed that ALA treatment significantly increased the expression of SIRT1, p-AMPK, p-ACC, p-SREBP-1, ATGL in the liver of mice, along with p-FoxO1, the expression of FAS was down.
The conclusions are as follows:
1.ALA regulates lipid metabolism in liver cells through activation of SIRT1/LKB1/AMPK pathway, and its downstream target molecules can be FoxO1/ATGL and SREBP-1/FAS..
2.ALA can increase the interaction between SIRT1 and Nrf2, activate Nrf2/ARE pathway, regulate the balance of redox system in liver, and then improve lipid metabolism in liver cells.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575

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