苦参碱致HepaRG细胞毒性研究

Study on the toxicity of matrine on HepaRG cell line

目的研究苦参碱对HepaRG细胞增殖、周期及凋亡的影响并初探其机制。方法采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethyl thiazol-2-yl-)-2,5-diphenyltetrazolium bromide,MTT]法检测不同浓度(0. 5,1,2,4 mg/mL)苦参碱对HepaRG细胞活力的影响;检测苦参碱给药后,HepaRG细胞培养上清液中的乳酸脱氢酶(lactate dehydrogenase,LDH)活力;采用4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)染色观察HepaRG细胞形态学变化;利用流式细胞仪检测苦参碱对HepaRG细胞内活性氧和线粒体膜电位变化;利用Annexin V-FITC/PI双染检测细胞凋亡变化;利用PI/RNase检测细胞周期变化。结果 MTT和LDH结果显示苦参碱能明显抑制HepaRG细胞活力,并呈时间与剂量依赖关系; DAPI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。流式结果表明苦参碱能使HepaRG细胞内活性氧升高,线粒体膜电位水平下降;与对照组相比,给药组HepaRG细胞出现明显的凋亡细胞,且细胞凋亡率随苦参碱浓度增大而逐渐升高;给药组HepaRG细胞处于G0/G1期的细胞数减少,S期的细胞增多,表明苦参碱能将HepaRG细胞的细胞周期阻滞在S期。结论苦参碱在体外对HepaRG细胞有明显的细胞毒性作用,其机制可能是通过增加活性氧,使线粒体功能受损,改变细胞周期分布,诱导细胞凋亡。

Objective To investigate the effects of matrine on inhibiting the growth,arresting the cell cycle and inducing the apoptosis of HepaRG cells and its mechanism.Methods The inhibitory effect of different concentration of matrine on the growth of HepaRG cells was measured by3-(4,5-dimethyl thiazol-2-yl-)-2,5-diphenyltetrazolium bromide,MTT.After matrine treatment,the culture supernatant was harvested,and lactate dehydrogenase(LDH)activity was measured with a commercial kit following the manufacturer's instructions.The nuclear morphological changes were observed by DAPI staining.Intracellular reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were measured by flow cytometry.Apoptotic cell death was analyzed by Annexin V-FITC/PI double staining.The cell cycle was detected by PI/RNase staining.Results Compared with the control group,the results of the MTT and LDH assay demonstrated that matrine significantly inhibited the viability of HepaRG cells in a dose and time dependent manner.DAPI staining showed the morphology of the cells changed significantly with the increase of drug concentration,such as condensation of chromatin,bright blue and apoptotic bodies with the increase of drug concentration.It provoked ROS generation and depolarization of MMP in HepaRG cells.When compared with control group,treatment with matrine significantly induced apoptosis in HepaRG cells in a dose dependent manner.It also induced S phase cell cycle arrest by increasing the cell number in S phase while significantly decreasing the cell number in G0/G1phase.Conclusion These results suggest that matrine possesses obvious cytotoxicity effect on HepaRG cells in vitro,most probably because of the generation of ROS and depolarization of MMP which will in turn change the cell cycle distribution and induction of apoptosis.