摘要
目的：探讨在肝损伤早期,酒精和HBV协同作用可能的分子机制；同时观察拉米夫定和水飞蓟素对早期肝损伤的保护作用,并从分子水平探讨二者对纤维化通路信号分子的影响。
方法：50只HBV转基因小鼠和20只普通小鼠,予以1周的适应性喂养。按以下两种方案进行实验动物分组,分组一：20只转基因小鼠和20只普通小鼠,各随机分为2组,每组10只：普通小鼠对照组和转基因小鼠对照组,以生理盐水灌胃,5 ml/kg,每天1次；普通小鼠酒精组和转基因小鼠酒精组,以体积分数为50%的白酒灌胃,5 ml/kg,每天1次；分组二：除外转基因小鼠对照组和转基因小鼠酒精组,另30只转基因小鼠随机分为3组,每组10只：拉米夫定组：转基因小鼠以体积分数为50%的白酒灌胃,5 ml/kg,每天1次；同时予拉米夫定(100 mg/kg)灌胃,每天1次；水飞蓟素组：转基因小鼠以体积分数为50%的白酒灌胃,5 ml/kg,每天1次；同时予水飞蓟素(100 mg/kg)灌胃,每天1次；拉米夫定+水飞蓟素组：转基因小鼠以体积分数为50%的白酒灌胃,5 ml/kg,每天1次；同时予拉米夫定(100 mg/kg)+水飞蓟素(100 mg/kg)灌胃,每天1次。连续处理10周后,检测各组小鼠血清ALT、AST水平,HBV DNA载量,肝脏病理组织学改变,肝组织TGF-β1、Smad3、Smad7、CTGF mRNA表达水平及TGF-β1、CTGF、α-SMA蛋白表达水平。
结果：酒精刺激对普通小鼠肝功能的水平没有影响,但能促进其肝组织中纤维化相关因子TGF-β1、Smad3、Smad7、CTGF、α-SMA的表达。酒精灌胃增加转基因小鼠体内HBV DNA病毒载量,同时引起转基因小鼠肝损伤较普通小鼠明显加重,表现为转基因小鼠血清ALT、AST水平升高,肝细胞病理损伤及肝组织炎症反应程度加重,但纤维化不明显；其肝组织TGF-β1、Smad3、Smad7、CTGF、α-SMA表达增加。拉米夫定、水飞蓟素分别单独使用或二者联用均不能降低酒精刺激的HBV转基因小鼠体内的病毒滴度；水飞蓟素单独使用或与拉米夫定二者联用可降低酒精刺激的转基因小鼠血清ALT、AST水平,但拉米夫定单独使用不能降低小鼠血清ALT、AST水平；拉米夫定、水飞蓟素分别单独使用或二者联用均可减轻酒精刺激的HBV转基因小鼠肝组织病理损伤,使小鼠肝组织内TGFβ1、Smad3、CTGF mRNA及TGF-β1、CTGF、α-SMA蛋白表达减少,同时进一步增加抑制肝纤维化的Smad7 mRNA的表达。
结论：1.酒精刺激对普通小鼠肝功能的水平没有影响,但能促进其肝组织中纤维化相关因子的表达。
2.酒精引起转基因小鼠肝损伤较普通小鼠明显加重,其肝组织中促纤维化相关因子的表达较普通小鼠增多,表明酒精和HBV对肝损伤有协同作用；其机制与增加TGF-β1、Smad3、CTGF表达,促进HSC活化以及TGF-β1/Smads通路信号分子表达比例失调有关。
3.在肝损伤早期,拉米夫定和水飞蓟素可通过调整TGF-β1/Smads信号通路,减少CTGF表达,抑制HSC活化,从而减少ECM的产生和蓄积等机制,阻断早期肝损伤的发生和进展。
Objective
To investigate synergistic effects and molecular mechanism of hepatitis B virus (HBV) and alcohol on liver injury in HBV transgenic mice (HBV-Tg mice).Meanwhile,to investigate the protective effect and mechanism of Lamivudine (LMV) and Silymarin (SIL) on liver injury induced by alcohol drinking in HBV-Tg mice.
Methods
The one program of animal grouping:20 HBV-Tg mice and 20 wild-type mice were randomly divided into 4 groups:alcohol-fed Tg mice group (n=10) and alcohol-fed Wt mice group (n=10),were given intragastric administration with alcohol;control Tg mice group (n=10) and control Wt mice group (n=10),were received intragastric administration with saline.The other program of animal grouping:30 HBV-Tg mice were randomly divided into 3 groups:LMV group (n=10), HBV-Tg mice were given intragastric administration with alcohol,at the same time,were administrated intragastricly with LMV(100 mg/kg);SIL group (n=10),HBV-Tg mice were given intragastric administration with alcohol,at the same time,were administrated intragastricly with SIL(100 mg/kg);LMV+SIL group (n=10),HBV-Tg mice were given intragastric administration with alcohol,at the same time,were administrated intragastricly with LMV(100 mg/kg) and SIL(100 mg/kg).All groups were rasied for 10 weeks.The levels of HBV DNA copy number, ALT,AST in serum,the degree of inflammation,the degree of fibrosis,the mRNA expression of TGF-β1,Smad3,Smad7,CTGF and the protein expression of TGF-β1,CTGF,a-SMA in liver tissue were detected.All the staining figures were scanned with electronic computer and all the data were analyzed with SPSS 13.0 software.
Results
The serum level of HBV DNA copies number,ALT and AST, the mRNA expression of TGF-β1,Smad3,Smad7,CTGF and the protein expression of TGF-β1,CTGF,a-SMA in liver all increased markedly in alcohol-fed Tg mice(vs control Tg mice,P<0.05).Alcohol consumption induced hepatocyte steatosis and hepatic inflammation in alcohol-fed Tg mice,but the change of liver fibrosis was not remarkable.Lamivudine and Silymarin single use respectively or drug combination failed to suppress the increased serum levels of HBV DNA copies number in alcohol-fed Tg mice.In alcohol-fed Tg mice,Silymarine single use or combined with Lamivudine could decrease the increased serum levels of ALT and AST(vs alcohol-fed Tg mice,P<0.05),but Lamivudine single use could not.Lamivudine and Silymarin single use respectively or drug combination could significantly relieve the histological injury and decrease the increased mRNA expression of TGF-β1,Smad3,CTGF and the protein expression of TGF-β1,CTGF,a-SMA mice(vs alcohol-fed Tg mice, P<0.05),but father improve the mRNA expression of Smad7(vs alcohol-fed Tg mice, P<0.05)in the liver.
Conclusions
1.Alcohol drinking have no effect on liver function, but enhancing the expression of TGF-β1,Smad3,Smad7, CTGF, a-SMA.
2.HBV and alcohol have synergistic effects on early liver injury, by enhancing the expression of TGF-β1,Smad3,CTGF, inducing unbalanced expression of Smads and motivating the activation of HSC.
3.Lamivudine and Silymarin single use respectively or drug combination could ruduce the serum level of ALT,AST;relieve the histological damage in the liver of alcohol-fed mice.The mechanism may be inhibiting the expression of TGF-β1,Smad3,CTGF,modulating the expression of Smads and suppressing the activation of HSC.
引文
[1]刘文有.乙型肝炎流行病学研究概况[J].应用预防医学,2006,8(12)：12-14.
[2]庄辉.乙型肝炎流行病学研究进展[J].国外医学流行病学传染病学分册,2004,31(3)：1331.
[3]骆抗先.乙型肝炎基础与临床[M].北京：人民卫生出版社,2006：422-431.
[4]MenonKV, GoresGJ, ShahVH. Pathogenesis, diagnosis and treatment of alcoholic liver disease [J].Mayo Clin Proc,2001,76(10):1021-1029.
[5]孙妩弋,魏伟.肝星状细胞信号转导机制及可能的抗肝纤维化药物作用新靶点[J].中国药理学通报,2006,22(12)：1433-1438.
[6]王要军,孙自勤.肝窦内皮细胞与肝纤维化[J].临床肝胆病杂志,1996,12(3)：115-117.
[7]Calabrese F,Valente M, Giacometi C, et al.Parenchymal transforming growth factor beta-1:its type Ⅱ receptor and Smad signaling pathway correlate with inflammation and fibrosis in chronic liver disease of viral etiology[J].J Gastroenterol Hepatol,2003,18(11):1302-1308.
[8]Valerie P, Delphine D, Michel V. Expression of Connective Tissue Growth Factor in Experimental Rat and Human Liver Fibrosis[J].Hepatol,1999,30 (4):968-976.
[9]Kim WH, Hong F, Jaruga B.Additive activation of hepatic NF-κB by ethanol and HBX or HCV core protein:involvement of TNF-a receptor Ⅰ-independent and-dependent mechanisms [J].The FASEB Journal,2001,15(13):2551-2553.
[10]Kim WH, Hong F, Jaruga B.Hepatitis B virus X protein sensitizes primary mouse hepatocytes to ethanol-and TNF-a-induced apoptosis by a Caspase-3 dependent mechanism [J].Cellular and Molecular Immunology,2005,2 (1):40-48.
[11]虞朝辉,陈卫星,陈韶华,等.嗜酒合并乙型肝炎病毒感染与转化生长因子pl表达水平的关系[J].中华消化杂志,2004,3(24)：871,971.
[12]Cohen A, Burczynski FJ, Rosser BG, et al.The effects of variousorgan preservation solutions on hepatocyte membrane potentials, intracellular calcium concentrations, and outcome following liver transplantation [J].Am J Surg,2000, 179(2):154-160.
[13]骆抗先.乙型肝炎基础与临床[M].北京：人民卫生出版社,2006：376-379.
[14]成军,肖琳.水飞蓟素在抗肝纤维化中的应用[J].国际消化杂志,2007,27(1)：69-71.
[15]Guha C, Mohan S, Roy-chowdhury N, et al.Cell culture and animal models of viral hepatitis. Part I:hepatitis B [J].Lab animal,2004,33(7):37-46.
[16]刘光泽,孔祥平,顾为望,等.高复制HBV转基因小鼠模型对抗乙型肝炎病毒药物的效应研究[J].中国病理生理杂志,2007,23(1)：99-102.
[17]刘建文.药理实验方法学[M].北京：化学工业出版社,2008：9-10.
[18]刘建文.药理实验方法学[M].北京：化学工业出版社,2008：210.
[19]Nouchi T, Tanaka Y, Tsukada T, et al.Appearance of a-smooth muscle actin positive cells in hepatic fibrosis[J].Liver,1991,11(2):100-105.
[20]刘光泽,熊一力,王洪敏,等.近交系高表达HBV转基因小鼠的建立及表达传代稳定性[J].中国兽医学报,2003,23(6)：580-582.
[21]胡莲美,李秀梅,黄黎珍,等.复制型乙型肝炎病毒转基因小鼠血清中病毒颗粒的免疫电镜检测[J].中华肝脏病杂志,2007,15(2)：145-146.
[22]程明亮,杨长青.肝纤维化的基础研究及临床[M].北京：人民卫生出版社,2002：21-85.
[23]Iredale JP.Hepatic stellate cell behavior during resolution of liver injury [J].Seminars in Liver Disease,2001,21(3):427-436.
[24]TahashiY, Matsuzaki K, Date M,et al.Diferential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury [J]. Hepatology,2002,35(1):49-61.
[25]Lewindon PJ,Pereira TN,Hoskina AC,et al.Role of hepatic stellate cells and transforming growth factor-β in cystic fibrosis liver disease[J].Am J Pathol,2002, 160(5):1705-1715.
[26]Blom IE, Goldschmeding R, Leask A.Gene regulation of connective tissue growth factor:new targets for antifibrotic therapy? [J].Matrix Biology,2002, (21): 473-482.
[27]Schnabl B,Kweon YO, Frederick JP,et al.The role of Smad3 in mediating mouse hepatic stellate cell activation [J].Hepatology,2001,34(1):89-100.
[28]Dooley S,Hamzavi J,Breitkopf K,et al.Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats [J].Gastoentenology,2003,125(1):178-191.
[29]Massague J,Chen YG.Controlling TGF-β signaling [J].Gene Development, 2000,14(6):627-644.
[30]Grotendorst GR. Connective tissue growth factor:a mediator of TGF-beta action on fibroblasts [J].Cytokine & Growth Factor Review,1997,8(3):171-179.
[31]Homles A, Abraham DJ, Sa S,et al.CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling [J].J Biol Chem,2001, 276(14):10594-10601.
[32]Li GM, Xie Q, Shi Y,et al. Inhibition of connective tissue growth factor by siRNA prevents liver fibrosis in rats [J].J Gene Med,2006,8(7):889-900.
[33]Murphy M, Godson C, Cannon S, et al.Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells[J].J Biol Chem,1999, 274(9):5830-5834.
[34]Abdel-Hadi AM, Talaat M.Histological assessment of tissue repair after treatment of human schistosomiasis [J].Acta Tropica,2000,77(1):91-96.
[35]Zhao WD,Zhu JY, Chen J, et al.Effects of ethanol on hepatitis B virus replication a gene expression in vivo [J].Clinical Medcine,2007,27(4):75-77.
[36]Larkin J, Clayton MM, Liu J, et al.Chronic ethanol consumption stimulates hepatitis B virus gene expression and replication in transgenic mice [J].Hepatology, 2001,34(4,pt.1):792-797.
[37]骆抗先.乙型肝炎基础与临床[M].北京：人民卫生出版社,2006：25-82.
[38]Sven K,Dirk JH,Markus B, et al.Concomitant endocrine and immune alterations during alcohol intoxication and acute withdrawal in alcohol-dependent subjects [J].Neuropsychobiology,2002,45(3):144-149.
[39]ParadisV,Dargere D,Bonvoust F,et al.Effects and regulation of connective tissue growth factor on hepatic stellate cells [J].Lab invest,2002,82 (6):767-775.
[40]Nada RT, Ivan VB,Dimitrije CB, et al.Activated liver stellate cells in chronic viral C hepatitis:histopathological and immunohistochemical study[J].J Gastrointestin Liver Dis,2009,18(2):163-167.
[41]Paradis V, Pelemuter G, Bonvoust F, et al. High glucose and hyperinsulinemia stimulate connective tissue growth factor expression:A potential mechanism involved in progression to fibrosis in nonalcoholic steatohapatitis [J]. Hepatology,2001,34 (4, pt.l):738-744.
[42]Crean JKG,Finlay D,Mruphy M,et al.The role of P42/44 MAPK and protein kinase B in connective tissue growth factor induced extrocellular matrix protein production, cell migration and actin cytoskeletal rearrangement in human mesangial cells [J].J Biol Chem,2002,277(46):44187-44194.
[43]Kwenon YO, Goodman ZD, Dienstag JL, et al.Decreasing fibrogenesis:an immunohistochemical study of paired liver biopsies following lamivudine therapy for chronic hepatitis B [J].Journal of Hepatology,2001,35(6):749-755.
[44]Saller R, Meier R, Brignoli R. The use of silymarin in the treatment of liver diseases [J].Drugs,2001,61(4):2035-2063.
[1]Zhao ZR, Huang Z. Clinical features of alcohol liver cirrhosis and hepatitis B liver cirrhosis [J].Beijing Yixue,2008,30 (4):197-201.
[2]He XH,Wang BY.Clinical features of excessive alcohol drinking and hepatitis B infections [J].J Clin Hepatol,2008,11(4):276-277.
[3]Nalpas B, Pol S,Thepot V,et al. Relationship between excessive alcohol drinking and viral infections [J].Alcohol and Alcoholism,1998,33(3):202-206.
[4]Giorgio B, Lucia M, Flora M, et al. Natural course of chronic HCV and HBV infection and role of alcohol in the general population:the Dionysos Study [J].The American Journal of Gastroenterology,2008,103 (9):2248-2253.
[5]Patrick M, Francoise P, Elisabeth DA, et al. Mortality related to chronic hepatitis B and chronic hepatitis C in France:Evidence for the role of HIV coinfection and alcohol consumption [J].Journal of Hepatology,2008,48 (2):200-207.
[6]Liu X. The effect of alcohol on chronic hepatitis B [J].Journal of Clinical and Experimental Medicine,2007,6(2):52-62.
[7]Niu FL, Hong SY, Wan MB.Impact of alcohol intake on liver inflammation and fibrosis in patients with chronic hepatitis B [J].World Chinese Journal of Digestology,2006,14 (26):2610-2612.
[8]Donato F,Tagger A,Chiesa R,et al.Hepatitis B and C virus infection, alcohol drinking, and hepatocellular carcinoma:A case-control study in Italy [J].Hepatology, 1997,26(3):579-584.
[9]Donato F, Tagger A, Gelatti U, et al.Alcohol and Hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women [J]. American Journal of Epidemiology,2002,155(4):323-331.
[10]Zhang Q, Zhang JL, Jia HB.Clinical observation of effect of alcohol intake on humoral immunity of hepatitis B virus [J].Chin J InternMed,2005,44(12):629.
[11]Zhao WD, Zhu JY, Chen J, et al.Effects of ethanol on hepatitis B virus replication an gene expression in vivo [J].Clinical Medcine,2007,27(4):75-77.
[12]NalPas B,Pourcel C, Feldmann G, et al.Chronic alcohol intoxication decreases the serum level of hepatitis B surface antigen in transgenic mice [J]. Journal of Hepatology,1992,15(1-2):118-124.
[13]Larkin J,Clayton MM, Liu J, et al.Chronic ethanol consumption stimulates hepatitis B virus gene expression and replication in transgenic mice [J].Hepatology, 2001,34(4Pt1):792-797.
[14]Sven K, Dirk JH, Markus B,et al.Concomitant endocrine and immune alterations during alcohol intoxication and acute withdrawal in alcohol-dependent subjects [J].Neuropsychobiology,2002,45(3):144-149.
[15]骆抗先.乙型肝炎基础和临床[M].第3版.北京：人民卫生出版社,2006：25-82.
[16]Ganne-Carie N,Krernsdorf D,GarTeau F,et al. Effects of ethanol on hepatitis B virus PreS/S human hepatocellular carcinoma derived HepG2 cell line gene expression in the hepatitis B DNA positive [J].Journal of Hepatology,1995, 23(2):153-159.
[17]Wu JM, Wu JS,Chen MX,et al. Effect of alcohol on replication and expression of HBV in vitro in 2.2.15 cells [J].Chin J Clin Pharmacol Ther,2005,10 (2):226-229.
[18]Nguyen VA.,Chen J, Hong F,et al.Interferons activate the p42/44 mitogen-activated protein-kinase and JAK-STAT (Janus kinase-signal transducer and activator transcription factor) signalling pathways in hepatocytes:differential regulation by acute ethanol via a protein kinase C-dependent mechanism [J].The Biochem J,2000,349(Pt2):427-434.
[19]Novick DM, Jenkins WJ,Karayiannis P,et al.A preliminary study of hepatitis B virus replication during short-term (7-day) social drinking [J].Alcoholism Clinical and Experimental Research,1987,11(6):556-558.
[20]Saito H, Masuda T, Tashita A, et al.Effect of ethanol on mouse hepatitis virus-induced cytotoxicity [J].Gastroenterol,1996,31(5):679-683.
[21]Chen J, ZhaoWD,Wang M,et al.Effect of hepatitis B on susceptivity of alcohol liver disease[J].Chin J InternMed,2005,44(86):611-612.
[22]Chen J, Xu JM, Wang M, et al.Effects of ethanol on HBV gene mutations in patients with chronic hepatitis B by gene chip [J].Chinese Journal of Pathophysiology,2008,24 (3):573-576.
[23]Kim WH,Hong FJaruga B.Additive activation of hepatic NF-κB by ethanol and HBX or HCV core protein:involvement of TNF-a receptor I independentand-dependent mechanisms [J].The FASEB Journal,2001,15(13):2551-2553.
[24]Kim WH, Hong F, Jaruga B.Hepatitis B virus X protein sensitizes primary mouse hepatocytes to ethanol-and TNF-a-induced apoptosis by a Caspase-3 dependent mechanism [J].Cellular and Molecular Immunology,2005,2(1):40-48.
[25]Yu ZH, Chen WX,Chen SH, et al.Relationship between TGF-β1 mRNA in PBMC and hepatitis B virus infection and alcohol drinking [J].Chinese Journal of Digestology,2004,3(24):871,971.
[26]Cohen A, Burczynski FJ, Rosser BG, et al. The effects of various organ preservation solutions on hepatocyte membrane potentials, intracellular calcium concentrations, and outcome following liver transplantation [J].Am J Surg 2000, 179(2):154-160.
