穿心莲内酯激活Nrf2抑制乙醇诱导肝细胞氧化应激损伤  

Andrographolide Activates Nrf2 to Inhibit Ethanol-induced Oxidative Stress Damage in Hepatic Cells

作　　者：曹衡玉[1] 欧阳征仁[1] 尹海辉[1] 张霞[1] 王剑[2] 曾慰[3] 

机构地区：[1]南华大学附属第二医院超声医学科,衡阳421001 [2]南华大学附属第一医院急诊科,衡阳421001 [3]南华大学附属第二医院胸心外科,衡阳421001

出　　处：《基因组学与应用生物学》2016年第11期2897-2902,共6页Genomics and Applied Biology

基　　金：湖南省卫生计生委科研基金(B2016136);湖南省分子靶标新药研究协同创新中心资助项目(湘教通2015351)共同资助

摘　　要：本研究为观察穿心莲内酯（AD）对乙醇诱导肝细胞氧化应激损伤的保护作用，体外培养肝细胞L-02，用不同浓度（0—30μmol／L）AD孵育1h，随后加入100mmol／L乙醇作用24h。ELISA测定AD处理前后细胞培养上清中谷丙转氨酶（ALT）和谷草转氨酶（AST）的含量；比色法检测丙二醛（MDA）和谷胱氨肽（GSH）的变化；荧光探针DCFH2-DA检测胞内活性氧（ROS）的产生；Western blotting和分析血红素氧合酶-1（HO—1）mRNA和蛋白的表达。电泳迁移率实验（EMSA）检测核转录因子Nrf2的DNA结合活性。结果表明，100mmol／L乙醇处理肝细胞后，可在不影响L-02活性的情况下显著增加培养上清中ALT和AST的含量，而AD预处理后可抑制ALT、AST、MDA和ROS的增加以及上调胞内GSH的水平。此外，Westem blotting和实时定量PCR结果也证实乙醇可降低肝细胞内源性HO-1的表达，而AD预处理后可增强转录因子Nrf2的活性并进一步上调HO-1的表达水平。AD可能通过激活Nrf2上调HO-1表达而发挥对乙醇所致肝细胞损伤的保护作用。To observe the hepatoprotective efficacy of Andrographolide against alcohol-induced oxidative stress, human hepatic cells were pretreated with andrographolide （0-30 μmol/L） for 1 h and then hepatotoxicity was stimulated by the addition of ethanol （100 mmol/L） for 24 h. The amount of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the culture supernatant were observed by ELISA. Lipid peroxidation and reduced glutathione （GSH） were measured by colorimetry. Intracellular ROS accumulation was detected by DCFH2-DA fluorescent Probe. Expression of hemoxygenase-1 （HO-1） mRNA and protein were observed by real- time PCR and Western blotting, respectively. The DNA binding activity of Nrf2 was determined by electrophoretic mobility shift assay （EMSA）. Results with response to ethanol-challenge, increased amount of ALT and AST release were observed, whereas Andrographolide pretreatment significantly inhibited the leakage of AST and ALT in L-02 cells without appreciable cytotoxic effects. We also found that Andrographolide pretreatment significantly decreased ethanol-induced malondialdehyde （MDA）, ROS and GSH depletion in L-02 cells. Furthermore, Western blotting and quantitative-PCR analyses showed that ethanol-exposure apparently down-regulated endogenous antioxidant HO-1 expression, whereas pretreatment with Andrographolide significantly up-regulated HO-1 expression followed by the transcriptional activation of Nrf-2. Andrographolide-mediated up-regulation of HO- 1 via Nrf-2 signaling pathway may provide a pivotal mechanism for its hepatoprotective action.

关 键 词：穿心莲内酯 乙醇 肝细胞 

分 类 号：R575[医药卫生—消化系统]