金丝桃素抗登革病毒活性研究

Study on inhibition of the dengue virus by hypericin

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(EC_(50))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。

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 20μmol/L hypericin and curcumin had no toxic effect on BHK-21 cells,the maximum drug concentration used in all experiments did not exceed 20μmol/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(EC_(50))of Hypericin was 3.544μmol/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 10μmol/L and 20μmol/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.