裙带菜茎硫酸多糖抗病毒活性及其作用机理的初步研究
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摘要
目的:
研究裙带菜茎硫酸多糖的抗病毒活性,并对其作用机理进行初步研究,为抗病毒作用机理的进一步研究奠定基础,并为新型的抗单纯疱疹病毒1型(herpes simplex virus type-1,HSV-1)药物的开发提供理论依据。
方法:
1.应用MTT法检测裙带菜茎硫酸多糖对细胞的毒性作用。
2.应用细胞病变效应(cytopathic effect,CPE)法,MTT法和空斑减数实验(plaqueredction assay,PRA)检测裙带菜茎硫酸多糖的抗病毒活性。
3.应用CPE法和PRA法,采用四种不同的加药方式在细胞水平探讨裙带菜茎硫酸多糖抗HSV-1的作用机理。
4.应用逆转录-聚核酶链反应(Reverse transcription polymerase chain reaction,RT-PCR)方法检测裙带菜茎硫酸多糖对HSV-1 gD糖蛋白基因US6和DNA聚合酶基因UL30的影响。
5.流式细胞仪分析裙带菜茎硫酸纯多糖对HSV-1感染导致的细胞周期紊乱的恢复情况。
6.CPE法初步检测裙带菜茎硫酸多糖对病毒突变株的选择情况。
结果:
1.裙带菜茎硫酸粗多糖(UPP1)和裙带菜茎硫酸纯多糖(UPP2)对Vero细胞的半数毒性浓度(50%toxic concentration,TC_(50))均远大于10000μg/ml,空斑减数实验测得其对HSV-1的半数抑制浓度(50%inhibiting concentration,IC_(50))分别为8.04μg/ml,13.54μg/ml,治疗指数(therapeutic index,TI)则分别大于1244,739。
2.UPP1、UPP2对柯萨奇B_3病毒(coxackie virus group B_3,CVB_3)、单纯疱疹病毒2型(herpes simplex virus type-2,HSV-2)所致的细胞病变都有明显的抑制作用,但是二者对伪狂犬病病毒(pseudorabies virus,PRV)所致的细胞病变均无明显的抑制作用。
3.四种不同加药方式判定裙带菜茎硫酸多糖抗病毒的作用阶段。结果显示UPP1无直接灭活病毒的作用,但是可干扰HSV-1对宿主细胞的吸附;而UPP2对HSV-1有一定的直接灭活作用,但对病毒的吸附没有影响;二者均不影响病毒的释放,但二者在高剂量对病毒DNA的复制均有一定的抑制作用。
4.与病毒对照组相比,UPP1能降低HSV-1 US6基因与UL30基因mRNA的表达水平,有显著性差异(p＜0.05),UPP2能降低UL30基因的mRNA的表达水平(p＜0.05),但对HSV-1US6基因的表达没有影响(p＞0.05)。
5.UPP2能部分恢复HSV-1感染导致的细胞周期的改变。
6.HSV-1在裙带菜茎硫酸纯多糖存在的情况下,连续扩增12代,未发现病毒突变株的出现。
结论:
本研究证实了裙带菜茎硫酸多糖具有抗HSV-1作用,其抗HSV-1作用机制有以下可能:(1)UPP1抑制病毒与细胞的吸附,抑制感染细胞中HSV-1 gD基因和DNA聚合酶基因的转录;(2)UPP2对病毒有直接灭活作用,抑制DNA聚合酶基因的转录;(3)UPP2能部分恢复病毒感染导致的紊乱的细胞周期。
Objective:
In order to examine the antiviral effect of sulfated polysaccharides from the caudex of Undaria pinnatifida against herpes simplex virus type-1(HSV-1) and assess its potential for clinical applications,antiviral activity and mechanism experiments of sulfated polysaccharides from the caudex of Undaria pinnatifida were carried out in this study.
Method:
1.MTT method was carded out to determine the 50%toxic concentration of sulfated polysaccharides from the caudex of Undaria pinnatifida.
2.CPE method and plaque reduction assay were used to determine the antiviral activity of sulfated polysaccharides from the caudex of Undaria pinnatifida.
3.CPE method and plaque reduction assay were applied to study the antiviral mechanism of sulfated polysaccharides from the caudex of Undaria pinnatifida by four action modes.
4.RT-PCR was applied to determine the effect of sulfated polysaccharides from the caudex of Undaria pinnatifida to the transcriptional expression of US6 and UL30 genes.
5.Flow cytometry was applied to analyse the turbulence to cell cycle by HSV-1 infection
6.CPE method was carded out to study resistance of HSV-1 to sulfated polysaccharides from the caudex of Undaria pinnatifida.
Results:
1.The TC_(50)(50%toxic concentration) value of UPP1 and UPP2 for Vero cells growth both were more than 10000μg/ml;the IC_(50)(50%inhibiting concentration) of UPP1 and UPP2 for HSV-1 yield reduction assay were 8.04μg/m,13.54μg/ml,respectively;and the therapeutic index(TI) were more than 1244,739,respectively.
2.CPE、MTT、PRA results showed that sulfated polysaccharides from the caudex of Undaria pinnatifida can inhibit CVB_3 and HSV-2.
3.Results indicated that UPP1 affected on viral adsorption and penetration;UPP2 inactivated HSV-1directly;both can inhibit the replication of the virus;but neither had effect on HSV-1 releasing from infected Vero cells to medium.
4.Contrast to HSV-1 control,UPP1 can depress the transcriptional expression of US6 and UL30 genes distinctly(p＜0.05),while UPP2 only depressed the transcriptional expression of UL30 gene(p＜0.05).
5.Flow cytometry results implied UPP2 can regulate the turbulence cell cycle by HSV-1 infection.
6.HSV-1 mutant strain did not find in the effect of the purfied sulfated polysaccharide from the caudex of Undaria pinnatifida.
Conclusion:
In brief,the pharmacological experiments confirmed that sulfated polysaccharides from the caudex of Undaria pinnatifida indeed showed strong anti-HSV-1 activity.The possible mechanism might include its effects on:(1) virus absorption;(2) virucidal activity;(3) HSV-1 gD gene and DNAP gene transcription;(4) rugulate the turbulence cell cycle infected by HSV-1.
研究裙带菜茎硫酸多糖的抗病毒活性,并对其作用机理进行初步研究,为抗病毒作用机理的进一步研究奠定基础,并为新型的抗单纯疱疹病毒1型(herpes simplex virus type-1,HSV-1)药物的开发提供理论依据。
方法:
1.应用MTT法检测裙带菜茎硫酸多糖对细胞的毒性作用。
2.应用细胞病变效应(cytopathic effect,CPE)法,MTT法和空斑减数实验(plaqueredction assay,PRA)检测裙带菜茎硫酸多糖的抗病毒活性。
3.应用CPE法和PRA法,采用四种不同的加药方式在细胞水平探讨裙带菜茎硫酸多糖抗HSV-1的作用机理。
4.应用逆转录-聚核酶链反应(Reverse transcription polymerase chain reaction,RT-PCR)方法检测裙带菜茎硫酸多糖对HSV-1 gD糖蛋白基因US6和DNA聚合酶基因UL30的影响。
5.流式细胞仪分析裙带菜茎硫酸纯多糖对HSV-1感染导致的细胞周期紊乱的恢复情况。
6.CPE法初步检测裙带菜茎硫酸多糖对病毒突变株的选择情况。
结果:
1.裙带菜茎硫酸粗多糖(UPP1)和裙带菜茎硫酸纯多糖(UPP2)对Vero细胞的半数毒性浓度(50%toxic concentration,TC_(50))均远大于10000μg/ml,空斑减数实验测得其对HSV-1的半数抑制浓度(50%inhibiting concentration,IC_(50))分别为8.04μg/ml,13.54μg/ml,治疗指数(therapeutic index,TI)则分别大于1244,739。
2.UPP1、UPP2对柯萨奇B_3病毒(coxackie virus group B_3,CVB_3)、单纯疱疹病毒2型(herpes simplex virus type-2,HSV-2)所致的细胞病变都有明显的抑制作用,但是二者对伪狂犬病病毒(pseudorabies virus,PRV)所致的细胞病变均无明显的抑制作用。
3.四种不同加药方式判定裙带菜茎硫酸多糖抗病毒的作用阶段。结果显示UPP1无直接灭活病毒的作用,但是可干扰HSV-1对宿主细胞的吸附;而UPP2对HSV-1有一定的直接灭活作用,但对病毒的吸附没有影响;二者均不影响病毒的释放,但二者在高剂量对病毒DNA的复制均有一定的抑制作用。
4.与病毒对照组相比,UPP1能降低HSV-1 US6基因与UL30基因mRNA的表达水平,有显著性差异(p＜0.05),UPP2能降低UL30基因的mRNA的表达水平(p＜0.05),但对HSV-1US6基因的表达没有影响(p＞0.05)。
5.UPP2能部分恢复HSV-1感染导致的细胞周期的改变。
6.HSV-1在裙带菜茎硫酸纯多糖存在的情况下,连续扩增12代,未发现病毒突变株的出现。
结论:
本研究证实了裙带菜茎硫酸多糖具有抗HSV-1作用,其抗HSV-1作用机制有以下可能:(1)UPP1抑制病毒与细胞的吸附,抑制感染细胞中HSV-1 gD基因和DNA聚合酶基因的转录;(2)UPP2对病毒有直接灭活作用,抑制DNA聚合酶基因的转录;(3)UPP2能部分恢复病毒感染导致的紊乱的细胞周期。
Objective:
In order to examine the antiviral effect of sulfated polysaccharides from the caudex of Undaria pinnatifida against herpes simplex virus type-1(HSV-1) and assess its potential for clinical applications,antiviral activity and mechanism experiments of sulfated polysaccharides from the caudex of Undaria pinnatifida were carried out in this study.
Method:
1.MTT method was carded out to determine the 50%toxic concentration of sulfated polysaccharides from the caudex of Undaria pinnatifida.
2.CPE method and plaque reduction assay were used to determine the antiviral activity of sulfated polysaccharides from the caudex of Undaria pinnatifida.
3.CPE method and plaque reduction assay were applied to study the antiviral mechanism of sulfated polysaccharides from the caudex of Undaria pinnatifida by four action modes.
4.RT-PCR was applied to determine the effect of sulfated polysaccharides from the caudex of Undaria pinnatifida to the transcriptional expression of US6 and UL30 genes.
5.Flow cytometry was applied to analyse the turbulence to cell cycle by HSV-1 infection
6.CPE method was carded out to study resistance of HSV-1 to sulfated polysaccharides from the caudex of Undaria pinnatifida.
Results:
1.The TC_(50)(50%toxic concentration) value of UPP1 and UPP2 for Vero cells growth both were more than 10000μg/ml;the IC_(50)(50%inhibiting concentration) of UPP1 and UPP2 for HSV-1 yield reduction assay were 8.04μg/m,13.54μg/ml,respectively;and the therapeutic index(TI) were more than 1244,739,respectively.
2.CPE、MTT、PRA results showed that sulfated polysaccharides from the caudex of Undaria pinnatifida can inhibit CVB_3 and HSV-2.
3.Results indicated that UPP1 affected on viral adsorption and penetration;UPP2 inactivated HSV-1directly;both can inhibit the replication of the virus;but neither had effect on HSV-1 releasing from infected Vero cells to medium.
4.Contrast to HSV-1 control,UPP1 can depress the transcriptional expression of US6 and UL30 genes distinctly(p＜0.05),while UPP2 only depressed the transcriptional expression of UL30 gene(p＜0.05).
5.Flow cytometry results implied UPP2 can regulate the turbulence cell cycle by HSV-1 infection.
6.HSV-1 mutant strain did not find in the effect of the purfied sulfated polysaccharide from the caudex of Undaria pinnatifida.
Conclusion:
In brief,the pharmacological experiments confirmed that sulfated polysaccharides from the caudex of Undaria pinnatifida indeed showed strong anti-HSV-1 activity.The possible mechanism might include its effects on:(1) virus absorption;(2) virucidal activity;(3) HSV-1 gD gene and DNAP gene transcription;(4) rugulate the turbulence cell cycle infected by HSV-1.
引文
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