褪黑素对梗阻性黄疸大鼠肝损伤保护作用的实验研究

Protective Effect of Melatonin on Rat Liver Injury Induced by Bile Duct Ligation

目的探讨氧化性损伤在大鼠梗阻性黄疸肝功能损害发生中的作用以及褪黑素对其的保护作用。方法成年雄性SD大鼠64只,采用完全随机化法随机分为正常对照组(CN组,n=16)、假手术组(SO组,n=16)、胆总管结扎组(BDL组,n=16)和胆总管结扎+褪黑素治疗组(BDL+MT组,n=16)。应用胆总管结扎法建立梗阻性黄疸模型,褪黑素治疗组大鼠手术前1 d至手术后7 d连续腹腔注射褪黑素0.5 mg(/kg.d),每日10∶00给药。分别于手术后第4 d和第8 d两个时间点采集标本,检测血浆中总胆红素(TBIL)、丙氨酸转氨酶(ALT)、门冬氨酸转氨酶(AST)、碱性磷酸酶(AKP)及γ-谷氨酰转肽酶(GGT)水平变化,比色法测定肝组织匀浆中丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)含量或活力变化,采用TUNEL法检测肝组织细胞凋亡,并计算肝细胞凋亡指数(AI)。结果与CN组和SO组比较,BDL组大鼠血浆TBIL、ALT、AST、AKP、GGT水平和肝组织MDA含量明显升高(P<0.05,P<0.01),SOD、CAT、GSH-Px活力或GSH含量显著降低(P<0.01),AI增加(P<0.01);褪黑素治疗可使血浆TBIL、ALT、AST、AKP、GGT和肝组织MDA含量显著降低,SOD、CAT、GSH-Px活力或GSH含量明显升高(P<0.01),AI减少(P<0.01)。BDL组肝组织MDA含量与血浆TBIL、ALT、AKP、AST、GGT水平均呈显著正相关(P<0.01),GSH、SOD、CAT、GSH-Px与血浆TBIL、ALT、AKP、ALT、AST水平分别均呈显著负相关(P<0.01);BDL组肝组织MDA含量的变化与AI呈正相关(P<0.01),而GSH含量及SOD、CAT、GSH-Px活力分别与AI呈负相关(P<0.01)。结论大鼠梗阻性黄疸时,肝组织自由基大量产生介导的氧化性损伤及其细胞凋亡,参与了肝功能损害的发生、发展。褪黑素对大鼠梗阻性黄疸肝功能损害有一定程度的保护作用,其机制可能与其拮抗肝组织过氧化和细胞凋亡有关。

Objective To investigate the protective effect of melatonin on rat liver injury induced by bile duct ligation.Methods Sixty-four rats were randomly divided into four groups：control group（CN group,n=16）,sham operation group（SO group,n=16）,bile duct ligation group（BDL group,n=16）,and bile duct ligation with melatonin injection（BDL＋MT group,n=16）.The model of obstructive jaundice was done by ligation of the common bile duct.Melatonin was injected daily（0.5 mg/kg）via peritoneal cavity from 1 d before the operation to 7 d following operation.On day 4 and 8 after the ligation,the plasma levels of total bilirubin（TBIL）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,gamma glutamyl transferase（GGT）,and alkaline phosphatase（AKP）were measured by routine methods.Malonaldehyde（MDA）,superoxide dismutase（SOD）,glutathione（GSH）,catalase（CAT）,and glutathione peroxidase（GSH-Px）in the liver tissue were determined by spectrophtometry,too.Hepatocytes apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated deoxynuridine triphosphate nick-end labeling（TUNEL） assay.Results Compared with the CN group and SO group,the levels of TBIL,ALT,AST,GGT,and AKP in the plasma,the content of MDA in the liver tissue,and the apoptosis index（AI）in the hepatocyte markedly increased（P 0.05,P0.01）,the content of GSH and the activities of SOD,CAT,and GSH-Px in the liver tissue markedly decreased（P0.01）in the BDL group.Compared with the BDL group,the levels of TBIL,ALT,AST,GGT,and AKP in the plasma,the content of MDA in the liver tissue,and the AI in the hepatocyte markedly decreased（P0.01）,the content of GSH and the activities of SOD,CAT,and GSH-Px in the liver tissue markedly increased（P0.01）in the BDL＋MT group.In the BDL group,the level of MDA in the liver tissue and the levels of TBIL,ALT,AST,GGT,and AKP were positively correlated（P0.01）,the content of GSH and the activities of SOD,CAT,and GSH-Px in the liver tissue and TBIL,ALT,AST,GGT,and AKP were negatively correlated（P0.01）.The level of MDA in the liver tissue and AI in the hepatocyte was positively correlated（P0.01）.The content of GSH and the activities of SOD,CAT,and GSH-Px in the liver tissue and AI were negatively correlated（P0.01）.Conclusions The participation of free radical of oxygen in the pathogenesis and severity of cholestasis produced by the acute obstruction of the extra-hepatic biliary duct is likely.The result of the present study indicates that melatonin exerts a protective effect on cholestatic liver injury in rats with BDL.The mechanism of melatonin’s protection on hepatocyte may be through its antioxidant action and by inhibiting hepatocyte apoptosis.