基于Nrf2/HO-1/NQO1信号通路探讨雷公藤内酯三醇对急性肝损伤大鼠的保护机制

Discussion on Protective Mechanism of Triptolide Against Acute Liver Injury in Rats Based on Nrf2/HO-1/NQO1 Signaling Pathway

目的:基于核因子E2相关因子2/血红素氧合酶/醌氧化还原酶1(Nrf2/HO-1/NQO1)信号通路探究雷公藤内酯三醇对四氯化碳(CCl4)诱导急性肝损伤大鼠的保护机制。方法:纳入40只SPF级雄性SD大鼠,适应性饲养7 d,而后随机分为4组(正常组、模型组及低剂量组、高剂量组),每组10只。正常组及模型组每日尾静脉注射等体积无菌0.9%氯化钠溶液,低剂量组、高剂量组分别注射100 mg/kg、200 mg/kg雷公藤内酯三醇,均连续注射7 d,于第7天注射雷公藤内酯三醇(模型组为无菌生理盐水)3 h后,对模型组、低剂量组、高剂量组大鼠腹腔注射1 mL/kg CCl4以进行急性肝损伤模型诱导(正常组腹腔注射等体积的橄榄油溶液)。在注射CCl416 h后取大鼠血液及肝脏标本,采用HE组织化学染色法观察大鼠肝组织切片的病理学改变,并检测4组血清肝功能指标水平及肝组织Nrf2、HO-1、NQO1蛋白水平及mRNA表达量。结果:肝组织病理检查结果显示,正常组肝组织细胞排列整齐,组织结构及形态正常,细胞界限明显,胞质完整。模型组在注射CCl416 h后,肝组织出现肝细胞边界不清晰、细胞排列紊乱等肝细胞坏死特征,且可见炎症细胞浸润及肝细胞严重水肿呈气泡样变。相比于模型组,低剂量组、高剂量组的肝细胞水肿程度及肝细胞坏死特征较轻,且高剂量组轻于低剂量组。模型组、低剂量组及高剂量组的肝指数大于正常组,血清天冬氨酸转氨酶(AST)、甲氨酸转氨酶(ALT)水平高于正常组(P<0.05);模型组肝指数大于低剂量组及高剂量组,且低剂量组大于高剂量组(P<0.05);模型组AST、ALT水平高于低剂量组及高剂量组,且低剂量组高于高剂量组(P<0.05)。模型组、低剂量组及高剂量组的肝组织Nrf2、HO-1、NQO1蛋白水平及Nrf2、HO-1、NQO1 mRNA表达量高于正常组,且模型组高于低剂量组及高剂量组,低剂量组高于高剂量组(P<0.05)。结论:雷公藤内酯三醇可减轻CCl4诱导的大鼠急性肝损伤,其作用机制可能与其能激活Nrf2/HO-1/NQO1信号通路以抑制肝细胞氧化损伤有关。

Objective:To explore the protective mechanism of triptolide against acute liver injury induced by carbon tetrachloride(CCl4)in rats based on nuclear factor E2 related factor 2/heme oxygenase/quinone oxidoreductase 1(Nrf2/HO-1/NQO1)signaling pathway.Methods:Forty SPF male SD rats were included and fed for seven days,and then were randomly divided into four groups(the normal group,the model group,the low-dose group and the high-dose group),with 10 rats in each group.The normal group and the model group were injected with equal volume of 0.9%sodium chloride solution daily through the tail vein,and the low-dose group and the high-dose group were respectively injected with 100 mg/kg and 200 mg/kg triptolide for seven days.Three hours after the injection of triptolide on the seventh day(sterile saline in the model group),rats in the model group,the low-dose group and the high-dose group were intraperitoneally injected with 1 mL/kg CCl4 to induce acute liver injury model(the normal group was intraperitoneally injected with equal volume of olive oil solution).Sixteen hours after CCl4 injection,the blood and liver samples of rats were collected,and the pathological changes of their liver sections were observed through HE histochemical staining,and the levels of serum liver function indexes,the protein levels and the mRNA expressions of Nrf2,HO-1 and NQO1 in liver tissues of the four groups were detected.Results:The histopathological examination showed that the liver cells in the normal group were arranged neatly,the structure and shape were normal,the cell boundaries were obvious,and that the cytoplasm was intact.In the model group,sixteen hours after CCl4 injection,the liver tissues showed the characteristics of hepatocyte necrosis such as unclear hepatocyte boundary and disordered cell arrangement,and the inflammatory cell infiltration and severe edema of hepatocytes showed bubble-like change.Compared with those in the model group,the degree of hepatocyte edema and the characteristics of hepatocyte necrosis in the low-dose and high-dose groups were lighter,and the degree in the high-dose group was lighter than that in the low-dose group.The liver indexes in the model group,the low-dose group and the high-dose group were higher than those in the normal group,and the AST and ALT levels were higher than those in the normal group(P<0.05);the liver indexes in the model group were higher than those in the low-dose group and the high-dose group,and the indexes in the low-dose group were higher than those in the high-dose group(P<0.05);the AST and ALT levels in the model group were higher than those in the low-dose and the high-dose groups,and the levels in the low-dose group were higher than those in the high-dose group(P<0.05).The protein levels and mRNA expressions of Nrf2,HO-1 and NQO1 in liver tissues of the model group,the low-dose group and the high-dose group were higher than those in the normal group;the above levels in the model group were higher than those in the low-dose group and the high-dose group,and the above levels in the low-dose group were higher than those in the high-dose group(P<0.05).Conclusion:Triptolide can alleviate CCl4-induced acute liver injury in rats,and its mechanism may be related to the activation of Nrf2/HO-1/NQO1 signaling pathway to inhibit oxidative damage of hepatocytes.