熊胆粉通过调节Keap1/Nrf2/ARE信号通路产生对急性酒精性肝损伤小鼠的肝保护作用

Protective Effects of Pulvis Fellis Ursi on Acute Alcohol-induced Liver Injury in Mice by Regulating Keap1/Nrf2/ARE Signaling Pathway

目的观察研究熊胆粉(Pulvis Fellis Ursi,PFU)对急性酒精性肝损伤小鼠的肝保护作用及其可能的机理。方法取昆明种小鼠50只,随机分为正常组、模型组、PFU低、中、高剂量组(剂量分别为150、300、600 mg·kg^(-1),溶于生理盐水),每组10只。正常组和模型组小鼠每天灌胃生理盐水(10 mL·kg^(-1)),PFU组每天灌胃相应剂量溶液,连续8天。末次给药结束后,模型组以灌胃的方法给予50%乙醇溶液(10 mL·kg^(-1)),每12 h一次,共6次,末次给予50%乙醇溶液4 h后,处死小鼠并收集血清及肝脏组织。分别用生化试剂盒检测血清中ALT、AST、TG、TC和肝脏组织中GSH、T-SOD和MDA水平;用酶联免疫法检测肝脏组织TNF-α、IL-1β和IL-6水平;用HE染色法观察肝脏组织显微结构变化情况;用免疫印迹法检测肝组织总Keap1的表达和肝组织核内Nrf2的表达,用免疫印迹和免疫组化法分别检测肝组织总HO-1和GCLC蛋白的表达。结果PFU能显著下调乙醇所致肝损伤小鼠血清中ALT、AST、TG和TC的异常升高,并改善乙醇诱导的肝脏显微结构变化。与肝损伤模型组比较,PFU组小鼠肝组织内MDA水平明显降低,GSH和T-SOD水平则明显增加。进一步研究表明,乙醇能诱导小鼠肝脏内炎症因子TNF-α、IL-1β和IL-6的上调,PFU可以剂量依赖性地缓解了上述病变。并且PFU预处理组下调了Nrf2的抑制因子Keap1的表达,从而促进了Nrf2蛋白的入核并上调了乙醇抑制的下游蛋白HO-1和GCLC的表达,从而发挥肝保护的作用。结论熊胆粉对小鼠急性酒精性肝损伤有一定的预保护作用,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。

Objective To observe the protective effects of Pulvis Fellis Ursi(PFU)on alcohol-induced liver injury in mice and possible underlying mechanisms.Methods Fifty Kunming mice were randomly divided into 5 groups,i.e.,normal group,liver injury model group,PFU low dosage group(150 mg·kg^(-1)),PFU medium dosage group(300 mg·kg^(-1))and PFU high dosage group(600 mg·kg^(-1)).Each group contained 10 mice.Normal group and liver injury model group were gavaged with normal saline(10 mL·kg^(-1))every day,and PFU groups were gavaged with corresponding dose of solution for 8 consecutive days.After the last administration,the model group was gavaged with 50%ethanol solution(10 mL·kg^(-1))once every 12 h,for 6 times in total.Four hours after the last administration of 50%ethanol solution,the mice were sacrificed and serum and liver tissues were collected.After the experiment we related physiological and biochemical parameters,including ALT,AST,TG,TC,GSH,MDA,T-SOD,TNF-α,IL-1βand IL-6 were determined.The micromorphology of the mouse liver was observed after HE staining.The expression of Keap1 in total of liver tissue and Nrf2 in nucleus of liver tissue were detected by immunoblot.The expression of HO-1,and GCLC were detected by immunoblot or immunohistochemistry.Results PFU significantly downed regulate the abnormal increase of alt,AST,TG and TC in serum of mice with ethanol induced liver injury,and improve the changes of liver microstructure induced by ethanol.Compared with the liver injury model group,the level of MDA in liver tissue of PFU group decreased significantly,while the levels of GSH and T-SOD increased significantly.Further studies showed that ethanol could induce the up-regulation of TNF-α,IL-1βand IL-6 in mice,PFU alleviated the above lesions in a dose-dependent manner.Moreover,PFU pretreatment group down regulated the expression of Nrf2 inhibitor Keap1,promoted the nuclear entry of Nrf2 protein,and up-regulated the expression of ethanol inhibited downstream proteins HO-1 and GCLC,so as to play a role in liver protection.Conclusion PFU considerably ameliorates alcohol-induced liver injury in mice,and its mechanism may be related to the regulation of Keap1/Nrf2/ARE signaling pathway in liver and recovery and upregulation of hepatic oxidative balance disrupted by ethanol.