乙醇对肝脏固醇调节原件结合蛋白及其降解机制的研究
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摘要
背景:我国近年来由酒精所致的肝损害逐年上升,酒精已成为继病毒后导致肝损害的第二大病因。典型的酒精性肝病临床表现为脂肪肝、酒精性肝炎、酒精性肝纤维化,最终将发展为不可逆性肝硬化。因此酒精性肝病的防治已经成为重要的医疗课题。国内、外关于酒精性肝病的研究日益增多,但研究热点大多集中于酒精本身或其代谢产物对肝细胞的直接毒性作用以及营养失调、脂质代谢异常和脂质过氧化等变化在酒精性肝病发病学中的作用。肝脏脂质代谢紊乱是酒精性脂肪肝的发病基础,固醇调节元件结合蛋白(sterol regulatory element-bindingproteins,SREBPs)可以促进多种脂质合成相关酶类如乙酰CoA羧化酶(acetylCoA carboxylase,ACC)、脂肪酸合成酶(fatty acid synthase,FAS)的基因转录,从而在分子水平调节脂质代谢。SREBPs在酒精性脂肪肝发病机制中的作用尚不完全明确,其降解机制国内外未有报道。本实验通过建立大鼠酒精性脂肪肝模型,研究了SREBPs在大鼠酒精性脂肪肝中表达及乙醇对其降解机制,有望对酒精性脂肪肝的进一步研究及临床治疗开辟新的途径。
目的:通过建立大鼠酒精性脂肪肝模型,检测大鼠血清中甘油三酯(Triglycerides,TG)的含量,分析肝脏中SREBP-1c及其靶基因ACC的表达,并进一步探讨乙醇对SREBP-1C降解机制,测定Fbw7αmRNA、蛋白的含量,以及SREBP-1c的磷酸化及乙酰化水平。
方法:健康雄性成年Wistar大鼠30只,体重(180±10g),正常喂养1周后随机分为2组:正常对照组、酒精性脂肪肝模型组(各15只)。模型组给予酒精灌胃,正常对照组予等量生理盐水。12周末处死大鼠,GPO-PAP酶法测定血清TG含量,HE染色观察肝脏病理学改变,PCR或Western blot检测大鼠肝内ACC、SREBP-1c及Fbw7α的表达含量,免疫沉淀分析SREBP-1c的磷酸化及乙酰化水平,并将各项与血清內TG做相关性分析。
结果:酒精性脂肪肝大鼠模型组相比对照组,模型组肝脏出现明显脂肪变,TG含量明显升高,SREBP-1c及其靶基因ACC明显上调,Fbw7αmRNA及蛋白水平降低,SREBP-1c磷酸化水平明显低于对照组,但SREBP-1c的乙酰化水平比对照组明显升高。各项与TG做相关性分析,SREBP-1c、ACC、SREBP-1c的乙酰化水平均与TG含量呈正相关,Fbw7α、SREBP-1c磷酸化水平与TG呈负相关。
结论:乙醇增加SREBP-1c及其靶基因ACC含量,导致肝脏脂质合成增加,TG蓄积,脂肪肝形成。而且乙醇不仅降低Fbw7αmRNA及蛋白的表达,还通过影响SREBP-1c翻译后修饰如下调磷酸化,上调乙酰化,导致SREBP-1c与Fbw7相互作用减弱,从而使SREBP-1c降解障碍,增加SREBP-1c含量,形成酒精性脂肪肝。
Background:The incidence of alcoholic liver disease(ALD)in our country has increased markedly and alcohol becomes the second reason of hepatic lesion after virus hepatitis in these years.The classical clinical manifestations of ALD are alcoholic fatty liver(AFL), alcoholic hepatitis and alcoholic liver fibrosis,which even can become inconvertible alcoholic cirrhosis.So the prevention and cure of ALD become more and more important in medical topic.The study and research of ALD has increasingly increased in world,but most of them are concentrating on the direct toxic action to liver cell by alcohol itself or its metabolic product and the contribution by the changes of nutritional disturbance,liver metabolism abnormality and lipid peroxidation in ALD.Lipid metabolism disorder is the basis of AFL and is controled by sterol regulatory element-binding proteins(SREBPs)in molecular mechanism for the increasing of transcription of many enzymes related with lipid synthsis,such as acetyl CoA carboxylase(ACC)and fatty acid synthase(FAS).The mechanism of SREBPs in AFL has not been well known,and there is no report about the degradation of SREBPs all over the word.So,our investigation of the expression and the degradation of SREBPs in rat models of AFL is important and helpful for the deeper reseach on the AFL and the treatment of AFL.
Objective:By setting up rat models of alcoholic fatty liver,to investigate the changes of triglycerides、SREBP-1c and ACC,and to evaluate the expressions of Fbw7αmRNA and protein、the phosphorylation and acetylation of SREBP-1c which affect the degradation of SREBP-1c.
Methods:After one-week acclimatization period,30 male Wistar rats(180±10g)were divided into two groups:a normal control group and an alcoholic fatty liver model group(n=15).The rats in the model groups were gavaged by intragastric ethanol infusion,and the rats in the control group were treated with saline of the same volume.At the end of 12th week of the experiment,the rats were sacrificed.Liver histology was detected by HE staining and the parameters of triglyceride contents were measured by GPO-PAP assay.Samples of the liver were subjected to PCR or Western blot for the expressions of ACC、SREBP-1c、Fbw7αand immunoprecipitation studies for the phosphorylation and acetylation of SREBP-1c.
Results:Pathology observation by light microscope showed liver steatosis in model group.The expression of TG、SREBP-1c、ACC in model group were dramatic higher than control group.Alcohol induced a decrease of Fbw7αmRNA、protein and the phosphorylation of SREBP-1c in the model group,which negatively correlated with the TG content,while increased SREBP-1c and its acetylation which positively correlated with the TG content.
Conclusion:The higher SREBP-1c、ACC by the alcohol administration result in a increased fatty synthesis and TG to form the alcoholic fatty liver.And alcohol increased SREBP-1c not only by decreasing the Fbw7α,but also by decreasing the phosphorylation of SREBP-1c and increasing acetylation of SREBP-1c which cause the weaker interactions between SREBP-1c/Fbw7 and the poor degradation of SREBP-1c.
目的:通过建立大鼠酒精性脂肪肝模型,检测大鼠血清中甘油三酯(Triglycerides,TG)的含量,分析肝脏中SREBP-1c及其靶基因ACC的表达,并进一步探讨乙醇对SREBP-1C降解机制,测定Fbw7αmRNA、蛋白的含量,以及SREBP-1c的磷酸化及乙酰化水平。
方法:健康雄性成年Wistar大鼠30只,体重(180±10g),正常喂养1周后随机分为2组:正常对照组、酒精性脂肪肝模型组(各15只)。模型组给予酒精灌胃,正常对照组予等量生理盐水。12周末处死大鼠,GPO-PAP酶法测定血清TG含量,HE染色观察肝脏病理学改变,PCR或Western blot检测大鼠肝内ACC、SREBP-1c及Fbw7α的表达含量,免疫沉淀分析SREBP-1c的磷酸化及乙酰化水平,并将各项与血清內TG做相关性分析。
结果:酒精性脂肪肝大鼠模型组相比对照组,模型组肝脏出现明显脂肪变,TG含量明显升高,SREBP-1c及其靶基因ACC明显上调,Fbw7αmRNA及蛋白水平降低,SREBP-1c磷酸化水平明显低于对照组,但SREBP-1c的乙酰化水平比对照组明显升高。各项与TG做相关性分析,SREBP-1c、ACC、SREBP-1c的乙酰化水平均与TG含量呈正相关,Fbw7α、SREBP-1c磷酸化水平与TG呈负相关。
结论:乙醇增加SREBP-1c及其靶基因ACC含量,导致肝脏脂质合成增加,TG蓄积,脂肪肝形成。而且乙醇不仅降低Fbw7αmRNA及蛋白的表达,还通过影响SREBP-1c翻译后修饰如下调磷酸化,上调乙酰化,导致SREBP-1c与Fbw7相互作用减弱,从而使SREBP-1c降解障碍,增加SREBP-1c含量,形成酒精性脂肪肝。
Background:The incidence of alcoholic liver disease(ALD)in our country has increased markedly and alcohol becomes the second reason of hepatic lesion after virus hepatitis in these years.The classical clinical manifestations of ALD are alcoholic fatty liver(AFL), alcoholic hepatitis and alcoholic liver fibrosis,which even can become inconvertible alcoholic cirrhosis.So the prevention and cure of ALD become more and more important in medical topic.The study and research of ALD has increasingly increased in world,but most of them are concentrating on the direct toxic action to liver cell by alcohol itself or its metabolic product and the contribution by the changes of nutritional disturbance,liver metabolism abnormality and lipid peroxidation in ALD.Lipid metabolism disorder is the basis of AFL and is controled by sterol regulatory element-binding proteins(SREBPs)in molecular mechanism for the increasing of transcription of many enzymes related with lipid synthsis,such as acetyl CoA carboxylase(ACC)and fatty acid synthase(FAS).The mechanism of SREBPs in AFL has not been well known,and there is no report about the degradation of SREBPs all over the word.So,our investigation of the expression and the degradation of SREBPs in rat models of AFL is important and helpful for the deeper reseach on the AFL and the treatment of AFL.
Objective:By setting up rat models of alcoholic fatty liver,to investigate the changes of triglycerides、SREBP-1c and ACC,and to evaluate the expressions of Fbw7αmRNA and protein、the phosphorylation and acetylation of SREBP-1c which affect the degradation of SREBP-1c.
Methods:After one-week acclimatization period,30 male Wistar rats(180±10g)were divided into two groups:a normal control group and an alcoholic fatty liver model group(n=15).The rats in the model groups were gavaged by intragastric ethanol infusion,and the rats in the control group were treated with saline of the same volume.At the end of 12th week of the experiment,the rats were sacrificed.Liver histology was detected by HE staining and the parameters of triglyceride contents were measured by GPO-PAP assay.Samples of the liver were subjected to PCR or Western blot for the expressions of ACC、SREBP-1c、Fbw7αand immunoprecipitation studies for the phosphorylation and acetylation of SREBP-1c.
Results:Pathology observation by light microscope showed liver steatosis in model group.The expression of TG、SREBP-1c、ACC in model group were dramatic higher than control group.Alcohol induced a decrease of Fbw7αmRNA、protein and the phosphorylation of SREBP-1c in the model group,which negatively correlated with the TG content,while increased SREBP-1c and its acetylation which positively correlated with the TG content.
Conclusion:The higher SREBP-1c、ACC by the alcohol administration result in a increased fatty synthesis and TG to form the alcoholic fatty liver.And alcohol increased SREBP-1c not only by decreasing the Fbw7α,but also by decreasing the phosphorylation of SREBP-1c and increasing acetylation of SREBP-1c which cause the weaker interactions between SREBP-1c/Fbw7 and the poor degradation of SREBP-1c.
引文
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[2]Cortez-Pinto H,Marques-Vidal P,Monteiro E.Liver disease-related admissions in Portugal:clinical and dentographie pattern[J].Eur J Gastroenterol Hepatol,2004,16:873-877.
[3]O'shea RS,McCul Iough AJ.Treatment of alcoholic hepatitis[J].Clin Liver Dis.2005,9:103-134.
[4]庄辉.酒精性肝病的流行病学[J].中华肝脏病杂志,2003,11(11):689.
[5]厉有名,陈卫星,虞朝辉等。浙江省酒精性肝病流行病学调查概况[J],中华肝脏病杂志,2003,11(11):647-649
[6]Bellentani S,Saccoccio G,Costa G,et al.Drinking habits as cofactors of risk for alcohol induced liver damage[J].The Dionysos Study Group.Gut,1997,41:845-850.
[7]Brittenham GM.Iron chelators and iron toxicity[J].Alcohol,2003,30:151-158.
[8]Said A,Williams J,Holden J,et al.The prevalence of alcohol induced liver disease and hepatitis C and their interaction in a tertiary care setting[J].Clin Gastroenterol Hepatol,2004,2:928-934.
[9]Betaller R,North KE,Brenner DA.Genetic polymorphisms and the progression of liver fibrosis:a critical appraisal[J]Hepatology,2003,37:493-503.
[10]Higuchi S.Polymorphisms of ethanol metabolizing enzyme genes and alcoholism[J].Alcohol,1994,2:29-34.
[11]G rove J,Daly AK,Bassendine MF,et al.Association of a tumor Beerosis factar promoter polymorphism with susceptibility to alcoholic steatohepatitis[J].Hepatology,1997,26;143-146.
[12]Toraita K,Azuma T,Kitamura N,et al.Pioglitazone prevents alcohol induced fatty liver in rats through up-regulation of Met [J].Gastroenteralogy, 2004,126: 873-885.
[13] Aisch H, ollmann H, Bornkessel S. Degradation of apoptotic cells and fragments in HL260 suspension cultures after induction of apoptosis by camptothecin and ethanol[J]. Cell Prolif , 1999 , 32: 303-19.
[14] Oikari S, Ahtialansaari T, Heinonen MV,et al.Downregulation of PPARs and SREBP by acyl-CoA-binding protein overexpression in transgenic rats[J]. European journal of physiology, 2008 (456): 369-77;
[15] Tsukamoto H, She H, Hazra S,et al.Fat paradox of steatohepatitis[J]. Journal of gastroenterology and hepatology ,2008. (23): 104-107
[16] Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor [J]. Cell,1997 (89) :331-340.
[17] Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver [J]. J Clin Invest , 2002 (109 ):1125- 1131.
[18] Shimano H. Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid regulators of lipid synthetic genes [J]. Prog Lipid Res , 2001 (40) :439-452.
[19].Eberle D, Hegarty B, Bossard P,et al. SREBP transcription factors: master regulators of lipid homeostasis [J].Biochimie , 2004 ; 86(11): 839-848.
[20] McPherson R, Gauthier A. Molecular regulation of SREBP function : the Insig-SCAP connection and isoform-specific modulation of lipid sythesis[J]. Biochem Cell Biol , 2004(82) :201 - 211.
[21] Edwards PA, Tabor D, Kast HR, et al. Regulation of gene expression by SREBP and SCAP[J].Molecular and Cell Biology of Lipids, 2000, 1529 (1-3): 103-113。
[22] MatsudaM, Korn BS, Hammer RE, et all .SREBP cleavage Oactivating protein ( SCAP) is required for increased lipid synthesis in liver induced by cholesterol dep rivation and insulin elevation [ J ]. GenesDev, 2001, 15 (10) : 12061
[23] Horton, J. D., Goldstein, J. L., Brown, M. S. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver [J]. J Clin Invest 2002(109), 1125-1131.
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