红背叶根体外抑制HCV亚基因复制子RNA表达活性部位筛选研究

Preliminary Screening of Activity Fraction of Alchornea trewioides Suppresses Expression of Subgenomic Hepatitis C Virus RNA in vitro

目的:筛选红背叶根体外抑制丙型肝炎病毒(HCV)复制的活性部位。方法:以HCV亚基因复制子体外培养体系CBRH7919(Jneo3-5B)为HCV复制模型,选用干扰素α联合利巴韦林为阳性对照药物,观察不同浓度红背叶根总提取物、石油醚部位、乙酸乙酯部位、正丁醇部位的抗HCV作用。采用荧光定量PCR检测HCV亚基因复制子RNA拷贝数,通过Western blot检测HCV功能蛋白NS3的表达,采用CCK-8法观察药物对细胞生长的影响。结果:红背叶根4种提取物中,乙酸乙酯部位对HCVRNA复制的抑制活性最强,对NS3蛋白表达有明显干预作用,对HCVRNA表达表现出剂量依赖的抑制作用(P<0.05)。其比活较初始总提取物提高5.71倍,对HCVRNA半数抑制浓度为14.60mg/L,对CBRH7919(Jneo3-5B)细胞半数细胞毒性浓度为40.30mg/L,治疗指数为2.76。结论:红背叶根乙酸乙酯部位可抑制HCV的复制,为抗HCVRNA表达的活性部位,在抗HCV方面有着潜在的用途。

Objective：To screen activity fraction of Alchornea trewioides which suppresses expression of subgenomic Hepatitis C Virus （HCV） RNA in vitro. Methods ： Anti-HCV effects in vitro were examined in an HCV subgenomic replicon cell culture system - CBRH7919 （Jneo3-5B）. The cells were exposed to different concentrations of A. trewioides initial ethanol extracts, portions of petroleum ether,ethyl acetate and n-butanol extracts with interferon α combined with ribavirin as positive control. The content of HCV RNA was examined by Quantitative PCR. The expression levels of functional proteins NS3 were examined in all groups by Western blot. Cell proliferation test with CCK-8 assay was used to evaluate the cytotoxicity of drugs. Results： The study showed that exposure of CBRH7919 （Jneo3-5B） cells to ethyl acetate extract of A. trewioides resulted in a concentration-dependent inhibition of subgenomic HCV RNA replication and NS3 protein expression ability among the four extracts （ P 〈 0. 05 ）. The activity of ethyl acetate extract was increased by 5.71 times than that of the initial ethanol extract. ICs0 to subgenomic HCV RNA was 14. 60 mg/L, CC50 to CBRH7919 （Jneo3-5 B）cells was 40. 30 mg/L and the treatment index（TI） was 2.76. Conclusion： The ethyl acetate extract of A. trewioides is the activity fraction which can significantly interfere with subgenomic HCV RNA replication and expression of NS3 protein in vitro. These data suggest that ethyl acetate extract isolated from A. trewioides may have potential use as an anti-HCV compound.