基于Keap1/Nrf2/ARE信号通路探讨葛花、枳椇子及其配伍对急性酒精性肝损伤小鼠抗氧化应激的作用机制

Flos Puerariae,Hoveniae Semen and Their Combinations Treat Oxidative Stress in Mice with Acute Alcoholic Liver Injury via Keap1/Nrf2/ARE Signaling Pathway

目的:观察葛花、枳椇子及其配伍对急性酒精性肝病的改善作用,为临床用药提供科学依据,并为开展其他酒精性相关疾病的研究提供思路和借鉴。方法:采用一次性灌胃给予56%(V/V)红星二锅头酒(12 mL·kg^(-1))建立小鼠急性酒精性肝损伤模型,120只雄性ICR小鼠按体质量随机分成空白组、模型组、水飞蓟宾组、葛花组、枳椇子组、葛花-枳椇子1∶1组、葛花-枳椇子1∶2组、葛花-枳椇子2∶1组,每组15只。各给药组按10 mL·kg^(-1)预防性灌胃相应药物3 d,除空白组外,其余各组小鼠按12 mL·kg^(-1)分别灌胃给予二锅头酒,于酒后12 h处死小鼠,并观察药物对小鼠肝功能、抗氧化应激能力的影响;苏木素-伊红(HE)染色观察肝脏病理变化;蛋白免疫印迹法(Western blot)检测各组小鼠Kelch样ECH相关蛋白1(Keap1)/核因子E2相关因子2(Nrf2)/抗氧化响应元件(ARE)信号通路相关蛋白表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测相关各指标mRNA表达。结果:与正常组比较,模型组小鼠血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)水平,肝组织中丙二醛(MDA)、活性氧(ROS)含量显著升高(P<0.01);谷胱甘肽(GSH)、超氧化物歧化酶(SOD)活性显著降低(P<0.01),Keap1 mRNA及蛋白表达明显升高(P<0.05,P<0.01),Nrf2 mRNA及蛋白表达显著降低(P<0.01);与模型组比较,葛花-枳椇子2∶1组小鼠ALT、AST和ALP水平显著减低(P<0.01),肝脏组织中MDA、ROS水平均显著减低(P<0.01),GSH、SOD水平均显著升高(P<0.01);肝脏脂肪变损伤均减轻,显著上调Nrf2 mRNA及蛋白表达(P<0.01),显著下调Keap1mRNA及蛋白表达(P<0.01)。结论:葛花、枳椇子及其配伍组合对小鼠急性酒精性肝损伤有一定的预防作用,其机制可能与调控肝脏内Keap1/Nrf2/ARE信号通路,恢复并上调由乙醇所破坏的肝脏氧化平衡有关,从而有效遏制酒精性肝病的发展。

Objective:To observe the effect of Hoveniae Semen,Flos Puerariae and their combinations on acute alcoholic liver disease and provide a scientific basis for the drug use in clinical practice and the research on other alcoholic diseases.Method:The acute alcoholic liver injury model of mice was established by one-time gavage with 56%(V/V)Hongxing Erguotou liquor(12 mL·kg^(-1)).One hundred and twenty male ICR mice were randomly assigned into blank group,model group,silybin group,Flos Puerariae group,Hoveniae Semen group,and Flos Puerariae-Hoveniae Semen combination groups(ratios of 1∶1,1∶2 and 2∶1,respectively),with 15 mice in each group.Each group was administrated with 10 mL·kg^(-1) corresponding preventive drugs for 3days by gavage.Except the blank group,the other groups were given Erguotou liquor by gavage at 12 mL·kg^(-1).The mice were sacrificed 12 h after drinking for the observation of liver function and oxidative stress.The pathological changes of liver were observed via hematoxylin-eosin(HE)staining.Western blot was employed to determine the expression levels of proteins in the Kelch-like Ech-associated protein 1(Keap1)/nuclear factor E_(2)-related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect mRNA levels of related genes.Result:Compared with control group,the modeling elevated the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and alkaline phosphatase(ALP)in the serum and the content of malondialdehyde(MDA)and reactive oxygen species(ROS)in liver tissue(P<0.01)and decreased the activities of glutathione(GSH)and superoxide dismutase(SOD)(P<0.01).The mRNA and protein expressions of Keap1 were significantly increased(P<0.05,P<0.01),mRNA and protein expressions of Nrf2 were significantly decreased(P<0.01).Compared with model group,Flos Puerariae-Hoveniae Semen 2∶1 lowered the levels of ALT,AST and ALP in the serum(P<0.01)and MDA and ROS in the liver(P<0.01),and increased the activities of GSH and SOD(P<0.01).Moreover,it alleviated the hepatic steatosis injury,up-regulated mRNA and protein levels of Nrf2(P<0.01),and down-regulated the mRNA and protein levels of Keap1(P<0.01).Conclusion:Flos Puerariae,Hoveniae Semen and their combinations may exert the pre-protective effect on acute alcoholic liver injury in mice by regulating the Keap1/Nrf2/ARE pathway in the liver and restoring the liver oxidative balance destroyed by ethanol to inhibit the development of alcoholic liver disease.