枸杞芽叶有效成分对急性酒精性肝损伤保护作用研究

Protective effect of active ingredients of Lycium barbarum leaves on acute alcoholic liver injury in mice

本文旨在研究枸杞芽叶有效成分(active ingredients of Lycium barbarum leaves,AILBL)的抗氧化能力及对急性酒精性肝损伤的保护作用。首先采用ABTS和DPPH法对AILBL的抗氧化活性进行检测;其次取48只雄性ICR小鼠随机分成6组,即正常对照组、酒精模型组、阳性药对照组水飞蓟素(silymarin)(200 mg/kg,i.g.)、AILBL低、中、高剂量组(30、100、300 mg/kg),连续灌胃1周,每天一次。末次给药12 h后以6 g/kg酒精灌胃,间隔12 h一次,共3次,建立小鼠急性酒精性肝损伤模型。检测分析小鼠血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和肝组织中丙二醛(MDA)与还原性谷胱甘肽(GSH)水平。采用苏木精-伊红(H&E)和免疫组化染色观察肝脏组织的病理学变化和氧化应激相关蛋白。体外ABTS和DPPH清除实验表明AILBL抗氧化能力强于芦丁,对DPPH·的清除优于Trolox。在酒精诱导的肝损伤模型小鼠中,AILBL预防给药能显著降低小鼠血清中ALT和AST水平,降低肝组织中MDA水平,升高肝组织中GSH水平,其作用效果呈剂量依赖关系,且中、高剂量组略优于阳性药水飞蓟素。HE组织染色分析表明AILBL能改善酒精引起的肝细胞坏死和炎症性病变。免疫组化分析显示AILBL能抑制酒精诱导的CYP2E1、HIF-1α和iNOS的表达,同时AILBL减少了乙醇刺激肝细胞诱导的ROS累积。由此可知,AILBL具有显著的体外抗氧化活性,且AILBL对急性酒精性肝损伤具有保护作用,其作用机制可能与AILBL中富含黄酮类抗氧化物进而提高小鼠机体内源性抗氧化活性、减少肝细胞脂质过氧化有关。

To investigate the antioxidant capacity of active ingredients of Lycium barbarum leaves(AILBL)and the protective effect of AILBL on acute alcoholic liver injury in mice and to reveal the antioxidant activity of AILBL.The antioxidant capacity of AILBL was determined by ABTS and DPPH methods in vitro.Furthermore,male ICR mice were randomly divided into 6 groups,control group,model group,positive control group silymarin(200 mg/kg,i.g.)and low-middle-and high-dose AILBL groups(30,100,300 g/kg,i.g.,once a day for 1 week).After pre-treatment of AILBL for 1 week,6 g/kg alcohol was administrated by gavage for 3 times to mimic acute alcoholic liver injury.The serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels,hepatic malondialdehyde(MDA)and glutathione(GSH)contents were determined.Liver histopathological changes were examined by H&E and immunohistochemical staining.ABTS as well as DPPH scavenging experiments in vitro showed that AILBL and was better than trolox on scavenging DPPH·.In the model of alcohol-induced liver injury mice,AILBL pretreatment dramatically reduced the serum ALT and AST levels,decreased hepatic MDA content and increased hepatic GSH content.In addition,AILBL alleviated the hepatocytes inflammation and necrosis induced by alcohol.Immunohistochemical analysis showed that AILBL inhibited the expression of CYP2E1,HIF-1αand iNOS induced by alcohol,and AILBL reduced the accumulation of ROS induced by ethanol in vitro.AILBL exerts strong antioxidant capacity in vitro and exhibits the protective effects on acute alcoholic liver injury in mice,and the mechanism of action is probably associated with the improvement of endogenous antioxidant activity and the reduction of lipid peroxidation of mice.