氧化苦参碱体外抗乙型肝炎病毒作用

Inhibitory Effect of Oxymatrine on Hepatitis B Virus in vitro

目的:体外观察氧化苦参碱(OM)抗乙型肝炎病毒(HBV)作用,并初步探讨其作用机制。方法:用125,250,500,1 000,2 000 mg.L-1OM连续作用于90%汇合度的HepG2.2.15细胞9 d,以MTT比色法观察药物细胞毒性;用酶联免疫法测定细胞上清液中乙型肝炎病毒e抗原(HBeAg),乙型肝炎病毒s抗原(HBsAg);采用荧光定量PCR法(FQ-PCR)测定细胞上清液中HBV DNA,细胞中HBV DNA和共价闭合环状DNA(cccDNA)。结果:OM对细胞内HBV DNA和cccDNA,以及细胞外HBV DNA均有抑制作用,随着浓度升高抑制作用加强,2 000 mg.L-1OM对细胞内HBV DNA,cccDNA和细胞外HBV DNA的抑制率分别为64.56%,52.12%,54.25%;对HBsAg和HBeAg的分泌也有抑制作用,且浓度越高、处理时间越长,抑制作用越明显;在相同条件下对HBsAg的抑制作用强于HBeAg,OM浓度为2 000 mg.L-1时对HBsAg和HBeAg的抑制率分别为51.59%,17.88%。结论:OM能有效抑制HepG2.2.15细胞中HBV复制,该作用是抑制病毒核酸复制和基因表达的结果。

Objective：To observe the antiviral activities of oxymatrine（OM） on hepatitis B virus（HBV） in vitro and initially explore the mechanism of it.Method： HepG2.2.15 cells（at 90% confluencerespectively） were treated with different concentrations（125,250,500,1 000,2 000 mg·L-1）of OM for 9 d.Cytotoxicity was observed with MTT colorimetric method.HBeAg and HBsAg in the culture supernatant were determined by ELISA assay.Fluorescence quantity PCR assay was used to assay the extracellular HBV DNA,intracellular HBV DNA and covalently closed circular DNA（cccDNA）.Result：After treatment with 2 000 mg·L-1 OM for 9 d the level of the extracellular HBV DNA,intracellular HBV DNA and（cccDNA）was significantly inhibited in a dose-dependent manner,the inhibitory rates were 54.25%,64.56%,52.12% respectively.It can also inhibit the level of HBeAg and HBsAg in culture supernatant and the inhibitory effect showed a dose-and time-dependent manner.In the same condition,the inhibitory effect on HBsAg is stronger than that of HBeAg.When OM concentration was 2 000 mg·L-1,the inhibitory rates on HBsAg and HBeAg were 51.59%,17.88%.Conclusion：OM can inhibit HBV DNA replication in HepG2.2.15 cells through inhibiting the reproduction of the viral nucleic acid and gene expression.