复方肃毒星提取物抑制乙型肝炎病毒cccDNA的作用  

Inhibitory effect of Suduxing extracts on covalently closed circular DNA of hepatitis B virus

作　　者：思兰兰 李晓东 李乐 牛明 王伽伯 兰金初 徐东平 刘妍 Si Lanlan;Li Xiaodong;Li Le;Niu Ming;Wang Jiabo;Lan Jinchu;Xu Dongping;Liu Yan(Viral Hepatitis Research Laboratory,Institute of Infectious Diseases and Research Center for Clinical and Translational Medicine,the Fifth Medical Centre of Chinese PLA General Hospital,Beijing 100039,China;Institute of Chinese Medicine,the Fifth Medical Centre of Chinese PLA General Hospital,Beijing 100039,China;Department of Chinese Traditional Medicine,Beijing Gulou Hospital of Chinese Medicine,Beijing 100009,China)

机构地区：[1]解放军总医院第五医学中心临床研究管理中心,北京100039 [2]解放军总医院第五医学中心中药研究所,北京100039 [3]北京鼓楼中医院中医科,北京100009

出　　处：《中华实验和临床感染病杂志（电子版）》2020年第4期265-271,共7页Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)

基　　金：国家自然科学基金面上项目(No.81573676);国家自然科学基金重点项目(No.81930110)。

摘　　要：目的研究复方肃毒星提取物(简称肃毒星)体外对乙型肝炎病毒(HBV)复制模板共价闭合环状DNA(cccDNA)的抑制作用。方法通过细胞计数试剂盒CCK-8检测肃毒星在恩替卡韦耐药HBV稳定复制细胞系(HepG2.A64)、野生HBV稳定复制细胞系(HepG2.2.15和HepAD38)以及肝癌细胞系(HepG2)中的毒性作用;选择安全有效的高、中、低浓度肃毒星分别处理3个细胞系,采用实时荧光定量PCR法检测细胞内HBV cccDNA水平。以高斯荧光素酶微环cccDNA(mc-cccDNA)转染HepG2细胞,同时选择高、中、低浓度的肃毒星在不同时间点作用转染mc-cccDNA的HepG2细胞,荧光素酶检测试剂盒分析相对荧光强度,计算对cccDNA的抑制率,评价cccDNA转录活性。结果肃毒星在HepG2.A64、HepG2.2.15、HepAD38和HepG2细胞中的半数毒性浓度分别为27.01μg/ml、29.36μg/ml、31.20μg/ml和52.80μg/ml。肃毒星对HepG2.A64、HepG2.2.15和HepAD38细胞中HBV cccDNA有抑制作用,最佳作用时间为5 d,5 d最大抑制率分别为(84.24±2.1)%、(52.02±4.74)%和(47.16±6.69)%,与未用药处理组差异有统计学意义(P均<0.05)。肃毒星对转染HepG2细胞后mc-cccDNA转录活性的最佳抑制率为(48.44±4.54)%,抑制作用优于干扰素对照组(P<0.05),略弱于特异性敲低mc-cccDNA的pLV-CRISPR处理对照组(P<0.05),差异有统计学意义。结论肃毒星对恩替卡韦耐药型和野生型细胞系HBV cccDNA的复制及转录均有很好的抑制作用,为慢性乙型肝炎功能学治愈新药的开发提供参考。Objective To investigate the inhibition effect of compound Suduxing extracts(hereinafter referred as Suduxing)on hepatitis B virus(HBV)covalently closed circular DNA(cccDNA).Methods The toxicity of Suduxing on entecavir(ETV)-resistant HBV-stably-replication cell line(HepG2.A64),wild-type HBV-stably-replication cell lines(HepG2.2.15 and HepAD38)and hepatocellular carcinoma cell line(HepG2)were assessed by cell counting kit-8(CCK-8)assay.The three HBV-stably-replication cells were treated with the high,medium and low concentrations of Suduxing at different time points,respectively,and HBV cccDNA were detected by the real-time fluorescent quantitative PCR.Gaussia luciferase microcircle cccDNA(mc-cccDNA)was transfected into HepG2 cells,and HepG2 cells were treated with high,medium and low concentrations of Suduxing at different time points.Then the relative fluorescence intensity was detected by luciferase assay kit,and the inhibition rate of the cccDNA was calculated to evaluate the transcriptional activity.Results The toxic concentration 50%of Suduxing in HepG2.A64,HepG2.2.15,HepAD38 and HepG2 cells were 27.01μg/ml,29.36μg/ml,31.20μg/ml and 52.80μg/ml,respectively.Compared with the untreated group,Suduxing had better inhibitory effect on HBV cccDNA in HepG2.A64,HepG2.2.15 and HepAD38 cells.The optimal treatment time was 5 days after-treatment,and the maximum inhibition rates were(84.24±2.1)%,(52.02±4.74)%and(47.16±6.69)%,respectively(all P<0.05).The optimal inhibition rate of Suduxing on the mc-cccDNA transcriptional activity in HepG2 cells was(48.44±4.54)%.The inhibitory effect was better than interferon-treated group(P<0.05),and slightly weaker than the pLVCRISPR group with specific knockdown of mc-cccDNA(P<0.05),with significant differences.Conclusions Suduxing could inhibit the replication and transcription of entecavir-resistant and wild-type HBV cccDNA.The findings provide a new reference strategy for drug development to functional cure of chronic hepatitis B.

关 键 词：肝炎病毒 乙型 共价闭合环状DNA 肃毒星提取物 转录活性 

分 类 号：R285[医药卫生—中药学]