早期生长反应因子1信号通路在大鼠急性肝内胆汁淤积性肝损伤中的作用

Role of early growth response factor-1 signal pathway in acute intrahepatic cholestatic hepatic injury in rats

目的 探讨早期生长反应因子1（Egr-1）信号通路在大鼠急性肝内胆汁淤积性肝损伤中的作用。方法 α-异硫氰酸萘酯50mg／kg一次灌胃建立大鼠急性肝内胆汁淤积性肝损伤模型，实时荧光定量PCR检测灌胃后24、48、72h肝组织中Egr-1、细胞因子诱导的中性粒细胞趋化因子1和巨噬细胞炎症蛋白2mRNA表达量，免疫组织化学法检测诱导型一氧化氮合酶蛋白表达，双抗体夹心酶联免疫吸附法检测肝组织匀浆上清液中肿瘤坏死因子Ⅸ和白细胞介素6含量，硫代巴比妥酸比色法、黄嘌呤氧化酶法和硝酸还原酶法分别检测肝组织匀浆上清液中丙二醛、超氧化物歧化酶和一氧化氮含量，比色法检测肝组织中髓过氧化物酶含量。结果24、48、72h模型组肝组织中，中性粒细胞趋化因子1和巨噬细胞炎症蛋白2mRNA表达量均高于对照组（P值均〈0．05），24、48h模型组肝组织中Egr1mRNA表达量均高于对照组（P值均〈0．05），72h模型组肝组织Egr-1mRNA表达量与对照组的差异无统计学意义（P〉0．05）；3个时间点模型组肝组织一氧化氮合酶吸光度值均低于对照组（P值均〈0．05），肿瘤坏死因子α、白细胞介素6、髓过氧化物酶、丙二醛均高于对照组（P值均〈0．05），一氧化氮、超氧化物歧化酶则低于对照组（P值均〈0．05）。结论肝内胆汁淤积性肝损伤存在以Egr-1为起始因子的信号传导通路，从而引起肝细胞损伤。诱导型一氧化氮合酶和一氧化氮在肝内胆汁淤积性肝损伤信号通路中可能发挥保护作用。

[Abstract] Objective To explore the role of early growth response factor-1 （Egr-1） signal pathway in acute intrahepatic cholestatic liver injury in rats. Methods A single dose （50 mg/kg） of ct- naphthylisothiocyanate （ANIT） was administered by gavage to each experimental rat to induce intrahepatic cholestasis. Liver tissue and serum specimens were collected from rats at 24, 48 and 72 h after the intoxication. The values of Egr-1, cytokine induced neutrophil chemoattractant I（CINC-1）, macrophage inflammatory protein-2 （MIP-2） mRNA, the protein expression of inducible nitricoxide synthase （iNOS） and the values of tumor necrosis factor alpha （TNF alpha） and interleukin 6 （IL-6） were assayed by real-time PCR, immunohistochemistry, and ELISA, respectively. The levels of MDA, SOD, NO and MPO were assayed by thiobarbitudc acid method, xanthine oxidase method, nitric acid deoxidizing assay, and colorimetric method, respectively. Results In the model group at 24, 48, 72 h after intoxication, the values of CINC-1 and MIP- 2 mRNA were higher than those of the controls （P 〈 0.05）. In the model group at 24, 48 h after intoxication, the value of Egr-1 mRNA was higher than that of the controls （P 〈 0.05）, but there was no significant difference at 72 h （P 〉 0.05）. Of the model group, the absorbance value of iNOS was lower than that of the controls at 24, 48 and 72 h （P 〈 0.05）. Of the model group at 24, 48, 72 h after intoxication, the values of TNF alpha, IL-6, MPO and MDA were higher than those of the controls （P 〈 0.05）, but the values of NO and SOD were lower than those of the controls （P 〈 0.05）. Conclusions Egr-1 signal pathway is involved in acute intrahepatic cholestatic liver injury induced by ANIT. After Egr-1 was activated, CINC-1 and MIP-2 are activated consequently and attract neutrophils into the liver. TNF alpha and IL-6 are activated at the same time, so neutrophils are activated and the resulting lipid peroxidation and MDA increased, injuring the liver, iNOS and NO may play a protective role in acute intrahepatic cholestatic liver injury induced by ANIT.