SIRT1在非酒精性脂肪肝病大鼠肝脏中的表达及姜黄素衍生物L6H4的干预作用
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摘要
目的:探讨SIRT1在非酒精性脂肪肝病(NAFLD)模型大鼠肝脏中的表达改变及姜黄素衍生物L6H4干预作用。方法:雄性SD大鼠24只,随机均分为正常组(control组)、模型组(NAFLD组)和治疗组(L6H4组)。control组给予基础饲料,其余2组全程给予高脂饲料,并从第9周开始用姜黄素衍生物L6H4干预治疗,于第20周末处死各组大鼠。取全血检测各组大鼠的TG、TC、LDL-C及HDL-C水平;取肝组织分别用于HE及Masson染色观察大鼠肝组织病理学改变;羟胺法测定T-SOD活性及硫代巴比妥酸法测定MDA含量;RT-PCR及Westernblot检测大鼠肝脏组织SIRT1、MFN2及DRP1m RNA和蛋白表达情况。结果:光镜下,NAFLD组大鼠肝组织可见明显脂肪变性及纤维化,L6H4组肝组织病理变化较NAFLD组明显减轻。与control组比,NAFLD组大鼠血清中TG、TC和LDL-C浓度及肝组织中MDA含量和DRP1的表达量均明显增加,而血清HDL-C浓度及肝组织中T-SOD活性,SIRT1和MFN2的表达量均显著降低,差异均具有统计学意义(P<0.05);与NAFLD组相比,L6H4组大鼠血清中TG、TC和LDL-C的浓度及肝组织中MDA含量,DRP1的表达量均显著降低,而血清HDL-C浓度及肝组织中T-SOD活性,SIRT1和MFN2的表达量均明显升高,差异均具有统计学意义(P<0.05)。结论:姜黄素衍生物L6H4对NAFLD具有保护作用,其机制可能是通过调高SIRT1的表达,从而减弱NAFLD中肝细胞对胰岛素的抵抗和脂质过氧化反应,减轻氧化应激损伤、抑制线粒体分裂。
Objective:To explore the expression changes of SIRT1 in rat liver with high-fat induced nonalcoholic fatty liver disease(NAFLD),and the effects of Curcumin derivatives L6H4 on it.Methods:Twentyfour male SD rats were randomly divided into normal control group(control group),model group(NAFLD group) and L6H4 treatment group(L6H4 group).Rats in the control group were fed with normal-diet while rats in NAFLD group and L6H4 group were fed with high-fat diet.At the same time,rats in L6H4 group were treated with Curcumin derivatives L6H4 from the 9~(th)week till the 20~(th) week of the experiment.At the end of the 20~(th) week,all rats were sacrificed.The serum triglyceride,total cholesterol,low-density lipoprotein cholesterol and high-density lipoprotein cholesterol levels were detected by biochemical method respectively.The pathological changes of liver tissues were observed by HE staining and Masson's trichrome staining.The total SOD activity was detected by hydroxylamine method while the content of MDA was analyzed by thiobarbituric acid method.The expressions of SIRT1,DRP1 and MFN2 m RNA and protein in liver tissues were detected by real-time PCR and Western blot respectively.Results:Obvious steatosis and fibrosis were observed in NAFLD group under microscope.The pathological changes were significantly reduced in L6H4 group compared with NAFLD group.Compared with control group,the serum triglyceride,total cholesterol,low-density lipoprotein cholesterol levels,the content of MDA and the expression of DRP1 m RNA and protein were increased significantly,while the serum high-density lipoprotein cholesterol level,the total SOD activity and the expressions of SIRT1 and MFN2 m RNA and protein were decreased significantly(P<0.05) in NAFLD group.Compared with NAFLD group,the serum triglyceride,total cholesterol,low-density lipoprotein cholesterol levels,the content of MDA and the expression of DRP1 m RNA and protein were decreased significantly,while the serum high-density lipoprotein cholesterol level,the total SOD activity and the expressions of SIRT1 and MFN2 m RNA and protein were increased significantly(P<0.05) in L6H4 group.Conclusion:Curcumin derivatives L6H4 has a protective effect on NAFLD,and its mechanism may be through increasing the expression of SIRT1,thereby reducing the resistance of hepatocyte to insulin and lipid peroxidation,attenuating oxidative stress damage and inhibiting mitochondrial fission.
Objective:To explore the expression changes of SIRT1 in rat liver with high-fat induced nonalcoholic fatty liver disease(NAFLD),and the effects of Curcumin derivatives L6H4 on it.Methods:Twentyfour male SD rats were randomly divided into normal control group(control group),model group(NAFLD group) and L6H4 treatment group(L6H4 group).Rats in the control group were fed with normal-diet while rats in NAFLD group and L6H4 group were fed with high-fat diet.At the same time,rats in L6H4 group were treated with Curcumin derivatives L6H4 from the 9~(th)week till the 20~(th) week of the experiment.At the end of the 20~(th) week,all rats were sacrificed.The serum triglyceride,total cholesterol,low-density lipoprotein cholesterol and high-density lipoprotein cholesterol levels were detected by biochemical method respectively.The pathological changes of liver tissues were observed by HE staining and Masson's trichrome staining.The total SOD activity was detected by hydroxylamine method while the content of MDA was analyzed by thiobarbituric acid method.The expressions of SIRT1,DRP1 and MFN2 m RNA and protein in liver tissues were detected by real-time PCR and Western blot respectively.Results:Obvious steatosis and fibrosis were observed in NAFLD group under microscope.The pathological changes were significantly reduced in L6H4 group compared with NAFLD group.Compared with control group,the serum triglyceride,total cholesterol,low-density lipoprotein cholesterol levels,the content of MDA and the expression of DRP1 m RNA and protein were increased significantly,while the serum high-density lipoprotein cholesterol level,the total SOD activity and the expressions of SIRT1 and MFN2 m RNA and protein were decreased significantly(P<0.05) in NAFLD group.Compared with NAFLD group,the serum triglyceride,total cholesterol,low-density lipoprotein cholesterol levels,the content of MDA and the expression of DRP1 m RNA and protein were decreased significantly,while the serum high-density lipoprotein cholesterol level,the total SOD activity and the expressions of SIRT1 and MFN2 m RNA and protein were increased significantly(P<0.05) in L6H4 group.Conclusion:Curcumin derivatives L6H4 has a protective effect on NAFLD,and its mechanism may be through increasing the expression of SIRT1,thereby reducing the resistance of hepatocyte to insulin and lipid peroxidation,attenuating oxidative stress damage and inhibiting mitochondrial fission.
引文
[1]CHALASANI N,YOUNOSSI Z,LAVINE J E,et al.The diagnosis and management of non-alcoholic fatty liver disease:Practice Guideline by the American Association for the Study of Liver Diseases,American College of Gastroenterology,and the American Gastroenterological Association[J].Hepatology,2012,107(6):811-826.
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[8]TOBITA T,GUAMAN-LEPE J,TAKEISHI K,et al.SIRT1disruption in human fetal hepatocytes leads to increased accumulation of glucose and lipids[J].PLo S One,2016,11(2):e0149344.
[9]COLAK Y,OZTURK O,SENATES E,et al.SIRT1 as a potential therapeutic target for treatment of nonalcoholic fatty liver disease[J].Med Sci Monitor,2011,17(5):HY5-HY9.
[10]ROLO A P,TEODORO J S,PALMEIRA C M.Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis[J].Free Radic Biol Med,2011,52(1):59-69.
[11]DING R B,BAO J,DENG C X.Emerging roles of SIRT1 in fatty liver diseases[J].Int J Biol Sci,2017,13(7):852-867.
[12]BEREITER-HAHN J,V?TH M.Dynamics of mitochondria in living cells:Shape changes,dislocations,fusion,and fission of mitochondria[J].Microsc Res Tech,1994,27(3):198-219.
[13]SUEN D F,NORRIS K L,YOULE R J.Mitochondrial dynamics and apoptosis[J].Genes Dev,2008,22(12):1577-1599.
[14]郑靖宇,吴欢,汤雯,等.Drp1和Mfn2在棕榈酸诱导大鼠肝细胞损伤中的作用机制及姜黄素衍生物L6H4的干预作用[J].温州医科大学学报,2016,46(7):469-475.
[15]VALERO T.Mitochondrial biogenesis:pharmacological approaches[J].Curr Pharm Design,2014,20(35):5507-5509.
[16]罗桂平,蹇朝,张华刚,等.Sirt1在缺氧条件下对心肌细胞线粒体融合与分裂中的作用[J].第三军医大学学报,2016,38(10):1121-1126.
[17]BIEL T G,LEE S,FLORES-TORO J A,et al.Sirtuin 1 suppresses mitochondrial dysfunction of ischemic mouse livers in a mitofusin 2-dependent manner[J].Cell Death Differ,2016,23(2):279-290.
[18]MIAO Y,ZHAO S,GAO Y,et al.Curcumin pretreatment attenuates inflammation and mitochondrial dysfunction in experimental stroke:The possible role of Sirt1 signaling[J].Brain Res Bull,2016,121:9-15.
[2]WANG L,LIU X,NIE J,et al.ALCAT1 controls mitochondrial etiology of fatty liver diseases,linking defective mitophagy to steatosis[J].Hepatology,2015,61(2):486-496.
[3]GAMBINO R,MUSSO G,CASSADER M.Redox balance in the pathogenesis of nonalcoholic fatty liver disease:mechanisms and therapeutic opportunities[J].Antioxid Redox Signal,2011,15(5):1325-1365.
[4]REN T,HUANG C,CHENG M.Dietary blueberry and bifidobacteria attenuate nonalcoholic fatty liver disease in rats by affecting SIRT1-mediated signaling pathway[J].Oxid Med Cell Longev,2014,2014:469059.
[5]王玲,徐梦菲,刘曦,等.姜黄素衍生物B06对2型糖尿病大鼠肝脏的保护作用[J].中国病理生理杂志,2014,30(2):328-332.
[6]MONJUR A.Non-alcoholic fatty liver disease in 2015[J].World J Hepatol,2015,7(11):1450-1459.
[7]WU T,LIU Y H,FU Y C,et al.Direct evidence of sirtuin downregulation in the liver of non-alcoholic fatty liver disease patients[J].Ann Clin Lab Sci,2014,44(4):410-418.
[8]TOBITA T,GUAMAN-LEPE J,TAKEISHI K,et al.SIRT1disruption in human fetal hepatocytes leads to increased accumulation of glucose and lipids[J].PLo S One,2016,11(2):e0149344.
[9]COLAK Y,OZTURK O,SENATES E,et al.SIRT1 as a potential therapeutic target for treatment of nonalcoholic fatty liver disease[J].Med Sci Monitor,2011,17(5):HY5-HY9.
[10]ROLO A P,TEODORO J S,PALMEIRA C M.Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis[J].Free Radic Biol Med,2011,52(1):59-69.
[11]DING R B,BAO J,DENG C X.Emerging roles of SIRT1 in fatty liver diseases[J].Int J Biol Sci,2017,13(7):852-867.
[12]BEREITER-HAHN J,V?TH M.Dynamics of mitochondria in living cells:Shape changes,dislocations,fusion,and fission of mitochondria[J].Microsc Res Tech,1994,27(3):198-219.
[13]SUEN D F,NORRIS K L,YOULE R J.Mitochondrial dynamics and apoptosis[J].Genes Dev,2008,22(12):1577-1599.
[14]郑靖宇,吴欢,汤雯,等.Drp1和Mfn2在棕榈酸诱导大鼠肝细胞损伤中的作用机制及姜黄素衍生物L6H4的干预作用[J].温州医科大学学报,2016,46(7):469-475.
[15]VALERO T.Mitochondrial biogenesis:pharmacological approaches[J].Curr Pharm Design,2014,20(35):5507-5509.
[16]罗桂平,蹇朝,张华刚,等.Sirt1在缺氧条件下对心肌细胞线粒体融合与分裂中的作用[J].第三军医大学学报,2016,38(10):1121-1126.
[17]BIEL T G,LEE S,FLORES-TORO J A,et al.Sirtuin 1 suppresses mitochondrial dysfunction of ischemic mouse livers in a mitofusin 2-dependent manner[J].Cell Death Differ,2016,23(2):279-290.
[18]MIAO Y,ZHAO S,GAO Y,et al.Curcumin pretreatment attenuates inflammation and mitochondrial dysfunction in experimental stroke:The possible role of Sirt1 signaling[J].Brain Res Bull,2016,121:9-15.