顺铂通过ROS促进乙型肝炎病毒复制的实验研究

Cisplatin contributes to hepatitis B virus replication via ROS:an experimental study

目的:探索顺铂(cisplatin)促进乙型肝炎病毒(hepatitis B virus,HBV)复制的分子机制。方法:采用不同浓度的顺铂处理稳定表达HBV的Hep AD38细胞,台盼蓝染色检测细胞活力,筛选出顺铂最适浓度,分别采用Real-time PCR和Southern blot检测HBV DNA水平,Western blot检测HBsAg和HBcAg蛋白表达。在顺铂诱导组加或者不加氧化应激抑制剂N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine,NAC),采用CellROX Orange Reagent染料在激光共聚焦显微镜下检测细胞内ROS水平,并且检测HBV DNA水平、HBsAg及HBcAg的蛋白水平。结果:台盼蓝染色筛选出顺铂的最适浓度为6μmol/L。Real-time PCR实验结果显示,PBS组,0.5、2.0、6.0μmol/L顺铂处理后HBV DNA拷贝数/细胞分别为515.152±18.924、1 215.758±49.001(P=0.000)、1 678.182±117.223(P=0.000)、2 110.606±143.640(P=0.000);Southern blot结果与Real-time PCR结果一致,顺铂以剂量依赖方式增加HBV DNA水平。Western blot结果表明,与PBS组相比,6.0μmol/L顺铂组HBsAg、HBcAg蛋白相对表达量分别为3.102±0.099(P=0.000)、4.001±0.200(P=0.000);顺铂可以促进HBV复制和病毒蛋白的表达。进一步采用NAC与顺铂联合处理HepAD38细胞,Real-time PCR实验结果表明,顺铂组、顺铂+NAC组HBV DNA拷贝数/细胞分别为2 180.444±82.573(P=0.000)、1 041.778±50.918(P=0.000),2组差异具有统计学意义。Western blot实验结果表明,顺铂组HBsAg、HBcAg蛋白相对表达量分别为3.139±0.061(P=0.000)、3.812±0.109(P=0.000),顺铂+NAC组HBsAg、HBcAg蛋白相对表达量分别为2.101±0.088(P=0.000)、1.613±0.073(P=0.000),2组之间HBsAg、HBcAg蛋白差异均具有统计学意义。结论:顺铂通过增强细胞内ROS水平,促进HBV复制,而NAC可以部分抑制顺铂刺激的HBV复制作用。

Objective:To investigate the molecular mechanism of cisplatin-induced hepatitis B virus(HBV) replication. Methods:Hep AD38 cells with stable expression of HBV were treated with different concentrations of cisplatin,and the optimal concentration of cisplatin was chosen according to trypan blue staining for cell viability. The level of HBV DNA was measured by real-time PCR and Southern blot,and the expression of HBsAg and HBcAg was measured by Western blotting. The cisplatin-induced group of HepAD38 cells were treated with or without N-acetyl-L-cysteine(NAC). Intracellular reactive oxygen species(ROS) levels were determined with CellROX Orange Reagent under laser scanning confocal microscopy,and the level of HBV DNA and expression of HBsAg and HBcAg proteins were measured. Results:The optimal concentration of cisplatin was 6 μmol/L according to trypan blue staining. The results of real-time PCR showed that compared with 515.152 ±18.924 in the PBS group,the copies(per cells) of HBV DNA were 1 215.758±49.001(P=0.000),1 678.182±117.223(P=0.000),and 2 110.606±143.640(P=0.000) after being treated with 0.5,2,and 6 μmol/L cisplatin,respectively. Moreover,cisplatin treatment increased HBV DNA levels in a dose-dependent manner as measured by Southern blot,the same as results of real-time PCR. The results of Western blot showed that compared with the PBS group,the 6 μmol/L cisplatin treatment group had a relative HBsAg expression level of 3.102±0.099(P=0.000) and a relative HBcAg expression level of 4.001±0.200(P=0.000),suggesting that cisplatin contributed to HBV replication and protein expression. After HepAD38 cells were treated with cisplatin and NAC,the results of real-time PCR showed that the copies(per cells) of HBV DNA in in the cisplatin group and cisplatin+NAC group were 2 180.444±82.573(P=0.000) and 1 041.778±50.918(P=0.000),respectively;there was a significant difference in the level of HBV DNA between the two groups. According to the results of Western blot,the cisplatin group had a relative HBsAg expression level of 3.139±0.061(P=0.000) and a relative HBcAg expression level of 3.812±0.109(P=0.000),and the cisplatin+NAC group had a relative HBsAg expression level of 2.101±0.088(P=0.000) and a relative HBcAg expression level of 1.613±0.073(P=0.000);there were significant differences in the relative expression levels of HBsAg and HBcAg between the two groups. Conclusion:Cisplatin enhances HBV replication by increasing intracellular ROS levels,and NAC can partially reverse cisplatin-induced HBV replication.