新型中药制剂肃毒星对乙型肝炎病毒体外复制的抑制作用研究

Inhibitory effect of herbal concoction Suduxing on hepatitis B virus replication in vitro

目的观察新型中药制剂肃毒星对野生和恩替卡韦(ETV)耐药乙型肝炎病毒(HBV)的抑制作用。方法以不同浓度(0、0.0005﹑0.001﹑0.005﹑0.01mg/ml)的肃毒星分别处理HepG2.2.15细胞(D基因型/ayw亚型野生HBV稳定复制细胞系)和HepG2.A64细胞(C基因型/adr亚型ETV耐药HBV稳定复制细胞系),作用时间4d。采用酶联免疫吸附试验检测细胞培养上清中HBsAg和HBeAg含量的变化;提取细胞内病毒核心颗粒,采用实时荧光定量PCR方法检测两种细胞内HBV DNA复制水平的变化。观察不同作用时间下,药物对HBV DNA的抑制作用。结果肃毒星对HepG2.2.15细胞和HepG2.A64细胞内HBV DNA复制和HBsAg、HBeAg的分泌均有明显抑制作用,且具有药物剂量和时间依赖性,最高浓度(0.01mg/ml)的肃毒星对HepG2.2.15细胞内病毒的抑制率达到80.83%,对HBsAg和HBeAg的抑制率分别达51.00%和64.05%;而对HepG2.A64细胞内HBV DNA抑制率为65.18%,对HBsAg和HBeAg的抑制率分别为32.85%和73.86%。随肃毒星作用时间延长,其对HepG2.2.15细胞HBV DNA的抑制效果增强,但未显示对HepG2.A64细胞内ETV耐药型HBV株病毒抑制率增强的效果。结论体外细胞培养实验中,肃毒星不仅可有效抑制野生HBV复制和抗原分泌,而且对ETV耐药HBV的复制和抗原分泌也具有很好的抑制作用。

Objective To investigate the inhibitory effect of Suduxing, a novel concoction of herbal extracts, on wild type and entecavir-resistant hepatitis B virus （HBV） in vitro. Methods Stable cell lines HepG2.2.15 （wild-type HBV, genotype D, serotype ayw） and HepG2.A64 （entecavir-resistant HBV, genotype C, serotype adr） were treated with various concentrations of Suduxing （0, 0.0005, 0.001, 0.005, 0.01mg/ml） for four days. Enzyme linked immunosorbent assay was used to determine the secreted HBsAg and HBeAg levels in the culture supernatant. Real-time PCR was used to detect the replication of HBV intermediate DNA. The inhibitory effect of Suduxing on the HBV DNA was observed at different time points. Results The replication of HBV DNA and the secretion of HBsAg and HBeAg in HepG2.2.15 cells and HepG2.A64 cells were effectively suppressed by Suduxing in a dose- and time-dependent manner. The inhibitory rate of HBV DNA, HBsAg and HBeAg at 0.01 mg/ml of Suduxing were 80.83%, 51.00%, 64.05% for HepG2.2.15 cells and 65.18%, 32.85%, 73.86% for HepG2.A64 cells respectively. The inhibitory effect of Suduxing on the HBV DNA in HepG2.2.15 cells increased with prolongation of time, while it did not in HepG2.A64 cells. Conclusion Suduxing, as a potential anti-HBV agent, can effectively suppress the replication of both wild type HBV and entecavirresistant HBV as well as the secretion of viral antigens.