抗纤丸激活Nrf2信号通路抗小鼠慢性肝损伤的作用和机制研究

Effect and mechanism of Kangxian Pills againstchronic liver injury in mice by activating Nrf2 signaling pathway

[目的]探讨抗纤丸对四氯化碳(CCL4)诱导的慢性肝损伤模型小鼠的保护作用及机制。[方法]BALB/c小鼠,分为正常组、模型组、甘草酸二铵组[60 mg/(kg·d)]、抗纤丸组[3 g/(kg·d)]。腹腔注射CCL4橄榄油溶液建立慢性肝损伤小鼠模型,正常组腹腔注射等体积橄榄油。造模成功后,灌胃给药4周,检测血清丙氨酸转氨酶(ALT),天冬氨酸转氨酶(AST)和水平;苏木精-伊红(HE)染色观察肝组织病理改变,Masson染色观察肝组织胶原沉积,免疫组化法观察肝组织中α-平滑肌动蛋白(α-SMA)的表达;检测肝组织中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)以及丙二醛(MDA)的含量;实时荧光定量PCR和Western Blot法检测肝组织中核因子相关因子2(Nrf2)、血红素氧合酶1(HO-1)、谷氨酸半胱氨酸连接酶催化亚基(Gclc)和醌氧化还原酶1(NQO1)的mRNA和蛋白表达水平。[结果]与正常组相比,模型组小鼠血清AST和ALT的水平显著升高;肝组织中胶原,α-SMA以及MDA的表达显著增加而SOD、GSH-Px的活性显著降低,此外,肝组织中Nrf2、HO-1、Gclc和NQO1的mRNA的表达水平以及Nrf2、Gclc和NQO1的蛋白表达水平均有不同程度的降低。与慢性肝损伤模型小鼠相比,抗纤丸组降低了血清AST和ALT的水平;减少肝组织中胶原的含量、抑制α-SMA的表达;降低肝组织中MDA的含量,同时升高肝组织中SOD、GSH-Px的活性;此外,能显著上调肝组织中Nrf2、HO-1、Gclc和NQO1的mRNA的表达水平以及Nrf2、Gclc和NQO1的蛋白表达水平。[结论]抗纤丸对CCL4诱导的慢性肝损伤模型小鼠具有保护作用,其作用机制可能是通过激活Nrf2信号通路抑制肝细胞氧化应激。

[Objective]To investigate the protective effect and mechanism of Kangxian Pills on CCl4 induced chronic liver injury in mice.[Methods]BALB/c mice were divided into normal group,model group,diammonium glycyrrhizinate group[60 mg/(kg·d)]and Kangxian pills group[3 g/(kg·d)].CCl4 was injected intraperitoneally to establish the model of chronic liver injury in mice,at the same time,the same volume of olive oil was injected intraperitoneally in the normal group.After the model was established successfully,the medicine were given for 4 weeks.We detected the serum AST and ALT levels,HE staining was used to observe the pathological changes of liver tissue,and the collagen deposition in liver tissue was observed by Masson staining;the expression ofα-SMA in liver tissue was observed by immunohistochemistry;the content of SOD,GSH-Px and MDA in liver tissue were detected,mRNA and protein expression of Nrf2,HO-1,Gclc and NQO1 in liver tissue were detected by qPCR and Western Blot.[Results]Compared with the normal group,the levels of serum AST and ALT in the model group were significantly increased;the expression of collagen,α-SMA and MDA in liver tissues were significantly increased while the activities of SOD and GSH-Px were significantly decreased.In addition,the mRNA expression levels of Nrf2,HO-1,Gclc and NQO1 and the protein expression levels of Nrf2,Gclc and NQO1 in liver tissues were reduced to different degrees.Compare with the mice with chronic liver disease,Kangxian Pills group could significantly decreased the activities of AST and ALT in serum,reduced the content of collagen in liver tissue and inhibitedthe expression ofα-SMA;reduced the content of MDA in liver and increased the content of SOD and GSH-Px in liver.In addition,the mRNA and protein expression of Nrf2,HO-1,Gclc and NQO1 in liver were also significantly up-regulated.[Conclusion]Kangxian Pills could protect CCl4induced chronic liver injury in mice,and its mechanism may be to activate of Nrf2 signaling pathway and inhibition of oxidative stress in hepatocytes.