6'-羟基爵床素A诱导人肝癌HepG2细胞凋亡及其机制  

Effect of 6'- hydroxy justicidin A on apoptosis and its mechanism in human hepatocelluar carcinoma Hep G2 cells

作　　者：宋维才[1] 贺晓丽[2] 路畅[2] 李凤金[1] 童元峰[3] 肖洪彬[1] 毕明刚[2] 

机构地区：[1]黑龙江中医药大学基础医学院,黑龙江哈尔滨150040 [2]中国医学科学院药用植物研究所,北京100193 [3]中国医学科学院药物研究所,北京100050

出　　处：《中国药理学与毒理学杂志》2015年第2期220-226,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：北京市自然科学基金(7142113);中央级公益性科研院所基本科研业务费专项资金(yz-13-09)~~

摘　　要：目的探讨6＇-羟基爵床素A（JR6）对人肝癌Hep G2细胞凋亡的诱导作用及其机制。方法体外培养人肝癌Hep G2细胞,采用MTT法观察不同浓度的JR6和5-氟尿嘧啶（5-FU）对Hep G2细胞存活的作用,荧光显微镜观察Hoechst33258染色后细胞核形态改变,用流式细胞仪检测AnnexinⅤ-FITC/PI双染后Hep G2细胞的凋亡率,JC-1荧光染色观察药物对细胞线粒体膜电位的影响,Western蛋白质印迹法检测细胞凋亡相关蛋白细胞色素c、Bax和Bcl-2蛋白的表达。结果 JR6 6.1~196μmol·L-1和5-FU 3.4~192μmol·L-1分别作用Hep G2细胞48 h,对Hep G2细胞存活具有抑制作用,IC50分别为74.90和49.75μmol·L-1。JR6 12.3,49和196μmol·L-1作用Hep G2细胞48 h,细胞凋亡率明显增加,由正常对照组的（6.9±2.0）%增加至（13.8±2.0）%,（18.6±4.3）%和（32.4±3.2）%（P〈0.05,P〈0.01）。Hoechst33258染色结果显示,JR6 49和196μmol·L-1作用Hep G2细胞48 h,部分细胞出现细胞核固缩和染色质凝集等凋亡变化。线粒体膜电位检测结果发现,JR6 49和196μmol·L-1作用48 h能使Hep G2细胞线粒体膜电位水平明显降低（P〈0.05,P〈0.01）。线粒体凋亡相关蛋白的检测结果表明,JR6 12.3,49和196μmol·L-1作用Hep G2细胞48 h,胞浆细胞色素c表达增加,促凋亡蛋白Bax表达增加,抗凋亡蛋白Bcl-2表达降低,且Bax/Bcl-2比值增加（P〈0.05,P〈0.01）。JR6 49μmol·L-1和5-FU组上述蛋白表达无明显差异。结论 JR6可能通过促进细胞色素c释放、打破Bcl-2/Bax平衡诱导Hep G2细胞凋亡。OBJECTIVE To investigate the effect of 6′-hydroxy justicidin A （JR6） on apoptosis and its mechanism in hepatocellular carcinoma HepG2 cells. METHODS HepG2 cells were cultured with JR6 or 5-fluorouracil（5-FU） at different concentrations for 48 h. The proliferation of HepG2 cells was tested by MTT assay. Hoechst33258 staining was employed to observe nucleus morphological changes. The apoptotic ratio and mitochondrial membrane potential were measured by AnnexinⅤ-FlTC/ Pl double staining and JC-1 staining, respectively. The protein expression of cytochrome c, Bax and Bcl-2 proteins was evaluated by Western blotting. RESULTS After HepG2 cells were treated with different concentrations of JR6 6.1-196 μmol.L-1 and 5-FU 3.4-192 μmol.L-1 for 48 h, cell proliferation was significantly inhibited, while the lC50 value was 74.90 and 49.75 μmol.L-1 ,respectively. JR6 12.3, 49 and 196 μmol.L-1 treat-ment for 48 h could induce apoptosis in HepG2 cells, the apoptosis rate of cells increased from （6.9±2.0）% to （13.8±2.0）%,（18.6±4.3）% and （32.4 ±3.2）% （P〈0.05, P〈0.01）. Hoechst33258 staining indicated cell shrinkage and nuclear condensation in HepG2 cells treated with JR6 49 and 196 μmol.L-1 for 48 h. JR6 49 and 196 μmol.L-1 treatment for 48 h significantly decreased the mitochondrial membrane potential （P〈0.05, P〈0.01）. Western boltting results showed that after JR6 12.3, 49 and 196 μmol.L-1 treatment for 48 h, the expression of cytochrome c increased in cytosolic fraction, the expression of Bax increased while that of Bcl-2 decreased, leading to an increase in the Bax / Bcl-2 ratio（P〈0.05, P〈0.01）. There was no significant difference between JR6 49 μmol.L-1 group and 5-FU group. CONCLUSION JR6 can significantly induce apoptosis in human hepatocellular carcinoma HepG2 cells, mainly by promoting cytochrome c release and breaking the balance of Bcl-2/ Bax.

关 键 词：6'-羟基爵床素A HEP G2细胞 细胞凋亡 线粒体 

分 类 号：R735.7[医药卫生—肿瘤]