Objectives: Liver disease is the most common extra-intestinal manifestation of ulcerative colitis (UC), but the underlying pathogenesis is still not clarified. It is well accepted that the occurrence of UC-related liver disease has close correlation with immune activation, intestinal bacterial liver translocation, inflammatory cytokine storm, and the disturbance of bile acid circulation. The occurrence of UC-related liver disease makes the therapy difficult, therefor study on the pathogenesis of UC-related liver injury is of great significance for its prevention and treatment. Glutathione (GSH) shows multiple physiological activities, such as free radical scavenging, detoxification metabolism and immune defense. The synthesis and the oxidation-reduction all contribute to GSH antioxidant function. It is reported that the deficiency in hepatic GSH antioxidant function participates in multiple liver diseases, but whether it participates in the pathogenesis of UC-related liver injury is still not clear. This study aims to investigate the feature and underlying mechanism of GSH synthesis and oxidation-reduction function during the development of UC, which will provide useful information for the pathogenesis study on UC-related liver injury.
Methods: UC model was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS)-ethanol solution (5 mg/0.8 mL per rat, 50% ethanol) via intra-colonic administration in rats, and the samples of serum, liver, and colon tissue of rats were collected at the 3rd, 5th, and 7th days post TNBS. The severity degree of colitis was evaluated by measuring the disease activity index, colonic myeloperoxidase activity, and histopathological score, and the degree of liver injury was evaluated by histopathological score and the serum content of alanine aminotransferase. Spearman correlation analysis was also conducted between the degree of colonic lesions and index of hepatic histopathological score as well as serum aspartate aminotransferase level to clarify the correlation between liver injury and colitis. To evaluate the hepatic antioxidant function of GSH in UC rats, hepatic GSH content, enzyme activity of GSH peroxidase (GSH-Px), and GSH reductase (GR) were determined in rats at the 3rd, 5th, and 7th days post TNBS, and the protein expressions of glutamine cysteine ligase (GCL), GSH synthase, GSH-Px, and GR in the liver of UC rats were also examined by Western blotting.
Results: Compared with the control, the disease activity index, colonic myeloperoxidase activity, and histopathological score were all significantly increased at the 3rd, 5th, and 7th days post TNBS (all P<0.01), the serum aspartate aminotransferase level and hepatic histopathologic score were also obviously elevated at the 7th day post TNBS (all P<0.05). There was a significant positive correlation between the degree of liver injury and the severity of colonic lesions (P=0.000 1). Moreover, compared with the control, hepatic GSH content and the activity of GSH-Px and GR were all significantly decreased at the 3rd and 5th days post TNBS (P<0.05 or P<0.01), and the protein expressions of GCL, GSH-Px, and GR were all obviously down-regulated at the 3rd, 5th, and 7th days post TNBS (P<0.05 or P<0.01).
Conclusions: There is a significant positive correlation between the degree of liver injury and the severity of colonic lesions, and the occurrence of reduced hepatic GSH synthesis and decreased GSH reduction function is obviously earlier than that of the liver injury in UC rats. The reduced hepatic expression of enzymes that responsible for GSH synthesis and reduction may contribute to the deficiency of GSH synthesis and oxidation-reduction function, indicating that the deficiency in GSH antioxidant function may participate in the pathogenesis of UC related liver injury.
目的: 肝病变是溃疡性结肠炎(ulcerative colitis，UC)最常见的肠外表现，但其发生机制目前尚未完全阐明，目前普遍认为其发生与免疫激活、肠道菌易位肝、炎症因子风暴及胆汁酸循环紊乱有关。UC相关性肝疾病的发生使其临床治疗更为困难，探究UC肝损伤的发病机制对于UC相关性肝疾病的防治意义重大。谷胱甘肽(glutathione，GSH)具有清除自由基、参与肝解毒代谢及免疫防御等多种生理功能，GSH的合成及氧化还原能力共同决定GSH的抗氧化功能。肝GSH抗氧化功能缺陷与多种肝疾病的发生密切相关，但其是否参与并介导UC相关性肝损伤的发生目前尚不明晰。本研究旨在探究UC发生中GSH合成及还原功能变化特征及其分子机制。方法: 应用2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid，TNBS)乙醇溶液(每只5 mg/0.8 mL，50%乙醇)灌注结肠制备UC大鼠模型，分别在TNBS灌注后第3、5和7天取材血清、肝及肠道组织，测定疾病活动指数、结肠组织髓过氧化物酶活性及结肠病理组织学评分以评价UC结肠的病变程度；以肝病理组织学评分、血清谷草转氨酶(AST)和谷丙转氨酶(ALT)水平为指标评估肝病变程度；并分别将UC造模7天组大鼠肝病理评分和血清AST水平与其结肠病变程度做Spearman相关分析以明确UC发生中肝病变与结肠病变的相关性。采用试剂盒动态测定UC造模后第3、5和7天组大鼠肝中GSH含量、GSH过氧化物酶(glutathione peroxidase，GSH-Px)及GSH还原酶(glutathione reductases，GR)活性；并应用蛋白质印迹法检测肝中GSH合成反应关键酶谷氨酰胺半胱氨酸连接酶(glutamine cysteine ligase，GCL)、GSH合成酶以及GSH-Px和GR蛋白质表达的变化。结果: 与正常对照组相比，UC造模后第3、5及7天组大鼠的疾病活动指数、结肠组织髓过氧化物酶活性及结肠病理组织学评分均显著高于正常对照组(均P<0.01)；UC造模后第7天组大鼠的血清AST水平及肝病理组织评分均显著高于正常对照组(均P<0.05)，且UC模型大鼠肝损伤程度与结肠病变程度呈显著正相关(P=0.000 1)。此外，与正常对照组相比，UC造模后第3天和第5天组大鼠肝组织中GSH总量、GSH-Px活性和GR活性均显著低于正常对照组(P<0.05或P<0.01)，且UC造模后第3、5及7天组大鼠肝组织中GCL、GSH-Px和GR蛋白质表达量均显著低于正常对照组(P<0.05或P<0.01)。结论: UC大鼠肝病变程度与结肠病变程度呈正相关，UC大鼠肝GSH合成减少及还原能力降低明显早于肝损伤的发生，调控肝GSH合成及还原反应的关键酶表达减少是导致GSH合成减少及还原能力降低的主要原因，提示GSH抗氧化功能缺陷参与并介导UC相关性肝损伤的发生。.
Keywords: antioxidant; glutathione; liver injury; ulcerative colitis.