论文部分内容阅读
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1 h,随后加入100 mmol/L乙醇作用24 h。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)m RNA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,100 mmol/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,Western 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。
This study was designed to observe the protective effect of andrographolide (AD) on hepatocellular oxidative stress induced by ethanol in vitro. L-02 cells were cultured in vitro and incubated with different concentrations of AD (0 ~ 30μmol / L) for 1 h. Then 100 mmol / L ethanol for 24 h. The contents of ALT and AST in the cell culture supernatants were measured by ELISA. The changes of malondialdehyde (MDA) and glutathione (GSH) were detected by colorimetric assay. The fluorescence intensity of DCFH2- DA was used to detect intracellular reactive oxygen species (ROS) production; Western blotting was used to detect the expression of HO-1 mRNA and protein. Electrophoretic mobility shift assay (EMSA) was used to detect DNA binding activity of nuclear transcription factor Nrf2. The results showed that 100 mmol / L ethanol treatment of liver cells could significantly increase the content of ALT and AST in the culture supernatant without affecting the activity of L-02, while AD pretreatment inhibited ALT, AST, MDA and ROS Increase and increase the level of intracellular GSH. In addition, Western blotting and real-time quantitative PCR results also confirmed that ethanol can reduce the expression of endogenous HO-1 in hepatocytes, while AD pretreatment can enhance the activity of transcription factor Nrf2 and further up-regulate the expression of HO-1. AD may play a protective role on ethanol-induced hepatocyte injury by activating Nrf2 up-regulation of HO-1 expression.