基于Keap1/Nrf2信号通路研究紫檀芪对对乙酰氨基酚诱导的小鼠急性肝损伤的保护作用

Protective Effect of Pterostilbene Against Acetaminophen-induced Acute Liver Injury in Mice Based on Keap1/Nrf2/Mitochondria Pathway

关键词:  紫檀芪  对乙酰氨基酚  急性肝损伤  Keap1/Nrf2  线粒体

Key words:  pterostilbene  acetaminophen  acute liver injury  Keap1/Nrf2  mitochondria

摘要:

目的 观察紫檀芪（pterostilbene，PTE）对对乙酰氨基酚（acetaminophen，APAP）诱导的小鼠急性肝损伤的保护作用，并探讨相关机制。方法 利用APAP （250 mg·kg^-1）制备小鼠急性肝损伤模型，PTE分别给予15，30，60 mg·kg^-1，连续灌胃15 d预保护。自动生化分析仪检测谷丙转氨酶（alanine aminotransferase，ALT）、谷草转氨酶（aspartate aminotransferase，AST）、总蛋白（total protein，TP）、白蛋白（albumin，Alb）并计算白球比（Alb/Glb，A/G）；苏木精-伊红（HE）染色法进行肝组织病理学检测并进行分级评价；试剂盒检测超氧化物歧化酶（superoxide dismutase，SOD）、还原型谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）水平；JC-1检测线粒体膜电位；TUNEL染色法检测肝细胞凋亡情况；Western blotting检测Bax、cytochrome C、转录因子NF-E2相关因子2（nuclear factor erythroid 2-related factor-2，Nrf2）、Kelch样环氧氯丙烷相关蛋白1（Keleh-like ECH-associated protein l，Keap1）的蛋白表达情况。结果 与APAP模型组相比，不同浓度PTE组小鼠血清ALT、AST值显著降低（P<0.01），Alb、TP及A/G比值显著升高（P<0.05或P<0.01）；肝组织病理损伤分级降低；肝组织SOD、GSH显著升高（P<0.01），MDA显著降低（P<0.01）；线粒体膜电位增加（P<0.01）；TUNEL染色结果显示PTE组小鼠肝细胞凋亡明显减少；PTE组与APAP模型组相比，细胞质中的Bax表达显著增加（P<0.05或P<0.01），cytochrome C表达显著降低（P<0.01），而对应的线粒体中Bax表达显著降低（P<0.01），cytochrome C表达显著增加（P<0.01），同时伴有肝组织总的Nrf2表达增加（P<0.01），Keap1表达降低（P<0.01），细胞质Nrf2表达增加（P<0.01），细胞核Nrf2表达增加（P<0.01）。结论 PTE可以保护APAP诱导的小鼠急性肝损伤，其机制可能与PTE上调Nrf2水平，抑制氧化应激，保护线粒体功能有关。

Abstract:

OBJECTIVE To observe the protective effect of pterostilbene(PTE) on acute liver injury induced by acetaminophen(APAP) in mice, and to explore related mechanisms. METHODS Acute liver injury model of mice was established by APAP(250 mg·kg^-1), PTE was given 15, 30, 60 mg·kg^-1 by intragastric for 15 d pre-protection. The serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TP) and albumin(Alb) were determined by automatic biochemical analyzer and the albumin/globulin(A/G) ratio was calculated. The liver tissue was stained by hematoxylin-eosin(HE) staining and liver histopathology detection and grading evaluation were calculated. The superoxide dismutase(SOD), reduced glutathione(GSH), malondialdehyde(MDA) level were assayed by related kits. The mitochondrial membrane potential was measured by JC-1. Apoptosis of hepatic cell were detected by TUNEL staining. The protein expressions of Bax, cytochrome C, nuclear factor erythroid 2-related factor-2(Nrf2) and Keleh-like ECH-associated protein 1(Keap1) were detected by Western blotting. RESULTS Compared with APAP model group, the serum ALT and AST values of mice in different concentrations of PTE group were significantly reduced(P<0.01), and Alb, TP and A/G ratio were significantly increased(P<0.05 or P<0.01); the histopathological grade of liver damage reduced; the SOD, GSH level of liver tissue significantly increased(P<0.01), and MDA level significantly decreased(P<0.01); moreover, the mitochondrial membrane potential increased(P<0.01); TUNEL staining results showed that apoptosis of hepatocytes was significantly reduced in PTE group. Compared with APAP model group, the protein expression of cytoplasmic Bax in PTE group was significantly increased(P<0.05 or P<0.01) while cytochrome C significantly decreased(P<0.01), the expression of Bax in the corresponding mitochondria was significantly decreased(P<0.01), cytochrome C significantly increased(P<0.01). The whole Nrf2 protein expression was increased(P<0.01) while the Keap1 decreased(P<0.01), whats more, the expression of nuclear Nrf2 increased(P<0.01) and the expression of cytoplasmic Nrf2 increased(P<0.01). CONCLUSION PTE can protect APAP-induced acute liver injury. The mechanism may be related to the up-regulation of Nrf2 level, inhibition of oxidative stress and protected the mitochondrial function by PTE.