红树林淡紫拟青霉EPS提取物体外抗HSV-1机制研究

The in vitro anti-HSV-1action of an EPS extract made from Paecilomyces lilacinus isolated from mangrove soil

目的探讨红树林淡紫拟青霉胞外多糖(extracellular polysaccharides,EPS)提取物体外抗单纯疱疹病毒Ⅰ型(HSV-1)感染的机制。方法以Vero细胞为病毒感染靶细胞,以不同浓度EPS提取物作用于HSV-1感染过程的各个阶段,MTT法测定提取物对Vero细胞的毒性作用和对HSV-1的直接灭活作用及对病毒吸附和生物合成的影响;采用RT-PCR检测该提取物对病毒gD基因US6和DNA聚合酶基因UL30表达的影响。结果 EPS提取物对Vero细胞无促进增殖作用,其对Vero细胞的半数有毒浓度(CC50)为1 300.17μg/ml,浓度在800μg/ml以下时细胞存活率达90%以上;在25~800μg/ml浓度范围内该提取物可抑制HSV-1吸附和生物合成,并表现出一定量效关系;浓度为800μg/ml时抑制率最高,分别为39.5%和59.7%,与病毒对照组相比差异有统计学意义(P<0.01),但并不能达到完全抑制。未发现该提取物对HSV-1有直接灭活作用。RT-PCR显示该提取物可干扰病毒US6和UL30基因转录,灰度扫描分析US6mRNA丰度与病毒对照组相比差异有统计学意义(P<0.05);UL30mRNA丰度与病毒对照组相比差异有统计学意义(P<0.01)。结论 EPS提取物对Vero细胞毒性较小,使用安全。该提取物具有一定的抗病毒作用,可抑制HSV-1吸附和生物合成,还可抑制US6和UL30基因转录。

Objective To examine the in vitro antiviral action of an extracellular polysaccharide（EPS）extract made fromPaecilomyces lilacinus isolated from mangrove soil against herpes simplex virus type 1（HSV-1）.Methods The maximum safe concentration of the extract to Vero cells was assessed based on its cytopathic effect（CPE）and an MTT assay.The action of polysaccharides in different concentrations was assessed during the HSV-1replication cycle.Virus infectivity（TCID50）was examined based on the CPE,and direct inactivation by the extract was observed using an MTT assay.Inhibition of HSV-1absorption and biosynthesis by the extract was examined with an MTT assay as well.The inhibitory action of the extract on US6 and UL30gene expression was examined with RT-PCR. Results The extract had little cytotoxic effect on Vero cells,with a CC50 value of 1 300.17μg/ml,and failed to stimulate proliferation of the cells.The extract inhibited HSV-1absorption and biosynthesis in Vero cells at doses of 25μg/ml-800μg/ml in somewhat of a dose-dependent manner,and the maximum level of US6 inhibition was 39.5% and that of UL30 was 59.7% at a dose of800μg/ml.The difference in inhibition in treated cells was significant compared to that in the virus control group（P〈0.01）.However,the extract did not directly inactivate HSV-1.US6 gene transcription was markedly inhibited in cells treated with the extract at a concentration of 800μg/ml concentration（P〈0.05）in comparison to the HSV-1control group.The US6/β-actin optical density was 0.77 while that in the viral control group was 0.86,and UL30 gene expression was inhibited by the extract（P〈0.01）.The UL30 optical density/β-action optical density ratio was 0.71 in cells treated with the extract at a concentration of 800μg/ml but 0.94 in the HSV-1control group. Conclusion The EPS extract caused little harm to Vero cells.The extract had antiviral action to an extent,and it inhibited HSV-1absorption and biosynthesis in a dose-dependent manner.It also inhibited US6 and UL30gene transcription.