【摘要】 目的 研究金丝桃素在细胞水平对2型登革病毒复制的抑制效应。方法 使用MTT法测定金丝桃素和姜黄素对BHK-21细胞的最大无毒剂量；空斑法检测细胞上清液病毒粒子；Western blot检测登革病毒包膜E蛋白的表达情况；实时荧光定量PCR检测细胞内病毒RNA的复制情况；间接免疫荧光法验证金丝桃素抑制登革病毒包膜E蛋白表达的效应。结果 MTT试验确定20μmol/L浓度的金丝桃素和姜黄素对BHK-21细胞无毒性作用，所有试验采用最高药物浓度不超过20μmol/L。实时荧光定量PCR结果显示金丝桃素和姜黄素均能抑制DENV-2 RNA的复制，并且随着药物浓度增加对病毒的抑制作用增强，金丝桃素的半数有效抑制浓度(EC₅₀)为3.544μmol/L。空斑试验显示金丝桃素能抑制DENV-2病毒粒子的释放；Western blot显示金丝桃素能够抑制DENV-2的包膜E蛋白的表达。间接免疫荧光验证药物处理组DENV-2的包膜E蛋白荧光强度下降。药物预处理试验显示在感染前加入10μmol/L和20μmol/L药物对病毒复制产生抑制作用。不同时间点添加药物对登革病毒复制的早期影响较大，以感染2～12 h内添加药物的作用效果更佳；感染后36h添加药物组病毒RNA的合成与病毒对照组相比差异无统计学意义（P>0.05）。结论 金丝桃素对2型登革病毒复制有抑制作用，主要通过影响RNA的合成抑制病毒的复制。更多还原

【Abstract】 Objective To investigate the effect of hypericin on DENV-2 in vitro. Methods The maximum non-toxic doses of hypericin and curcumin on BHK-21 cells were determined using Methyl thiazolyl tetrazolium（MTT） assay; The inhibitory effect of hypericin of DENV-2 of cell supernatant viral particleswas detected using the plaque assay; Western blot detection of dengue virus envelope E protein expression; The replication of intracellular viral RNA was detected by real-time fluorescence quantitative PCR;Indirect immunofluorescence assay was used to verify the inhibitory effect of hypericin on dengue virus envelope E protein expression Results MTT assay showed that 20 μmol/L hypericin and curcumin had no toxic effect on BHK-21 cells, the maximum drug concentration used in all experiments did not exceed 20 μmol/L;Real-time fluorescence quantitative PCR results showed that hypericin and curcumin inhibited DENV-2 RNA replication, with the increase of drug concentration, the inhibitory effect on virus was enhanced. The 50% effective inhibitory concentration(EC₅₀) of Hypericin was 3.544 μmol/L;the plaque assay showed that hypericin could inhibit the release of DENV-2 virus particles. Western blot showed that hypericin could inhibit the expression of envelope E protein of DENV-2;indirect immunofluorescence assay showed that the fluorescence intensity of envelope E protein of DENV-2 decreased in the hypericin treatment group. The pretreatment test showed that the addition of 10 μmol/L and 20 μmol/L drugs before infection produced an inhibitory effect on viral replication; The results showed that the addition of hypericin at different times after infection DENV-2 had an effect on RNA synthesis, and the best effect was observed when the drug was added at 2 to 12 hours. When the drug was added at 36 hours after infection, there was no statistical difference in RNA synthesis compared with the virus control group（P>0.05）. Conclusion Hypericin inhibits the replication of dengue virus type 2 in vitro.更多还原