强效抗乙肝病毒化合物金丝桃素对抗病毒新靶点pgRNA的作用及机制研究

Hepatitis B virus (HBV) infection is still a major world health problem despite the availability of an effective vaccine. Currently, there is no treatment that completely eliminates the infection in all chronically infected patients. The approved nucleoside drugs inhibit virus replication by targeting the viral DNA polymerase, However，development of drug-resistant virus becomes problematic after elongated treatment. Therefore, the search for compounds with novel antiviral targets and mechanisms is urgent. HBV carries a partial double-stranded DNA genome and replicates through an RNA intermediate(pregenomic RNA, pgRNA). pgRNA is translated to the HBc, HBe proteins and serves as the template for HBV DNA polymerase to reverse transcribe. It is an important intermediate in the process of HBV replication and is a hot novel antiviral target which is verified in gene therapy but not in compounds. Our previous study which was supported by natural science foundation of Yunnan Province confirmed that compound hypericin had powerful anti-HBV effect and impact on the fresh spot pgRNA. pgRNA is transcribed by C open reading frame of HBV virus genome. This process is mainly modulated by liver enrichment transcription factor HNF3, HNF4 and PPAR α/PXR α. Therefore, further examination is formulated to evaluate the effects of hypericin on pgRNA. The study is carried out at different levels which includes both in vitro and in vivo studies. This research will focus on the classical cell line HepG2(2.2.15) which secretes the activated hepatitis B virus particles, as well as other cell lines rtM204V and rtV180M which include the drug-resistant mutational sitemutation site to ADR and 3TC. The expression of live enrichment transcription factor HNF3,HNF-4 and PPARα/PXRα was realized by a variety of molecular biology methods in order to analyse the mechanism of hypericin on pgRNA. Our research will elucidate the role of hypericin on pgRNA and provide the fundamental understanding for its future clinical application, This study will be of great value in developing new higher-efficiency and lower-toxicity antiviral medicines. This study will provide morein depth insights for future design of better HBV therapy.

现有核苷类化合物由于抗乙肝病毒靶点相同产生病毒耐药性而导致治疗效果很不理想。pgRNA是抗乙肝病毒的新靶点，在基因治疗中获得验证，但目前尚无化合物对pgRNA作用的报道。我们前期研究已经证实强效抗乙肝病毒化合物金丝桃素（hypericin）作用于抗病毒新靶点pgRNA。pgRNA由Cp启动子转录HBV 病毒基因组C开放阅读框生成，该过程主要受肝富集转录因子HNF3、HNF4和PPARα/PXRα调控。因此，我们计划通过体外和体内实验检测hypericin对pgRNA的作用来评价其抗乙肝病毒复制的作用，并通过进一步检测hypericin对Cp及调控因子HNF3、HNF-4、PPARα/PXRα的作用来揭示其对pgRNA的具体作用机制。对这些机理的深入研究不仅使hypericin对pgRNA的作用及抗病毒机制得到阐明，为其将来的临床应用提供实验基础，而且还为将来设计抗乙肝病毒新药提供新的思路。

现有核苷类化合物由于抗乙肝病毒靶点相同产生病毒耐药性而导致治疗效果很不理想。pgRNA是抗乙肝病毒的新靶点，由Cp启动子转录HBV 病毒基因组C开放阅读框生成，该过程主要受肝富集转录因子HNF3、HNF4和PPARα/PXRα调控。因此，我们计划通过体外和体内实验检测hypericin对pgRNA的作用来评价其抗乙肝病毒复制的作用，并通过进一步检测hypericin对Cp及调控因子HNF3、HNF-4、PPARα/PXRα的作用来揭示其对pgRNA的具体作用机制。以金丝桃素为实验组，拉米夫定为对照组，去离子水为空白组进行分组给药。选择不同细胞株及乙肝动物实验模型分组为实验对象，采用Southern blot及荧光定量PCR检测病毒HBV-DNA的复制水平；ELISA 检测HBsAg 和HBeAg 的抑制率；采用Northern blot与荧光定量PCR检测pgRNA的表达水平；Western blot以及荧光定量PCR检测调控因子HNF3β,HNF4α,PPARα/PXRα的表达水平。分别将构建好的带有病毒启动子的双荧光素酶报告基因系统及含有1.3倍HBV基因组的质粒转染进293T、L02及HepG2细胞中，并对细胞进行分组给药。给药48 h之后，收集细胞进行双荧光素酶指标检测及pgRNA的表达水平的荧光定量PCR检测。结果细胞及动物实验均显示金丝桃素与拉米夫定相比，HBV DNA及HBsAg、HBeAg的表达有明显抑制作用。与空白对照组相比，金丝桃素可显著减少pgRNA的表达，而拉米夫定无明显变化。与空白对照组与拉米夫定组相比，金丝桃素对调控因子HNF3β与HNF4α的表达有显著影响，但对PPARα和PXRα的表达无影响。在病毒启动子活性检测的结果中，金丝桃素组与拉米夫定组和空白对照组的结果一致，启动子活性变化不明显。而在pgRNA表达水平检测结果中，较空白对照组及拉米夫定组，金丝桃素组在非肝源细胞株293T细胞中表达水平并无显著性变化，但在肝源细胞株L02、HepG2中，其pgRNA的表达水平较其他两组有较明显下降。目前已证实金丝桃素通过作用于pgRNA而具有强效抗乙肝病毒活性，但其具体作用机理并非通过干扰Cp启动子活性作用。该研究为其将来的临床应用提供实验基础，而且还为将来设计抗乙肝病毒新药提供新的思路。