水芹总酚酸体外抗HBV作用及其机制探讨

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摘要目的观察水芹总酚酸体外对乙型肝炎病毒（HBV）的抑制作用并探讨其作用机制。方法水芹总酚酸对Hep AD38细胞的毒性实验分为水芹总酚酸组（细分为1600、800、400、200、100 μg/mL浓度组）和空白对照组。体外培养Hep AD38细胞，用MTT法检测水芹总酚酸的毒性范围。水芹总酚酸体外抗HBV作用实验分为水芹总酚酸组（细分为200、100、50、25 μg/mL浓度组）、阳性对照组[恩替卡韦（ETV） 10 μmol/L]和病毒对照组。用无毒浓度以下的水芹总酚酸药液处理Hep AD38细胞，连续6 d，收取上清和细胞，酶联免疫吸附测定（ELISA）法检测上清中HBsAg、HBeAg滴度；实时荧光定量PCR方法检测细胞内HBV DNA载量。水芹总酚酸对鸭乙型肝炎病毒（DHBV）DNA聚合酶的影响实验分为水芹总酚酸组（细分为400、200、100、50、25 μg/mL浓度组）、阴性对照组和DHBV DNA聚合酶对照组。从感染鸭乙肝病毒的鸭肝中提取DHBV DNA聚合酶，分别给予不同浓度水芹总酚酸药液，采用ELISA法检测水芹总酚酸对DHBV DNA聚合酶活性的影响。结果水芹总酚酸对Hep AD38细胞的无毒浓度为400 μg/mL。与病毒对照组比较，水芹总酚酸组中的200、100、50 μg/mL浓度组的HBsAg、HBeAg滴度和HBV DNA载量均明显降低，差异有高度统计学意义（P ＜ 0.01）；与DHBV DNA聚合酶对照组比较，水芹总酚酸组中的400、200、100 μg/mL浓度组可抑制DHBV DNA聚合酶活性，差异有高度统计学意义（P ＜ 0.01）。结论水芹总酚酸体外具有良好的抗HBV作用，其作用机制包括抑制HBV DNA聚合酶的活性。

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	刘青川1 王盼丽2 杨新波1 王仁杰1 王蒙蒙1 黄正明1

关键词 ：水芹总酚酸, Hep AD38细胞, 乙型肝炎病毒, 乙肝病毒DNA聚合酶

Abstract：Objective To observe the inhibitory effect of total phenolics acid from Oenanthe Javanica on hepatitis B virus (HBV) in vitro and explore its mechanism. Methods The toxicity of total phenolics acid from Oenanthe Javanica to Hep AD38 cells was divided into the total phenolics acid from Oenanthe Javanica group (subdivided into 1600, 800, 400, 200, 100 μg/mL concentration groups) and the blank control group. Hep AD38 cells were cultured in vitro and the toxicity range of total phenolics acid from Oenanthe Javanica was determined by MTT assay. In vitro anti-HBV effects of total phenolics acid from Oenanthe Javanica were divided into the total phenolics acid from Oenanthe Javanica group (subdivided into 200, 100, 50 and 25 μg/mL concentration groups), the positive control group [Entecavir (ETV) 10 μmol/L] and the virus control group. Hep AD38 cells were treated with total phenolics acid from Oenanthe Javanica solution with a concentration below the non-toxic concentration for 6 consecutive days. The supernatant and cells were collected. The titer of HBsAg and HBeAg in the supernatant was detected by enzyme linked immune sorbent assay (ELISA). The intracellular HBV DNA load was detected by real-time fluorescent quantitative PCR. The effects of total phenolics acid from Oenanthe Javanica on duck hepatitis B virus (DHBV) DNA polymerase were divided into the total phenolics acid from Oenanthe Javanica group (subdivided into 400, 200, 100, 50 and 25 μg/mL concentration groups), the negative control group and the DHBV DNA polymerase control group. DHBV DNA polymerase was extracted from duck liver infected with duck hepatitis B virus, different concentrations of total phenolics acid from Oenanthe Javanica solution were given, and the effects of total phenolics acid from Oenanthe Javanica on DHBV DNA polymerase activity were detected by ELISA. Results The non-toxic concentration of total phenolics acid from Oenanthe Javanica in Hep AD38 cells was 400 μg/mL. Compared with the virus control group, HBsAg, HBeAg titer and HBV DNA load were significantly decreased in the 200, 100 and 50 μg/mL concentration groups of the total phenolics acid from Oenanthe Javanica group, the differences were highly statistically significant (P ＜ 0.01). Compared with the DHBV DNA polymerase control group, DHBV DNA polymerase activity was inhibited in the 400, 200, and 100 μg/mL concentration groups of the total phenolics acid from Oenanthe Javanica group, the differences were highly statistically significant (P ＜ 0.01). Conclusion Total phenolics acid from Oenanthe Javanicahas a good anti-HBV effect in vitro, and its mechanism includes inhibition of HBV DNA polymerase activity.

Key words： Total phenolics acid from Oenanthe Javanica Hep AD38 cells Hepatitis B viral Hepatitis B viral DNA polymerase

基金资助:中国医药教育协会孙思邈中医药研究专项课题（协科字[2017第（03号）]）。

通讯作者: 黄正明（1947.2-），男，教授，主要从事新药药理学研究。

作者简介: 刘青川（1986.5-），男，硕士，主要从事新药药理学研究。

引用本文:

刘青川1 王盼丽2 杨新波1 王仁杰1 王蒙蒙1 黄正明1. 水芹总酚酸体外抗HBV作用及其机制探讨[J]. 中国医药导报, 2019, 16(28): 12-16,34.
LIU Qingchuan1 WANG Panli2 YANG Xinbo1 WANG Renjie1 WANG Mengmeng1 HUANG Zhengming1. Study on anti-HBV effect of total phenolics acid from Oenanthe Javanica in vitro and its mechanism. 中国医药导报, 2019, 16(28): 12-16,34.

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http://www.yiyaodaobao.com.cn/CN/ 或 http://www.yiyaodaobao.com.cn/CN/Y2019/V16/I28/12

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