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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 investigate the mechanism of extracellular polysaccharides (EPS) extract from Paecilomyces lilacinus in vitro against herpes simplex virus type 1 (HSV-1) infection. Methods Vero cells were used to infect target cells with different concentrations of EPS extract in all stages of HSV-1 infection. MTT assay was used to determine the toxicity of extract on Vero cells and the direct inactivation of HSV-1 Virus adsorption and biosynthesis. The effect of the extract on the expression of US6 and UL30 gene of the DNA gD gene was detected by RT-PCR. Results EPS extract had no effect on the proliferation of Vero cells. The CC50 value of Vero cells was 1 300.17 μg / ml. The cell viability was above 90% at 800 μg / ml and 25 ~ 800 μg / ml, the extract could inhibit HSV-1 adsorption and biosynthesis, and showed a dose-effect relationship; the highest inhibitory rate was 39.5% and 59.7% at the concentration of 800μg / ml, which was significantly lower than that of the virus control group There was statistical significance (P <0.01), but it did not reach complete inhibition. No direct inactivation of HSV-1 was found. The results of RT-PCR showed that the extract could interfere with the transcription of US6 and UL30 genes. The intensity of US6 mRNA by gray scale scanning was significantly lower than that of the virus control group (P <0.05). The UL30 mRNA abundance was significantly lower than that of the virus control group There was statistical significance (P <0.01). Conclusion EPS extract is less cytotoxic to Vero cells and safe to use. The extract has a certain antiviral effect, can inhibit HSV-1 adsorption and biosynthesis, but also inhibit US6 and UL30 gene transcription.