四氯化碳诱导小鼠急性肝损伤中Nrf2信号通路的变化

Changes of Nrf2 pathway in acute liver injury induced by CCl4 in mice

目的探讨Nrf2信号通路在CCl4诱导的小鼠急性肝损伤中的表达变化。方法 40只C57BL/6小鼠随机分正常对照组和CCl4实验组,腹腔注射CCl4油溶液(0.1 ml/10 g)建立小鼠急性肝损伤模型。24 h后检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)含量;苏木素-伊红(HE)染色观察肝组织形态学变化;RT-PCR和Western blot检测Nrf2相关基因和蛋白的表达。结果与正常对照组比较,5μl/kg·bw CCl4组血清中ALT和AST含量无明显变化,而当CCl4浓度分别增加至10、20和30μl/kg·bw时,小鼠血清中ALT和AST含量明显升高(P<0.01);HE染色结果显示,5μl/kg·bw CCl4组未出现肝病理损伤,而随着CCl4剂量分别增加至10、20和30μl/kg·bw时,肝组织出现明显的病理损伤,且随着CCl4浓度升高肝组织损伤加重;RT-PCR结果显示,CCl4处理后肝组织中Nrf2、Keap1、Ho-1和Nqo1 mRNA的表达均明显增加(P<0.05),其中20和30μl/kg·bw CCl4组,Nrf2、Keap1和Gclc mRNA的表达降低(P<0.05);Western blot结果显示:CCl4各实验组中NRF2、KEAP1蛋白表达无明显变化,而HO-1蛋白表达在20和30μl/kg·bw CCl4组中明显增加(P<0.05),NQO1蛋白表达在10和20μl/kg·bw CCl4组中明显增加,差异有统计学意义(P<0.05)。结论在不同剂量CCl4诱导的小鼠急性肝损伤中,20μl/kg·bw CCl4建立的急性肝损伤模型更有利于进行与Nrf2相关的分子机制研究。

Objective To investigate the changes of nuclear factor erythroid 2-related factor 2(Nrf2) in CCl4-induced hepatotoxicity in mice. Methods Forty male C57/BL6 mice were randomly divided into four groups(eight in each group): control group and experimental groups were treated with different doses of CCl4. Experimental groups were intraperitoneally injected CCl4 olive oil solution(0.1 ml/10 g) to establish the acute liver injury model. Blood and liver samples were collected 24 h after the treatment. Serum alanine transaminase(ALT), aspartate aminotransferase(AST), and histopathology(using HE staining) were determined as the sign of liver injury. RT-PCR and Western blot analysis were employed to measure the expression of Nrf2-targeted molecules. Results Compared with the normal control group, the contents of ALT and AST in serum of 5 μl/kg·bw CCl4 group had no significant change, but when the concentration of CCl4 increased to 10, 20 and 30 μl/kg·bw, the contents of ALT and AST in serum of mice increased significantly(P<0.01). The morphology of hepatocytes in the control group was normal, however, experimental groups showed pathological lesions. The expressions of Nrf2, Keap1, Ho-1 and Nqo-1 mRNA were significantly increased in all experimental groups(P<0.05), however, the expressions of Nrf2, Keap1 and Gclc mRNA decreased in a dose-dependent manner. For the Western blot result, NRF2 and KEAP1 protein expression levels were not changed, HO-1 and NQO1 protein expression levels were increased in 20 and 30 μl/kg·bw CCl4-treated group(P<0.05). NQO1 protein expression level was increased in the liver specimens of 10 and 20 μl/kg·bw CCl4 group(P<0.05). Conclusion The establishment of acute liver injury model by using 20 μl/kg·bw CCl4 is more conducive to the study of acute liver injury releted with the on Nrf2 signaling pathway.