葛菊护肝片调节NF-κB和Bcl-2/Bax信号通路改善酒精所致的小鼠肝脏损伤

Geju Hugan Tablets Ameliorate Alcohol-induced Liver Injury in Mice by Regulating NF-κB and Bcl-2/Bax Signaling Pathways

目的:探究葛菊护肝片对酒精性肝损伤小鼠肝脏的保护作用,并基于核转录因子-κB p65(NF-κB p65)和B细胞淋巴瘤-2(Bcl-2)蛋白/Bcl-2相关X蛋白(Bax)信号通路探讨其作用机制。方法:SPF级雄性ICR小鼠60只,按体质量随机分为正常组、模型组、复方益肝灵片组、葛菊护肝片低、中、高剂量组。葛菊护肝片低、中、高剂量组分别灌胃0.2、0.4、0.8 g·kg^(-1)葛菊护肝片;复方益肝灵片组灌胃剂量0.16 g·kg^(-1)的复方益肝灵片,连续28 d,每天给药1次;正常组及模型组灌胃等体积纯水。第29天除正常组外,其余各组小鼠灌胃53°白酒,灌胃2次,灌胃间隔6 h;第30天取材。苏木素-伊红(HE)染色观察肝组织病理改变;透射电镜观察肝细胞超微结构改变;酶联免疫吸附测定法(ELISA)检测肝组织丙二醛(MDA)、还原型谷胱甘肽(GSH)、甘油三酯(TG)、血清丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)含量;蛋白免疫印迹法(Western blot)检测肝组织NF-κB p65、磷酸化核因子抑制蛋白α(p-IκBα)、Bcl-2、Bax蛋白表达。结果:与正常组比较,模型组血清ALT、AST含量显著升高(P<0.01),肝组织MDA、TG含量显著升高(P<0.01),GSH含量显著下降(P<0.01),肝脏HE染色、油红O和电镜肝损伤评分显著升高(P<0.01);肝组织NF-κB、p-IκBα、Bax蛋白表达量均明显升高(P<0.05,P<0.01),Bcl-2蛋白表达量明显降低(P<0.05)。与模型组比较,葛菊护肝片低、中、高剂量组血清ALT、AST含量显著下降,肝组织MDA、TG含量显著下降,GSH含量显著增加(P<0.01);葛菊护肝片中、高剂量组HE和电镜评分显著降低(P<0.01);葛菊护肝片中、高剂量组小鼠肝组织NF-κB、p-IκBα、Bax蛋白表达量显著降低(P<0.01),葛菊护肝片低、中、高剂量组Bcl-2蛋白表达量显著升高(P<0.01)。结论:葛菊护肝片对ALD小鼠肝脏有一定的保护作用,其机制可能是通过下调NF-κB信号通路减轻肝组织炎症,下调Bax蛋白表达,上调Bcl-2蛋白表达抑制肝细胞凋亡实现的。

Objective:To investigate the protective effect of Geju Hugan tablets on the liver of mice with alcohol-induced liver injury,and explore the underlying mechanism based on nuclear factor-κB p65(NF-κB p65)and B-cell lymphoma-2(Bcl-2)/Bcl-2-associated X protein(Bax)signaling pathways.Method:According to the body weight,60 SPF-grade male ICR mice were randomized into normal,model,Compound Yiganling tablets(0.16 g·kg^(-1)),and low-,medium-,and high-dose(0.2,0.4,0.8 g·kg^(-1),respectively)Geju Hugan tablets groups.The drugs were administrated at the corresponding doses by gavage,and the normal and model groups with equal volume of pure water once a day for 28 consecutive days.On day 29,the mice in other groups except the normal group were administrated with liquor(53%Vol)by gavage twice a day at the doses of 20,10 mL·kg^(-1) and with the interval of 6 h.Samples were harvested on day 30.The histopathological changes in the liver were observed by hematoxylin-eosin(HE)staining,and the ultrastructural changes in hepatocytes were observed by transmission electron microscopy.The enzyme-linked immunosorbent assay was employed to measure the levels of malonaldehyde(MDA),reduced glutathione(GSH),and triglycerides(TG)in the liver tissue and the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the serum.Western blotting was employed to determine the protein levels of NF-κB p65,phosphorylated p-inhibitor kappa B alpha(p-IκBα),Bcl-2,and Bax in the liver tissue.Result:Compared with the normal group,the model group showed increases in the ALT,AST,MDA,and TG levels,a decrease in the GSH level,and increases in the liver injury scores evaluated based on the HE,oil red O,and transmission electron microscopy(P<0.01).Moreover,the model group showed up-regulated expression of NF-κB,p-IκBα,and Bax(P<0.05,P<0.01)and down-regulated expression of Bcl-2(P<0.05)in the liver tissue.Compared with the model group,Geju Hugan tablets of all the doses lowered the ALT,AST,MDA,and TG levels and elevated the GSH level(P<0.01).The liver injury scores assessed based on HE staining and transmission electron microscopy in the medium-and high-dose Geju Hugan tablets groups were lower than those in the model group(P<0.01).Compared with the model group,medium-and high-dose Geju Hugan tablets down-regulated the protein levels of NF-κB,p-IκBα,and Bax(P<0.01)and all doses of Geju Hugan tablets up-regulated the protein level of Bcl-2(P<0.01).Conclusion:Geju Hugan tablets protect mice from alcohol-induced liver injury by downregulating NF-κB signaling pathway to alleviate inflammation in the liver tissue and down-regulating the expression of Bax and up-regulating the expression of Bcl-2 to inhibit hepatocyte apoptosis.