棒曲霉素对人肝细胞L-02的毒性效应及作用机制

摘要

OBJECTIVE To investigate the toxicological effect of patulin(PAT)on the growth of human normal liver cells L-02 and its possible mechanisms. METHODS After cells were treated with PAT 1.25, 2.5,5,10 and 20μmol·L-1 for 24 or 48 h,cell viability was examined using MTT assay. L-02 cells were treated with PAT 5 and 10 μmol · L- 1 for 24 h ,respectively. Cytomorphology and mitochondrial membrane potential (MMP) were observed under a fluorescence microscope. Apoptosis,MMP and reactive oxygen species (ROS)were analyzed by flow cytometry. Mitochondria apoptosis pathways were detected by Western blotting. RESULTS PAT exhibited a strong inhibitory effect on L-02 in a concentration-dependent and time-dependent manner. IC50 of PAT treatment for 24 or 48 h was 6.61 and 2.78 μmol · L-1,respectively. MMP was decreased,while the percentage of low MMP cells increased from(9.2±2.3)%in controls to(23.4±4.5)%( PAT 5μmol·L-1)and(47.1±5.5)%(PAT 10μmol·L-1), respectively. Compared to untreated cells,the early apoptosis population increased from(3.8±1.1)%to(29.8±4.5)%( PAT 5μmol·L-1)and (24.1±6.2)%(PAT 10μmol·L-1)(P<0.01),respectively. Further⁃more,the accumulation of ROS was also observed. The effect of PAT on ROS and cell viabilities could be attenuated by glutathione. CONCLUSION PAT can significantly inhibit the growth of L-02 and induce apoptosis via ROS-dependent mitochondria pathways.%目的：研究棒曲霉素（PAT）对人肝细胞L-02的毒性作用，并探讨其作用的分子机制。方法 PAT 1.25，2.5，5，10和20μmol·L-1分别作用于L-02细胞24和48 h，采用噻唑蓝（MTT）法检测PAT对L-02细胞增殖的抑制作用；PAT 0，5和10μmol·L-1作用L-02细胞24 h后，Hoechst33258和JC-1染色，通过荧光显微镜进行细胞形态及线粒体膜电位（MMP）观察；利用流式细胞术对细胞凋亡、MMP及细胞内活性氧（ROS）水平进行定量分析；Western蛋白印迹法检测PAT对线粒体凋亡通路的影响。结果 PAT明显抑制L-02细胞的生长，24和48 h的IC50值分别为6.61和2.78μmol·L-1；与正常对照组低MMP比率（9.2±2.3）%相比，PAT 5和10μmol·L-1组低MMP比率显著升高（P<0.01），分别升高至（23.4±4.5）%和（47.1±5.5）%；PAT 5和10μmol · L-1处理后L-02细胞早期凋亡率较正常对照组（3.8±1.1）%升高（P<0.01），分别增至（29.8±4.5）%和（24.1±6.2）%；PAT引起L-02细胞内ROS增加，使用外源性谷胱甘肽预孵育细胞后，阻断了PAT诱导的ROS升高和增殖抑制的作用。结论 PAT可显著影响L-02的生长，并通过线粒体途径诱导细胞凋亡，其机制与胞内ROS水平升高有关。