State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academic of Sciences, Wuhan 430071, Hubei Province, China.
World J Gastroenterol. 2011 Mar 21;17(11):1507-14. doi: 10.3748/wjg.v17.i11.1507.
To analyze the antiviral mechanism of Epigallocatechin gallate (EGCG) against hepatitis B virus (HBV) replication.
In this research, the HBV-replicating cell line HepG2.117 was used to investigate the antiviral mechanism of EGCG. Cytotoxicity of EGCG was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hepatitis B virus e antigen (HBeAg) and hepatitis B virus surface antigen (HBsAg) in the supernatant were detected by enzyme-linked immunosorbent assay. Precore mRNA and pregenomic RNA (pgRNA) levels were determined by semi-quantitative reverse transcription polymerase chain reaction (PCR) assay. The effect of EGCG on HBV core promoter activity was measured by dual luciferase reporter assay. HBV covalently closed circular DNA and replicative intermediates of DNA were quantified by real-time PCR assay.
When HepG2.117 cells were grown in the presence of EGCG, the expression of HBeAg was suppressed, however, the expression of HBsAg was not affected. HBV precore mRNA level was also down-regulated by EGCG, while the transcription of precore mRNA was not impaired. The synthesis of both HBV covalently closed circular DNA and replicative intermediates of DNA were reduced by EGCG treatment to a similar extent, however, HBV pgRNA transcripted from chromosome-integrated HBV genome was not affected by EGCG treatment, indicating that EGCG targets only replicative intermediates of DNA synthesis.
In HepG2.117 cells, EGCG inhibits HBV replication by impairing HBV replicative intermediates of DNA synthesis and such inhibition results in reduced production of HBV covalently closed circular DNA.
分析表没食子儿茶素没食子酸酯（EGCG）抗乙型肝炎病毒（HBV）复制的抗病毒机制。
本研究采用 HBV 复制细胞系 HepG2.117 研究 EGCG 的抗病毒机制。用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法分析 EGCG 的细胞毒性。用酶联免疫吸附试验检测上清液中的乙型肝炎病毒 e 抗原（HBeAg）和乙型肝炎病毒表面抗原（HBsAg）。用半定量逆转录聚合酶链反应（PCR）法检测前核心 mRNA 和前基因组 RNA（pgRNA）水平。用双荧光素酶报告基因检测法测定 EGCG 对 HBV 核心启动子活性的影响。用实时 PCR 法检测 HBV 共价闭合环状 DNA 和 DNA 复制中间体。
当 HepG2.117 细胞在 EGCG 存在的情况下生长时，HBeAg 的表达受到抑制，而 HBsAg 的表达不受影响。EGCG 也下调 HBV 前核心 mRNA 水平，而前核心 mRNA 的转录不受影响。HBV 共价闭合环状 DNA 和 DNA 复制中间体的合成均被 EGCG 处理显著减少，但 EGCG 处理并不影响染色体整合 HBV 基因组转录的 HBV pgRNA，表明 EGCG 仅靶向 DNA 合成的复制中间体。
在 HepG2.117 细胞中，EGCG 通过损害 HBV 复制中间体的 DNA 合成来抑制 HBV 复制，从而减少 HBV 共价闭合环状 DNA 的产生。
