溶酶体在2,2',4,4'-四溴联苯醚诱导HepG2细胞凋亡中的作用
Possible Involvement of Lysosomes in 2, 2', 4, 4'-Tetrabromodiphenyl Ether Mediated Apoptosis in HepG2 Cells
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摘要:
目的 探讨2,2',4,4'-四溴联苯醚(BDE 47)致人肝癌HepG2 细胞凋亡机制。 方法 不同浓度BDE 47(0.00、6.25、12.50、25.00、50.00、100.00 μmol/L)染毒HepG2 细胞24 h,采用四氮唑盐比色法(MTT 法)检测细胞存活率;用2',7'-二氢二氯荧光素(DCFH-DA)探针检测活性氧水平;用吖啶橙(AO)探针及罗丹明(Rh123)荧光探针分别检测溶酶体膜通透性和线粒体膜电势,并通过溶酶体组织蛋白酶B 特异性抑制剂环氧酶琥珀酰肽甲基酯(CA-074)验证溶酶体在BDE 47 细胞毒性的作用。 结果 与对照组比较,50.00、100.00 μmol/L BDE 47 染毒组HepG2 细胞存活率明显降低(P< 0.01);各BDE 47 染毒组HepG2 细胞凋亡率明显升高(P< 0.01),呈现剂量-效应关系(R2=0.981);各BDE 47染毒组HepG2 细胞活性氧含量明显升高(P< 0.01),≥ 12.50 μmol/L BDE 47 染毒组HepG2 细胞内溶酶体膜通透性明显升高(P< 0.05;P< 0.01),各BDE 47 染毒组HepG2 细胞内线粒体膜电势明显降低(P< 0.01),上述3 项指标与染毒浓度均呈剂量-效应关系(R2=0.918,R2=0.636,R2=0.678)。25 μmol/L CA-074 能够明显干预50 μmol/L BDE 47 对细胞的毒性作用使细胞存活率升高,细胞调亡减少(均P< 0.05)。 结论 BDE 47 可能通过溶酶体介导线粒体途径诱导HepG2 细胞凋亡。 Abstract:Objective To explore the possible involvement of lysosomes in 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE 47) induced apoptosis in HepG2 cells. Methods HepG2 cells were exposed to different levels of BDE 47 (0.00, 6.25, 12.50, 25.00, 50.00, 100.00 μmol/L) for 24 h, and the effects on cytotoxicity, reactive oxygen species (ROS), lysosomal and mitochondrial membrane stability were examined. Results The data demonstrated that the 50.00 and 100.00 μmol/L BDE 47 statistically reduced cell viability (P< 0.01), compared with the control group. The BDE 47 exposure induced apoptosis in HepG2 cells significantly (P< 0.01) and in a dose-dependent manner (R2=0.981) and statistically increased ROS generation in HepG2 cells (P< 0.01, R2=0.918). The treatment with 12.50 μmol/L and higher doses of BDE 47 also led to elevated lysosomal membrane permeabilization (P< 0.05; P< 0.01) along with mitochondrial membrane potential disturbance (P< 0.01), both in a dose-effect manner (R2=0.918, R2=0.636, R2=0.678). The toxic effect of 50 μmol/L BDE 47 was significantly alleviated by 25 μmol/L CA-074 (P< 0.05), a specific inhibitor for cathepsin B, for which increased cell viability and decreased apoptosis were demonstrated. Conclusion The findings indicate that the apoptotic event induced by BDE 47 in HepG2 might be initiated through a lysosomalmitochondrial pathway.
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