大黄素诱导肝细胞L-02凋亡的作用机制

Study on the mechanism of emodin-induced apoptosis of hepatocyte L-02

目的:研究大黄素对L-02正常肝细胞增殖、周期及凋亡的影响并初探其机制。方法:以大黄素为研究对象,L-02细胞为体外模型,采用MTT法检测不同浓度(20、40、60μmol/L)的大黄素对细胞增殖的影响;采用Hoechst染色观察L-02细胞形态学变化;采用FITC AnnexinV/PI双染法,检测不同浓度的药物处理后细胞凋亡情况;采用PI/RNase法检测药物对细胞周期的影响;利用流式细胞仪检测大黄素对L-02细胞内活性氧和线粒体膜电位的影响;采用荧光定量PCR技术检测肝细胞中Sirt1的mRNA表达水平,采用Western Blot检测凋亡相关蛋白Sirt1、Bax、Bcl-2、Caspase-3的表达,探讨大黄素细胞毒性的作用机制。结果:大黄素抑制L-02细胞增殖,且呈浓度相关性;Hoechst染色发现随着药物浓度升高,细胞形态发生明显变化,出现核固缩,呈亮蓝色,形成凋亡小体等;实验结果表明大黄素能使L-02细胞内活性氧升高,线粒体膜电位水平下降,细胞中谷胱甘肽含量降低;与对照组比较,3个大黄素组L-02细胞出现明显的凋亡细胞,且细胞凋亡率随大黄素浓度增大而逐渐升高;40、60μmol/L大黄素组L-02细胞处于G0/G1期的细胞数减少,S期的细胞增多,表明大黄素能将L-02细胞的细胞周期阻滞在S期。PCR结果显示3个大黄素组能够使Sirt1的mRNA表达水平不同程度降低;Western Blot结果显示3个大黄素组可以不同程度地降低Sirt1、Bax、Caspase-3蛋白的表达,同时升高Bcl-2蛋白的表达。结论:大黄素在体外对L-02肝细胞的增殖具有明显抑制作用,其机制可能是通过降低Sirt1的表达,使线粒体功能受损,增加活性氧的产生,从而引起细胞凋亡。

Objective:To investigate the effects of emodin on the proliferation,cycle and apoptosis in L-02 normal hepatocytes and explore its mechanism.Methods:With emodin as the research object and L-02 cells as the model in vitro,the effect of different concentrations of emodin on the proliferation of L-02 cells was evaluated by MTT.The nuclear morphological changes of L-02 cells were observed by Hoechst staining.The apoptosis after different concentration of drugs treatment was detected by FITC AnnexinV/PI double staining.The effect of drugs on cell cycle was detected by PI/RNase staining.Effect of emodin on intracellular reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)in L-02 cells were detected by flow cytometer.The mRNA expression of Sirt1 in L-02 cells was detected by quantitative real-time PCR and the expression of apoptosis-related proteins Sirt1,Bax,Bcl-2,Caspase-3 were measured by western blot.Results:Emodin significantly inhibited the proliferation of L-02 cells in a concentration-dependent manner.DAPI staining revealed the clear morphological changes,such as nuclear pyknosis,bright blue and apoptotic bodies with the increase of drug concentration.The results showed that emodin could increase reactive oxygen species,decrease mitochondrial membrane potential and glutathione content in L-02 cells.When compared with the control group,treatment with emodin significantly induced apoptosis in L-02 cells,and the apoptotic rate gradually increased with the increase of emodin concentration.In the administration group,the number of L-02 cells in G0/G1 phase decreased and the number of cells in S phase increased,suggesting that emodin could block the cell cycle of L-02 cells in S phase.The results of PCR showed emodin significantly inhibited the expression of mRNA of Sirt1.Western blot results showed that the protein expression of Sirt1,Bax and Caspase-3 decreased and Bcl-2 increased at a certain concentration of emodin.Conclusion:These results suggest that emodin had a significant inhibitory effect on the proliferation of L-02 cells in vitro,most probably by reducing the expression of Sirt1,impairing mitochondrial function and increasing the production of reactive oxygen species,thereby causing cell apoptosis.