万涛, 晏彪, 洪亮. 磷酸化胞外信号调节激酶1/2在邻苯二甲酸二丁酯致雄性小鼠肝损伤中的作用[J]. 环境与职业医学, 2020, 37(6): 622-627. DOI: 10.13213/j.cnki.jeom.2020.19733
引用本文: 万涛, 晏彪, 洪亮. 磷酸化胞外信号调节激酶1/2在邻苯二甲酸二丁酯致雄性小鼠肝损伤中的作用[J]. 环境与职业医学, 2020, 37(6): 622-627. DOI: 10.13213/j.cnki.jeom.2020.19733
WAN Tao, YAN Biao, HONG Liang. Role of phosphorylated extracellular signal-regulated kinase 1/2 in dibutyl phthalate-induced hepatic damage in male mice[J]. Journal of Environmental and Occupational Medicine, 2020, 37(6): 622-627. DOI: 10.13213/j.cnki.jeom.2020.19733
Citation: WAN Tao, YAN Biao, HONG Liang. Role of phosphorylated extracellular signal-regulated kinase 1/2 in dibutyl phthalate-induced hepatic damage in male mice[J]. Journal of Environmental and Occupational Medicine, 2020, 37(6): 622-627. DOI: 10.13213/j.cnki.jeom.2020.19733

磷酸化胞外信号调节激酶1/2在邻苯二甲酸二丁酯致雄性小鼠肝损伤中的作用

Role of phosphorylated extracellular signal-regulated kinase 1/2 in dibutyl phthalate-induced hepatic damage in male mice

  • 摘要: 背景

    邻苯二甲酸二丁酯(DBP)具有潜在的肝毒性。胞外信号调节激酶1/2(ERK1/2)的活化形式磷酸化ERK1/2(p-ERK1/2)被证实与肝脏损伤有关。但尚不清楚DBP暴露是否通过p-ERK1/2导致肝损伤。

    目的

    探讨氧化应激介导的p-ERK1/2在DBP所致小鼠肝损伤中的作用。

    方法

    SPF级雄性KM小鼠28只,随机分为4组:对照组、50 mg·kg-1·d-1 DBP组、2.5 mg·kg-1·d-1 U0126(一种非三磷酸腺苷竞争性的抑制剂,可阻断ERK1/2的磷酸化和激活)组、50 mg·kg-1·d-1 DBP+2.5 mg·kg-1·d-1 U0126组,实验周期28 d。实验结束后测定小鼠肝脏的脏器系数,HE染色观察肝组织病理学变化,采用二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光染料检测各组小鼠肝组织的活性氧(ROS)水平,硫代巴比妥酸比色法检测丙二醛(MDA)含量,化学比色法检测总抗氧化能力(T-AOC),蛋白质印迹法检测肝组织ERK1/2及其磷酸化(p-ERK1/2)蛋白表达,全自动生化分析仪测定各组小鼠血清中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、白蛋白(ALB)含量。

    结果

    染毒28 d后,与对照组相比,DBP组小鼠肝脏的脏器系数(4.97±0.14)%下降,ALT/AST(2.02±0.25)上升,ALB含量(18.54±2.64)U·L-1下降,ROS荧光强度(6 387.0±84.80)增加、MDA含量(1.65±0.10)μmol·g-1(以蛋白计)上升,T-AOC水平(0.55±0.05)U·mg-1下降,p-ERK1/2蛋白表达水平(1.29±0.02)上升(P < 0.05)。经U0126处理后,与DBP组比较,DBP+U0126组小鼠肝脏的脏器系数(5.36±0.33)%上升,ALT/AST(1.40±0.17)下降,ALB含量(28.92±0.69)U·L-1上升,ROS荧光强度(5 934.8±38.07)减弱、MDA含量(1.10±0.08)μmol·g-1(以蛋白计)下降,p-ERK1/2蛋白表达水平(0.90±0.13)下调(P < 0.05)。

    结论

    DBP可提高雄性小鼠肝组织的氧化应激水平,上调p-ERK1/2蛋白表达,引起肝功能障碍;而U0126可通过抑制ERK1/2的磷酸化,改善DBP诱导的肝损伤,提示p-ERK1/2参与了DBP诱导的肝损伤。

     

    Abstract: Background

    Dibutyl phthalate (DBP) has potential hepatotoxicity. The activated form of extracellular signal-regulated kinase (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), has been confirmed to be associated with liver injury. It is unclear whether DBP exposure can lead to hepatic damage through p-ERK1/2.

    Objective

    This study explores the role of p-ERK1/2 mediated by oxidative stress in DBP-induced hepatic damage in mice.

    Methods

    SPF male KM mice (n=28) were randomly divided into four groups:control group, 50 mg·kg-1·d-1 DBP group, 2.5 mg·kg-1·d-1 U0126 (a non-adenosine triphosphate competitive specific inhibitor of ERK1/2) group, and 50mg·kg-1·d-1 DBP+2.5 mg·kg-1·d-1 U0126 group. After 28 days of experiment, the organ coefficient of mice liver was measured; the liver pathological changes were observed after HE staining; the level of reactive oxygen species (ROS) in liver was detected with 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA); the level of malondialdehyde (MDA) was detected by thiobarbituric acid colorimetric method; the total antioxidative capacity (T-AOC) was detected by chemical colorimetric assay; the expressions of ERK1/2 and p-ERK1/2 in liver tissues were detected by Western blotting; the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) in serum were determined with automatic biochemical analyzer.

    Results

    After 28 days of designed treatment, compared with the control group, the DBP group showed decreased liver organ coefficient(4.97±0.14)%, increased ALT/AST (2.02±0.25), decreased ALB level(18.54±2.64) U·L-1, increased fluorescence intensity of ROS (6 387.0±84.80), increased MDA level(1.65±0.10) μmol·g-1 (in protein), decreased T-AOC level(0.55±0.050) U·mg-1, and increased p-ERK1/2 protein expression level (1.29±0.02) (P < 0.05). After the U0126 treatment, compared with the DBP group, the DBP+U0126 group showed reversed effects, including increased liver organ coefficient(5.36±0.33)%, decreased ALT/AST (1.40±0.17), increased ALB level(28.92±0.69) U·L-1, decreased fluorescence intensity of ROS (5 934.8±38.07), decreased MDA level(1.10±0.08) μmol·g-1 (in protein), and decreased expression level of p-ERK1/2 (0.90 ±0.13) (P < 0.05).

    Conclusion

    DBP could increase the level of oxidative stress and up-regulate the expression of p-ERK1/2 protein in the liver tissues of male mice, resulting in liver dysfunction; while U0126 could repair the DBP-induced hepatic damage by inhibiting the phosphorylation of ERK1/2, suggesting that p-ERK1/2 is involved in the hepatic damage caused by DBP.

     

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