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目的研究核因子NF-E2相关因子-抗氧化反应元件(Nrf2-ARE)信号通路在二氯甲烷(DCM)致小鼠亚急性肝脏氧化损伤的作用,探讨DCM致小鼠肝脏毒性的作用机制。方法将32只健康SPF级雄性昆明小鼠随机分为4组,分别为空白对照组、溶剂对照组、DCM染毒组和tBHQ干预组,每组8只。DCM染毒组和tBHQ干预组采用呼吸道动式染毒DCM,染毒剂量为12.9 g/m~3,每次2 h,每天1次,连续28 d;tBHQ干预组和溶剂对照组采用经口灌胃方式给药,染毒容量为10 ml/kg,tBHQ干预组给予tBHQ溶液(1.25 mg/ml),溶剂对照组给予玉米油;空白对照组正常喂养。检测血清谷氨酸氨基转移酶(AST)、天冬氨酸氨基转移酶(ALT)和碱性磷酸酶(ALP)的活力及肝组织匀浆活性氧簇(ROS)、丙二醛(MDA)含量和谷胱甘肽-S-转移酶(GST)、血红素加氧酶-1(HO-1)活力,采用免疫组化法检测Nrf2蛋白在肝细胞的定位及半定量表达水平。结果 DCM染毒组小鼠血清AST、ALT、ALP活力高于空白对照组、溶剂对照组和tBHQ干预组,差异均有统计学意义(P<0.05);而空白对照组、溶剂对照组和tBHQ干预组小鼠血清AST、ALT、ALP的活力间比较,差异均无统计学意义(P>0.05)。与空白对照组比较,溶剂对照组和tBHQ干预组小鼠肝组织匀浆GST、HO-1活力均升高;DCM染毒组小鼠肝组织匀浆ROS、MDA含量及GST、HO-1活力均升高,差异均有统计学意义(P<0.05)。与DCM染毒组比较,tBHQ干预组小鼠肝组织匀浆ROS、MDA含量均下降,而GST、HO-1活力均升高,差异均有统计学意义(P<0.05)。DCM染毒组和tBHQ干预组小鼠肝组织Nrf2蛋白的表达水平均高于空白对照组,差异均有统计学意义(P<0.05)。tBHQ干预组小鼠肝组织Nrf2蛋白的表达水平高于DCM染毒组,差异有统计学意义(P<0.05)。DCM染毒组和tBHQ干预组Nrf2蛋白的表达量增加,在胞核内表达量增多。结论 Nrf2-ARE信号通路在DCM致小鼠亚急性肝脏氧化损伤中发挥一定抗氧化代偿作用,在一定程度上减轻了DCM对肝脏的损伤。
Objective To investigate the role of Nrf2-ARE signaling pathway in oxidative damage induced by methylene chloride (DCM) in mice and explore the mechanism of liver toxicity caused by DCM in mice. Methods Thirty-two healthy Kunming mice were randomly divided into 4 groups: control group, solvent control group, DCM group and tBHQ intervention group, with 8 rats in each group. In the DCM and tBHQ intervention groups, the animals were treated with respiratory tract dynamic exposure with DCM at a dose of 12.9 g / m 3 for 2 h each for 28 days. In the tBHQ intervention group and the solvent control group, The rats were given gavage by intragastric administration, the exposure volume was 10 ml / kg, tBHQ solution (1.25 mg / ml) was given to the tBHQ intervention group, and the corn oil was given to the solvent control group. The blank control group was fed normally. The activities of serum glutamate aminotransferase (AST), aspartate aminotransferase (ALT) and alkaline phosphatase (ALP) and the levels of reactive oxygen species (ROS), malondialdehyde (MDA) (GST) and heme oxygenase-1 (HO-1) were measured. The localization and semi-quantitative expression of Nrf2 protein in hepatocytes were detected by immunohistochemistry. Results The serum levels of AST, ALT and ALP in DCM-treated mice were significantly higher than those in the blank control group, solvent control group and tBHQ intervention group (P <0.05), while the blank control group, solvent control group and tBHQ There was no significant difference in the serum AST, ALT and ALP between intervention group mice (P> 0.05). Compared with the blank control group, the activity of GST and HO-1 in the liver homogenate of the solvent control group and the tBHQ intervention group were increased; the content of ROS and MDA and the activity of GST and HO-1 Were increased, the differences were statistically significant (P <0.05). Compared with the DCM group, the levels of ROS and MDA in the liver homogenate of the mice in the tBHQ intervention group decreased, but the activities of GST and HO-1 in the tBHQ intervention group were significantly increased (P <0.05). The levels of Nrf2 protein in liver tissue of DCM-treated and tBHQ-treated mice were significantly higher than those of blank control group (P <0.05). The level of Nrf2 protein expression in tBHQ intervention group was higher than that in DCM group (P <0.05). Nrf2 protein expression was increased in DCM-treated and tBHQ-treated groups, and increased in nucleus. Conclusion The Nrf2-ARE signaling pathway may play a role in anti-oxidative compensation in the sub-acute oxidative damage of liver induced by DCM, which may reduce the damage of DCM to some extent.