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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 role of oxidative damage in the development of hepatic dysfunction induced by obstructive jaundice in rats and the protective effect of melatonin on it. Methods Sixty-four adult male Sprague Dawley rats were randomly divided into normal control group (CN group, n = 16), sham operation group (n = 16), common bile duct ligation group = 16) and common bile duct ligation + melatonin treatment group (BDL + MT group, n = 16). Obstructive jaundice model was established by common bile duct ligation, and melatonin 0.5 mg (/ kg.d) was intraperitoneally injected in the melatonin treatment group from 1 d before operation to 7 d after operation. The specimens were collected at the 4th and 8th day after surgery to detect the levels of total bilirubin (TBIL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP) and γ-glutamyltranspeptidase (GGT) were measured by enzyme-linked immunosorbent assay (ELISA). Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) , Glutathione (GSH) and glutathione peroxidase (GSH-Px) were measured. The apoptosis of hepatic tissue was detected by TUNEL method and the apoptosis index (AI) was calculated. Results Compared with CN group and SO group, the levels of plasma TBIL, ALT, AST, AKP, GGT and MDA in liver tissue of BDL group were significantly increased (P <0.05, P <0.01) The levels of MDA, MDA, GSH-Px, GSH-Px, GSH-Px and GSH-Px in plasma were significantly decreased Vigor or GSH content was significantly increased (P <0.01), AI decreased (P <0.01). The content of MDA in liver tissue of BDL group was significantly and positively correlated with the level of plasma TBIL, ALT, AKP, AST and GGT (P <0.01), and the level of serum TBIL, ALT, AKP, ALT and AST (P <0.01). The content of MDA in liver tissue of BDL group was positively correlated with AI (P <0.01), while the content of GSH and the activities of SOD, CAT and GSH-Px were negatively correlated with AI <0.01). Conclusion In rats with obstructive jaundice, a large number of free radicals in liver tissue produce oxidative damage and apoptosis, which is involved in the occurrence and development of liver dysfunction. Melatonin has a protective effect on liver dysfunction induced by obstructive jaundice in rats, and its mechanism may be related to antagonizing liver tissue peroxidation and apoptosis.