联合使用表没食子儿茶素没食子酸酯、牛磺酸和三羟基异黄酮对大鼠酒精性肝纤维化的保护作用
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摘要
目的:研究联合使用表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG),牛磺酸(taurine)和三羟基异黄酮(genistein)对大鼠酒精性肝纤维化的保护作用及其作用机制。方法:Wistar雄性大鼠随机分成6组即正常组,模型组,秋水仙碱组(0.5 mg·kg~(-1)),联合用药低剂量15.625 mg·kg~(-1)组(牛磺酸12.5 mg·kg~(-1)+EGCG 1.875 mg·kg~(-1)+三羟基异黄酮1.25 mg·kg~(-1)),中剂量31.25 mg·kg~(-1)组(牛磺酸25 mg·kg~(-1)+EGCG 3.75 mg·kg~(-1)+三羟基异黄酮2.5 mg·kg~(-1)),高剂量62.5 mg·kg~(-1)组(牛磺酸50 mg·kg~(-1)+EGCG 7.5 mg·kg~(-1)+三羟基异黄酮5 mg·kg~(-1))。除正常组外,其余各组ig乙醇5.0~9.5 g·kg~(-1),正常组ig给予等量生理盐水,每天1次,连续24周。末次给药24 h后,取血,处死大鼠,采集肝组织;检测血清中丙氨酸转氨酶(ALT),天冬氨酸转氨酶(AST),碱性磷酸酶(ALP),谷氨酰转移酶(GGT),透明质酸(HA),层黏连蛋白(LN)和三型前胶原(PCⅢ)水平的变化,检测肝组织中丙二醛(MDA),羟脯氨酸(Hyp),谷胱甘肽过氧化物酶(GSH-Px),超氧化物歧化酶(SOD),采用免疫组化法检测各组肝组织中α-平滑肌肌动蛋(α-SMA),转化生长因子-β1(TGF-β1)及人母亲DPP同源物3(Smad 3)蛋白表达情况,同时做组织学检查,观察肝组织的病理变化。结果:与正常组比较,模型组大鼠ALT,AST,ALP,GGT,MDA,Hyp,HA,LN,PCⅢ,α-SMA,TGF-β1和Smad3显著升高(P<0.05),SOD和GSH-Px显著降低(P<0.05);与模型组相比,联合用药可显著降低ALT,AST,ALP,GGT,MDA,Hyp,HA,LN,PCⅢ,α-SMA,TGF-β1和Smad3的表达(P<0.05),显著升高SOD和GSH-Px的活性(P<0.05)。结论:联合使用表没食子儿茶素没食子酸酯、牛磺酸和三羟基异黄酮用药对乙醇所致的大鼠肝纤维化具有一定的保护作用,其机制可能与加快自由基清除,减轻脂质过氧化损伤,下调α-SMA,TGF-β1,Smad3表达有关。
Objective: To study the protective effect of the compounds of taurine,epigallocatechin-3-gallate( EGCG) and genistein on chronic alcohol-induced hepatic fibrosis, and to explore its underlying mechanism. Method: Wistar male rats were randomly divided into six groups: normal group,model group,colchicine group,combination therapy low( taurine 12. 5 mg·kg~(-1)+ EGCG 1. 875 mg·kg~(-1)+ genistein 1. 25mg·kg~(-1)),middle( taurine 25 mg·kg~(-1)+ EGCG 3. 75 mg·kg~(-1)+ genistein 2. 5 mg·kg~(-1)) and high( taurine 50mg·kg~(-1)+ EGCG 7. 5 mg·kg~(-1)+ genistein 5 mg·kg~(-1)) dose group. The rats except normal group were tablished by intragastric administration of alcohol( 5. 0-9. 5 g·kg~(-1)) once a day for 24 weeks and the normal group were given normal saline. Twenty-four hours after the last administration of drugs,all rats were killed,and the hepatic tissue were taken. The contents of hydroxyproline( Hyp), superoxide dismutase( SOD), malondialdehyde( MDA) in hepatic tissue and serum biochemical indicators were measured,as well as the related key cytokines/proteins. The damage of liver tissues was observed by light microscope. Result: Compared with normal group,the activities of alanine aminotransferase( ALT),aspartate aminotransferase( AST),alkaline phosphatase( ALP),gamma glutamyl transpeptidase( GGT),MDA,Hyp,hyaluronic acid( HA),laminin( LN),collagen type Ⅲ( PC Ⅲ),α-smooth muscle actin( α-SMA),transforming growth factor-β1( TGF-β1) and mothers against decapentaplegic homolog 3( Smad3) increased( P < 0. 05) and SOD, glutathione peroxidase( GSH-Px)decreased significantly( P < 0. 05) in model group; compared with model group,the levels of ALT,AST,ALP,GGT,MDA,Hyp,HA,LN,PC Ⅲ,α-SMA,TGF-β1and Smad3 decreased( P < 0. 05) and SOD,GSH-Px increased significantly( P < 0. 05) in combination therapy group. Conclusion: The compounds of taurine,EGCG and genistein can relieve chronic hepatic fibrosis induced by alcohol. Its mechanism may involve scavenging free radica,alleviating the lipid peroxidation,inhibiting the expression of α-SMA,TGF-β1and Smad3.
Objective: To study the protective effect of the compounds of taurine,epigallocatechin-3-gallate( EGCG) and genistein on chronic alcohol-induced hepatic fibrosis, and to explore its underlying mechanism. Method: Wistar male rats were randomly divided into six groups: normal group,model group,colchicine group,combination therapy low( taurine 12. 5 mg·kg~(-1)+ EGCG 1. 875 mg·kg~(-1)+ genistein 1. 25mg·kg~(-1)),middle( taurine 25 mg·kg~(-1)+ EGCG 3. 75 mg·kg~(-1)+ genistein 2. 5 mg·kg~(-1)) and high( taurine 50mg·kg~(-1)+ EGCG 7. 5 mg·kg~(-1)+ genistein 5 mg·kg~(-1)) dose group. The rats except normal group were tablished by intragastric administration of alcohol( 5. 0-9. 5 g·kg~(-1)) once a day for 24 weeks and the normal group were given normal saline. Twenty-four hours after the last administration of drugs,all rats were killed,and the hepatic tissue were taken. The contents of hydroxyproline( Hyp), superoxide dismutase( SOD), malondialdehyde( MDA) in hepatic tissue and serum biochemical indicators were measured,as well as the related key cytokines/proteins. The damage of liver tissues was observed by light microscope. Result: Compared with normal group,the activities of alanine aminotransferase( ALT),aspartate aminotransferase( AST),alkaline phosphatase( ALP),gamma glutamyl transpeptidase( GGT),MDA,Hyp,hyaluronic acid( HA),laminin( LN),collagen type Ⅲ( PC Ⅲ),α-smooth muscle actin( α-SMA),transforming growth factor-β1( TGF-β1) and mothers against decapentaplegic homolog 3( Smad3) increased( P < 0. 05) and SOD, glutathione peroxidase( GSH-Px)decreased significantly( P < 0. 05) in model group; compared with model group,the levels of ALT,AST,ALP,GGT,MDA,Hyp,HA,LN,PC Ⅲ,α-SMA,TGF-β1and Smad3 decreased( P < 0. 05) and SOD,GSH-Px increased significantly( P < 0. 05) in combination therapy group. Conclusion: The compounds of taurine,EGCG and genistein can relieve chronic hepatic fibrosis induced by alcohol. Its mechanism may involve scavenging free radica,alleviating the lipid peroxidation,inhibiting the expression of α-SMA,TGF-β1and Smad3.
引文
[1]张莎莎,吕文良,张旭,等.肝纤维化的治疗研究进展[J].浙江中医药大学学报,2012,36(4):471-475.
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[6]陈永欣,黄权芳,林兴,等.满天星异荭草苷对大鼠酒精性肝纤维化保护作用的实验研究[J].中国中药杂志,2013,38(21):3726-3730.
[7]石慧,肖和杰.肝纤维化分子研究进展及其治疗[J].中西医结合肝病杂志,2012,22(5):318-320.
[8]李志钢,杨晋翔,张伟,等.肝心宁对大鼠肝纤维化SMA,MMP-13及TIMP-1蛋白表达的影响[J].北京中医药,2009,28(7):556.
[9]朱碧红,张慧芳,陈永平.大鼠肝纤维化组织中TGF-β1的研究[J].中国微生态学杂志,2013,25(2):150-152
[10]Wicks S,Grocott T,Haros K,et al.Reversible ubiquitination regulates the Smad/TGF-beta signalling pathway[J].Biochem Soc Trans,2006,34(5):761-763.
[2]廖明,李彦,舒伟,等.鸡尾酒疗法对大鼠肝星状细胞的增殖及Ⅰ型胶原,TIMP-1 mRNA表达的影响[J].世界华人消化杂志,2010,18(9):932-936.
[3]Zhen M,Wang Q,Huang X,et al.Green tea polyphenol epigallocatechin-3-gallate inhibits oxidative damage and preventive effects on carbon tetrachlorideinduced hepatic fibrosis[J].J Nutr Biochem,2007,18(12):795-805.
[4]廖明,林兴,陈兆霓,等.鸡尾酒疗法对肝纤维化大鼠肝组织中TGF-β1,COL I及COLⅢ表达的影响[J].世界华人消化杂志,2010,18(18):1867-1872.
[5]邵祥强,肖华胜.肝纤维化发病机制与临床诊断的进展研究[J].世界华人消化杂志,2011,19(3):268-274.
[6]陈永欣,黄权芳,林兴,等.满天星异荭草苷对大鼠酒精性肝纤维化保护作用的实验研究[J].中国中药杂志,2013,38(21):3726-3730.
[7]石慧,肖和杰.肝纤维化分子研究进展及其治疗[J].中西医结合肝病杂志,2012,22(5):318-320.
[8]李志钢,杨晋翔,张伟,等.肝心宁对大鼠肝纤维化SMA,MMP-13及TIMP-1蛋白表达的影响[J].北京中医药,2009,28(7):556.
[9]朱碧红,张慧芳,陈永平.大鼠肝纤维化组织中TGF-β1的研究[J].中国微生态学杂志,2013,25(2):150-152
[10]Wicks S,Grocott T,Haros K,et al.Reversible ubiquitination regulates the Smad/TGF-beta signalling pathway[J].Biochem Soc Trans,2006,34(5):761-763.