Yang Huan, Liu Bing-Fang, Xie Fu-Jia, Yang Wei-Lin, Cao Nong
Department of General Surgery, First Hospital of Lanzhou University, Lanzhou, Gansu 730000, P.R. China.
Department of Nuclear Magnetic Resonance, The Second Clinical Medical College of Lanzhou University, Lanzhou, Gansu 730000, P.R. China.
Exp Ther Med. 2020 Mar;19(3):2179-2187. doi: 10.3892/etm.2020.8464. Epub 2020 Jan 22.
The aim of the current study was to investigate luteolin-induced apoptosis and the molecular mechanisms underlying it in HT29 cells. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the cytotoxicity of luteolin on HT29 cells, and a dichloro-dihydro-fluorescein diacetate assay was used to measure cellular levels of reactive oxygen species (ROS). The effects of luteolin on the mitochondrial membrane potential were also evaluated. Bax and Bcl-2 mRNA expression were determined using reverse transcription-quantitative PCR. Additionally, western blot analysis was performed to assess changes in cytochrome c and caspase-3 protein expression. Localization of nuclear factor erythroid 2-related factor 2 (Nrf2) in the nucleus was also assessed using immunofluorescence. Luteolin exhibited cytotoxicity on HT29 cells in a time- and concentration-dependent manner. Additionally, ROS production was indicated to be increased and ROS scavenging was decreased, which resulted in a significant increase in the levels of ROS in the cells. The mitochondrial membrane potential was indicated to decrease following luteolin treatment. At the molecular level, luteolin significantly increased the mRNA expression of Bax and the protein expression of cytochrome c, caspase-3, p47 and p22. The results revealed that luteolin decreased Bcl-2 protein expression and inhibited the nuclear localization of Nrf2. In conclusion, the current study indicated that luteolin inhibited HT29 cell proliferation and induced apoptosis via the mitochondrial pathway.
本研究的目的是探讨木犀草素诱导HT29细胞凋亡及其潜在的分子机制。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法评估木犀草素对HT29细胞的细胞毒性，采用二氯二氢荧光素二乙酸酯法检测细胞内活性氧(ROS)水平。还评估了木犀草素对线粒体膜电位的影响。采用逆转录定量PCR法检测Bax和Bcl-2 mRNA表达。此外，进行蛋白质印迹分析以评估细胞色素c和半胱天冬酶-3蛋白表达的变化。还采用免疫荧光法评估核因子红细胞2相关因子2(Nrf2)在细胞核中的定位。木犀草素对HT29细胞具有时间和浓度依赖性的细胞毒性。此外，ROS生成增加而ROS清除减少，导致细胞内ROS水平显著升高。木犀草素处理后线粒体膜电位降低。在分子水平上，木犀草素显著增加Bax的mRNA表达以及细胞色素c、半胱天冬酶-3、p47和p22的蛋白表达。结果显示，木犀草素降低Bcl-2蛋白表达并抑制Nrf2的核定位。总之，本研究表明木犀草素通过线粒体途径抑制HT29细胞增殖并诱导凋亡。
