發(fā)布時(shí)間：2018-04-30 05:31

  本文選題：硫辛酸 + FoxO1��； 參考：《山東大學(xué)》2014年博士論文

【摘要】：因能量過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)日益成為全世界公眾最關(guān)注的健康問題。NAFLD的主要發(fā)病機(jī)制至今尚未完全明確,目前許多理論都停留在假說階段,其中以“二次打擊假說”最具有說服力。該學(xué)說認(rèn)為胰島素抵抗(insulin resistance,IR)導(dǎo)致肝臟脂肪沉積成為NAFLD發(fā)病過程中的第一次打擊,而在肝臟脂肪沉積基礎(chǔ)上所發(fā)生的氧化應(yīng)激和脂質(zhì)過氧化損傷則形成第二次打擊,第二次打擊增加了肝細(xì)胞對凋亡和壞死的易感性,進(jìn)一步促進(jìn)肝纖維化和肝硬化的發(fā)生和發(fā)展,并最終導(dǎo)致NAFLD的發(fā)生。由此可見,肝臟脂質(zhì)代謝穩(wěn)態(tài)的變化是形成脂肪肝的基礎(chǔ)。在內(nèi)外環(huán)境因素作用下肝細(xì)胞對脂肪酸的攝取、合成多于氧化及分泌,可促使脂肪酸在肝內(nèi)積聚�，F(xiàn)階段對NAFLD的臨床治療,主要是通過合理控制體重、給予胰島素增敏劑、降血脂藥物、抗氧化劑、肝臟保護(hù)劑等等。但是,但大樣本的薈萃分析發(fā)現(xiàn),上述大部分藥物均不能有效緩解肝組織學(xué)炎癥病變程度。 SIR (silence information regulator)基因家族是一種保守的NAD+(煙酰腺嘌呤二核苷酸)依賴的組蛋白/非組蛋白去乙�；�,該家族成員基因結(jié)構(gòu)高度保守。沉默信息調(diào)節(jié)蛋白2(silencing information regulator2, SIRT2)為染色體的異源結(jié)構(gòu)組分,具有獨(dú)特的生物學(xué)性質(zhì)和功能,因其具有將機(jī)體能量代謝、氧化狀況與基因表達(dá)調(diào)控相耦聯(lián)的重要作用而成為調(diào)控壽命基因研究的熱點(diǎn)。沉默信息調(diào)節(jié)因子1(silencing information regulator1, SIRT1)是在哺乳動(dòng)物細(xì)胞中發(fā)現(xiàn)的與SIRT2同源性最高的同系物。研究發(fā)現(xiàn)SIRT1不僅參與了細(xì)胞的能量代謝,而且還能通過各種途徑改善肝臟的胰島素敏感性、調(diào)節(jié)脂質(zhì)代謝、減輕炎性反應(yīng)和氧化應(yīng)激損傷。因此,其已成為NAFLD治療的新靶點(diǎn)及尋找新一代NAFLD防治新藥的重要途徑。 AMP-activated protein kinase (AMPK)是一種重要的能量代謝調(diào)節(jié)因子,主要生物學(xué)效應(yīng)是通過感受胞漿內(nèi)AMP/ATP比值的變化,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)調(diào)節(jié)激活,進(jìn)而影響細(xì)胞物質(zhì)代謝的多個(gè)環(huán)節(jié)以維持細(xì)胞能量供求平衡。文獻(xiàn)報(bào)告以白藜蘆醇為代表的多酚類化合物能夠增強(qiáng)SIRT1去乙�；傅幕钚�,促進(jìn)其下游LKB1和AMPK-p的表達(dá)與活化并通過SIRT1/LKB1/AMPK通路進(jìn)行脂質(zhì)代謝調(diào)節(jié)�；罨蟮腟IRT1/LKB1/AMPK通路主要是利用去乙�；蛄姿峄饔眯揎椶D(zhuǎn)錄因子p53,FoxO, NF-kB, PGC-α以及SREBP-1,加速脂肪酸氧化、抑制脂肪酸的從頭合成過程從而在肝臟脂代謝過程中發(fā)揮核心調(diào)節(jié)作用。 nuclear factor E2-related factor2(Nrf2)在阻止肝臟氧化應(yīng)激及調(diào)節(jié)肝臟代謝、脂肪肝方面有極大的保護(hù)作用。研究表明,SIRT1與AMPK及Nrf2可能存在著內(nèi)在聯(lián)系,從而在調(diào)節(jié)能量代謝過程尤其是脂代謝過程中起著重要的調(diào)控作用。硫辛酸(Alpha-lipoic acid, ALA)是一種內(nèi)源性的強(qiáng)抗氧化劑,以輔酶的形式參與機(jī)體能量代謝。本研究通過體內(nèi)、體外兩部分實(shí)驗(yàn)驗(yàn)證ALA對肝臟內(nèi)SIRT1表達(dá)及活性的直接調(diào)節(jié)作用；通過SIRT1的siRNA及化學(xué)激動(dòng)劑、抑制劑實(shí)驗(yàn),證實(shí)ALA活化SIRT1/AMPK、SIRT1/Nrf2通路調(diào)節(jié)肝臟脂質(zhì)代謝,改善NAFLD。希望通過上述研究明確ALA對肝臟脂代謝調(diào)節(jié)的基本機(jī)制,為拓展和深化ALA的臨床用藥提供理論基礎(chǔ)。 研究方法： 本研究首先采用不同劑量ALA處理人肝癌細(xì)胞株HepG2細(xì)胞,通過NAD+/NADH比值測定及SIRT1去乙�；富钚詼y定初步判斷ALA對SIRT1分子表達(dá)及活性調(diào)節(jié)的可能機(jī)制。ALA處理HepG2細(xì)胞后應(yīng)用免疫共沉淀方法明確SIRT1與LKB1分子的相互作用。運(yùn)用蛋白免疫印跡實(shí)驗(yàn)證實(shí)ALA對AMPK、ACC的磷酸化表達(dá)調(diào)節(jié)作用。利用SIRT1化學(xué)抑制劑(NA),AMPK化學(xué)抑制劑(CC)以及AMPK化學(xué)激動(dòng)劑(AICAR)孵育實(shí)驗(yàn),驗(yàn)證SIRT1對AMPK、ACC的直接調(diào)節(jié)作用以及對肝細(xì)胞內(nèi)甘油三酯合成、儲(chǔ)存的影響。運(yùn)用RNAi技術(shù)確定SIRT1對肝細(xì)胞內(nèi)甘油三酯合成、分解途徑限速酶FAS、ATGL表達(dá)的直接調(diào)節(jié)作用。 ALA處理HepG2細(xì)胞,同時(shí)分別給予NA、CC、AICAR孵育,運(yùn)用免疫熒光技術(shù)檢測FoxO1、SREBP-1的表達(dá)及核遷移情況。ALA處理被SIRT1化學(xué)抑制劑NA孵育的HepG2細(xì)胞,運(yùn)用蛋白免疫印跡法檢測SIRT1、核內(nèi)Nrf2、抗氧化酶SOD、Catalase、HO-1的表達(dá)變化。進(jìn)而運(yùn)用RNAi技術(shù)抑制SIRT1的細(xì)胞內(nèi)表達(dá),再次檢測上述蛋白質(zhì)的表達(dá)變化。運(yùn)用高脂飲食持續(xù)喂養(yǎng)C57BL/6J小鼠建立NAFLD模型,檢測小鼠體重、血糖、血脂水平變化。通過攝食量、體重以及肝臟內(nèi)甘油三酯含量測定,顯示各組小鼠肝臟脂代謝基本狀況。利用組織學(xué)手段確定肝臟組織內(nèi)脂滴堆積情況。運(yùn)用蛋白免疫印跡方法檢測SIRT1/AMPK下游脂代謝相關(guān)信號分子的表達(dá)變化。運(yùn)用超氧化陰離子熒光免疫探針DHE進(jìn)行各組小鼠肝組織內(nèi)活性氧含量測定。 研究結(jié)果： 1.ALA對HepG2細(xì)胞內(nèi)SIRT1活性有顯著激活作用,這種作用有明顯的劑量效應(yīng)關(guān)系,且與NAD+/NADH比值增加有相關(guān)性。免疫共沉淀實(shí)驗(yàn)證實(shí),ALA對AMPK的活化作用依賴SIRT1/LKB1的互作效應(yīng)�；瘜W(xué)激動(dòng)劑、抑制劑實(shí)驗(yàn)以及SIRT1RNAi實(shí)驗(yàn)證實(shí),ALA對肝細(xì)胞內(nèi)脂代謝的調(diào)節(jié)效應(yīng)依賴SIRT1/LKB1/AMPK信號通路。 2.SIRT1、AMPK的化學(xué)激動(dòng)劑、抑制劑孵育HepG2細(xì)胞并輔以ALA處理,運(yùn)用免疫熒光實(shí)驗(yàn)證實(shí)ALA減弱了胞漿內(nèi)p-FoxO1表達(dá),增強(qiáng)了甘油三酯脂肪酶ATGL的表達(dá)。同樣的處理?xiàng)l件顯著增強(qiáng)了胞漿內(nèi)p-SREBP-1的表達(dá),抑制了脂肪酸合成酶FAS的表達(dá)。 3.SIRT1化學(xué)抑制劑NA孵育HepG2細(xì)胞輔以ALA處理,蛋白免疫印跡實(shí)驗(yàn)顯示SIRT1、核內(nèi)Nrf2、以及抗氧化酶SOD、Catalase、HO-1的表達(dá)顯著下降,SIRT1siRNA實(shí)驗(yàn)結(jié)果與之吻合。ALA長期處理對于高脂持續(xù)喂養(yǎng)誘導(dǎo)的NAFLD小鼠脂肪重量有顯著降低作用,同時(shí)對于體重和攝食量也體現(xiàn)明顯抑制效應(yīng)(P0.05)。各組小鼠肝臟重量沒有明顯差異,但是ALA處理組的肝內(nèi)甘油三酯含量較高脂模型組明顯降低(P0.05),且油紅O染色顯示,該組小鼠肝細(xì)胞內(nèi)脂滴積存現(xiàn)象顯著改善。ALA處理組血糖、血脂指標(biāo)都顯著優(yōu)于高脂誘導(dǎo)組(P0.05)。高脂喂養(yǎng)并輔以ALA處理的C57BL/6J小鼠,肝臟組織內(nèi)ROS含量少于高脂組小鼠。蛋白免疫印跡實(shí)驗(yàn)顯示,ALA處理明顯增加小鼠肝臟內(nèi)SIRT1、p-AMPK、p-ACC、 p-SREBP-1、ATGL的表達(dá),并伴隨p-FoxO1、FAS的表達(dá)下調(diào)。 研究結(jié)論： 1.ALA對肝細(xì)胞內(nèi)脂代謝的調(diào)節(jié)作用通過SIRT1/LKB1/AMPK通路的活化加以實(shí)現(xiàn),其下游靶分子可為FoxO1/ATGL以及SREBP-1/FAS。 2.ALA可通過增強(qiáng)SIRT1與Nrf2的相互作用,活化Nrf2/ARE通路,調(diào)節(jié)肝臟內(nèi)氧化還原體系平衡,進(jìn)而改善肝細(xì)胞內(nèi)脂代謝。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R575

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