皱瘤海鞘抗单纯疱疹病毒Ⅱ型活性及作用机制研究
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摘要
海鞘(Ascidian)是尾索动物亚门(Uro-chordata)海鞘纲(Ascidiacea)的尾索动物,主要分布在热带及亚热带海域,海鞘中含有许多有生理活性的化合物,这些活性化合物有抗肿瘤、抗病毒、抗菌等作用。皱瘤海鞘Styela plicata在我国南海有大量分布,研究皱瘤海鞘的药用价值,对开发利用我国的这一丰富资源具有一定的现实意义。
研究目的
课题组前期研究发现皱瘤海鞘在体内外对乙型肝炎病毒(HBV)都有一定的抑制作用,显示出良好的研究和开发前景。因此,本课题拟对皱瘤海鞘对同是具有包膜的DNA病毒单纯疱疹病毒Ⅱ型(HSV-2)药效活性和作用机制进行研究。为进一步了解皱瘤海鞘的抗病毒谱,为新型抗病毒药物的研究和开发提供理论依据,为皱瘤海鞘的进一步深入研究和开发利用提供科学的理论基础。
研究方法1.皱瘤海鞘体外抗HSV-2药效学研究
运用HSV-2在非州绿猴肾细胞(Vero细胞)上的增殖模型,通过观察细胞病变(CPE法)和四甲基偶氮唑盐比色法(MTT比色法)检测细胞活性,以病毒抑制率和治疗指数(TI)为指标对皱瘤海鞘体外抗HSV-2的药效活性作用进行评价。
2.皱瘤海鞘体外抗HSV-2病毒作用机制研究
运用HSV-2在Vero细胞上的增殖模型,在HSV-2复制的不同阶段用药物进行干预,运用CPE法结合MTT比色法检测细胞存活率,以病毒抑制率和TI为指标考察了药物在体外对HSV-2病毒直接杀伤作用、对HSV-2吸附的阻断作用、对HSV-2病毒生物合成的抑制作用和在不同时间点加入药物对HSV-2复制的抑制作用；运用荧光定量PCR方法,从分子水平上探讨了皱瘤海鞘对HSV-2DNA双链复制的影响；运用RT-PCR方法进一步研究皱瘤海鞘对HSV-2DNA聚合酶基因表达的影响。
3.皱瘤海鞘对阴道感染HSV-2小鼠的保护作用
运用小鼠阴道感染HSV-2的致病模型,研究了皱瘤海鞘对小鼠生殖器疱疹的治疗作用。以小鼠外阴病变程度评分和小鼠阴道粘膜组织病理学检查为指标评价皱瘤海鞘对生殖器疱疹的治疗作用。4.皱瘤海鞘体内抗HSV-2作用机制研究
运用小鼠阴道感染HSV-2的致病模型,研究了皱瘤海鞘对小鼠免疫细胞的的调节作用。在治疗周期结束后,小鼠处死,取小鼠外周血抗凝,流式细胞术测定CD4, CD8阳性T细胞百分比,比较用药后各组小鼠外周血T细胞亚群的差异,评价皱瘤海鞘对小鼠的免疫功能的调节作用。
运用小鼠阴道感染HSV-2的致病模型,研究了皱瘤海鞘对小鼠Th细胞因子的调节作用。在治疗周期结束后,小鼠处死,取小鼠外周血,收集血清,用酶联免疫吸附分析法测定血清中IL-2, IFN-y, IL-4, IL-10水平。比较用药后各组小鼠外周血IL-2, IFN-y, IL-4和IL-10的差异,评价皱瘤海鞘对小鼠的免疫功能的调节作用。
5.皱瘤海鞘抗HSV-2活性有药效部位的筛选
运用HSV-2在Vero细胞上的增殖模型,以CPE法和MTT比色法检测细胞活性。以细胞存活率和半数细胞毒性浓度(CC50)为指标考察药物的细胞毒性；以病毒抑制率为指标,对皱瘤海鞘氯仿部位、正丁醇部位,水部位体外抗HSV-2的药效活性作用进行评价；以病毒抑制率为指标考察了皱瘤海鞘水部位在体外对HSV-2病毒直接杀伤作用、对HSV-2吸附的阻断作用以及对HSV-2病毒生物合成的抑制作用。
6.统计学处理
本文所有计量资料数据均采用单因素方差分析方法进行,先进行方差齐性检验,若方差齐,则应用One-way ANOVA方法进行整体检验,用LSD方法进行组间多重比较,若方差不齐,则应用Welch方法进行整体检验,用DunnettT3方法进行组间多重比较。等级资料数据应用多个独立样本非参数检验进行分析。P<0.05,可认为组间比较具有统计学意义,数据用SPSS13.0软件处理。
研究结果
1.药物对Vero细胞毒性
实验结果显示,与细胞对照组比较,皱瘤海鞘各组吸光度值均无统计学意义(P>0.05), ACV组在1 mg/ml浓度以下各组均无统计学意义(P>0.05)。皱瘤海鞘对Vero细胞的毒性作用小,在10mg/ml浓度下细胞存活率仍有74.88%,其对Vero细胞的CC50值大于10mg/ml,阳性对照药ACV的CC50为1.57mg/ml。
2.药物对HSV-2致细胞病变的抑制作用
实验结果显示,皱瘤海鞘各组吸光度值与病毒对照组比较均具有统计学意义(P<0.01),阳性对照药阿昔洛韦(acylovir, ACV) 0.01～0.04 mg/ml组吸光度与病毒对照组比较均具有统计学意义(P<0.01)。皱瘤海鞘对HSV-2的半数有效浓度(EC50)为0.40 mg/ml, TI>25,对HSV-2感染Vero细胞显示出较强的保护作用；ACV50%病毒抑制率药物浓度EC50=0.0067mg/ml,治疗指数TI=224,对HSV-2显示出较强的抑制作用。
3.皱瘤海鞘对HSV-2直接杀伤活性
应用MTT法测定细胞活性,计算皱瘤海鞘对HSV-2病毒的抑制率。结果显示,皱瘤海鞘在1 mg/ml时吸光度与病毒对照组比较具有统计学意义(P<0.01),在2和1 mg/ml时,其对病毒抑制率分别达74.19,64.52%,EC50为0.63mg/ml, TI值大于15.87。结果表明,皱瘤海鞘对病毒具有一定的直接杀伤活性。
4.皱瘤海鞘对HSV-2增殖的影响
运用MTT法测定细胞活性,计算皱瘤海鞘对HSV-2病毒的抑制率。实验结果显示,皱瘤海鞘2和1mg/ml组吸光度与病毒对照组比较均具有统计学意义(P<0.05),在2和1mg/ml时,其对病毒的抑制率分别达82.98%和65.96%,在0.25mg/ml时,其对病毒抑制率也达29.79%。其ECso值为0.66mg/ml, TI值大于15.15,实验结果提示,皱瘤海鞘对病毒复制具有一定的抑制作用。
5.皱瘤海鞘对HSV-2吸附的阻断作用
运用MTT法测定细胞活性,计算皱瘤海鞘对HSV-2病毒的抑制率。实验测得皱瘤海鞘各组吸光度值与病毒对照比较均无统计学意义(P>0.05),经计算求得,其在2mg/ml时,其对病毒抑制率达只有23.08%,没有超过50%。实验结果提示皱瘤海鞘对病毒的吸附没有阻断作用。
6.在病毒感染前后不同时间点加入皱瘤海鞘对HSV-2病毒的影响
运用MTT法测定在不同时间点加入皱瘤海鞘对病毒复制的抑制作用,计算皱瘤海鞘对HSV-2病毒的抑制率。结果显示,在病毒感染前2小时和感染后0,2,4,6小时加入药物,最后测得的吸光度值与病毒对照比较均具有统计学意义(P<0.01)；病毒在感染后0,2小时加入药物对病毒抑制率达65%以上,对病毒显示出明显的抑制作用。在感染后4,6小时加入药物,对病毒抑制率分别是41.27%,34.92%,在感染后10小时加入药物,对病毒复制没有抑制作用。
7.荧光定量PCR方法测定皱瘤海鞘对HSV-2病毒DNA复制的影响
实验结果显示,与病毒对照组比较,皱瘤海鞘2,1,0.5 mg/ml组能使HSV-2DNA拷贝数下降2-3个数量级,差异具有统计学意义(P<0.01)。实验结果提示,皱瘤海鞘对HSV-2 DNA链复制有一定抑制作用。
8. RT-PCR法检测皱瘤海鞘对HSV-2 DNA聚合酶UL30基因表达的影响
RT-PCR扩增40个循环后,所有样品均能扩增出特异性的β-actin片段,在皱瘤海鞘(2mg/ml)组和ACV组的均能检测到DNA聚合酶UL30 mRNA的表达,但是与病毒对照组相比,表达量显著降低,提示皱瘤海鞘对HSV-2DNA聚合酶的基因表达具有一定的干扰作用。
9.皱瘤海鞘对小鼠生殖器疱疹的治疗作用
病毒攻击后,经过连续7天治疗。实验结果显示,海鞘可明显减轻阴道感染HSV-2小鼠外阴肿胀,缩短病程。与模型组比较,阳性药ACV组,海鞘高、低剂量组小鼠外阴病变评分都较小,差异具有统计学意义(P<0.05)。小鼠阴道组织病理学检查,光镜下,正常组的阴道粘膜完整,没有炎细胞浸润,没有损伤表现。模型组小鼠阴道粘膜损伤严重,有大量的出血点,有大量炎细胞浸润；ACV组,皱瘤海鞘高、低剂组阴道粘膜损伤较轻,只有少量炎细胞浸润,没有出血点。结果表明,皱瘤海鞘对小鼠生殖器疱疹具有一定治疗作用。
10.皱瘤海鞘对感染HSV-2小鼠外周血T细胞亚群的影响
病毒攻击后,连续给药治疗7天。实验结果显示,与空白对照组相比较,模型对照组外周血中CD4+T细胞,CD8+T细胞百分比均具有显著差异(P<0.01),可认为造模后两者均显著减少,免疫功能受到一定抑制。ACV治疗组、海鞘治疗组和模型对照组相比,小鼠外周血中CD4+T细胞,CD8+T细胞百分比均有显著差异(P<0.01),可认为经治疗后,CD4+T细胞和CD8+T细胞数量有了显著提高。与空白对照组相比较,模型组外周血CD4+T细胞/CD8+细胞比值有显著差异(P<0.01),可认为模型组CD4+T细胞和CD8+细胞平衡失调。ACV治疗组、皱瘤海鞘治疗组和模型对照组相比,外周血CD4+T细胞/CD8+细胞比值均具有显著性差异(P<0.01,P<0.05),可认为经治疗后,小鼠T细胞平衡失调得到明显改善；与正常组比较,皱瘤海鞘治疗组CD4+T细胞/CD8+细胞比值无显著差异(P>0.05),ACV治疗组CD4+T细胞/CD8+细胞比值有显著差异(P<0.01)。
11.皱瘤海鞘对小鼠IL-2, IFN-γ细胞因子水平的影响
病毒攻击后,连续给药治疗7天。实验结果显示,与空白对照组相比较,模型对照组小鼠血清IL-2水平显著降低(P<0.01)；与模型组比较,皱瘤海鞘高、低剂量组小鼠血清IL-2水平均有显著增加(P<0.01),可认为皱瘤海鞘能提高小鼠IL-2的表达水平；与模型组比较,阳性药(ACV)组小鼠血清IL-2水平有升高趋势,但无统计学意义(P>0.05)；与模型组比较,各组小鼠血清IFN-y水平均无显著性差异(P>0.05)。
12.皱瘤海鞘对小鼠IL-4,IL-10细胞因子水平的影响
病毒攻击后,连续给药治疗7天。实验结果显示,与空白对照组相比较,模型对照组小鼠血清IL-4, IL-10水平无显著性差异(P>0.05)；与模型组比较,阳性药(ACV)组小鼠血清IL-4,IL-10水平均无显著性差异(P>0.05)；皱瘤海鞘高、低剂量组与模型组比较,小鼠血清IL-4, IL-10水平均具有显著性差异(P<0.01,P<0.05),可认为皱瘤海鞘能够提高致病小鼠IL-4,IL-10水平。
13.皱瘤海鞘不同部位对Vero细胞毒性
MTT法测定皱瘤海鞘不同部位对Vero细胞毒性。实验测得皱瘤海鞘氯仿部位、正丁醇部位和水部位0.08～10 mg/ml组吸光度值与细胞对照组比较均具有统计学意义(P<0.01,P<0.05)；皱瘤海鞘氯仿部位对Vero细胞的CC50为4.62mg/ml,皱瘤海鞘正丁醇部位对Vero细胞的CCso大于10mg/ml,皱瘤海鞘水部位对Vero细胞的CC50大于10mg/ml。
14.皱瘤海鞘氯仿部位对HSV-2致细胞病变抑制效应
MTT法测定皱瘤海鞘氯仿对HSV-2致细胞病变抑制效果,结果显示,各浓度组吸光度值与病毒对照组比较均无统计学意义(P>0.05),2mg/ml皱瘤海鞘氯仿部位对HSV-2抑制率为19.23%,没有达到50%。实验结果表明,皱瘤海鞘氯仿部位对HSV-2无抑制作用。
15.皱瘤海鞘正丁醇部位对HSV-2致细胞病变抑制效应
MTT法测定皱瘤海鞘正丁醇部位对HSV-2致细胞病变抑制效果,结果显示,各浓度组吸光度值与病毒对照组比较均无统计学意义(P>0.05),2mg/ml皱瘤海鞘正丁醇部位HSV-2抑制率为11.54%。实验结果表明,皱瘤海鞘正丁醇部位对HSV-2无抑制作用。
16.皱瘤海鞘水部位对HSV-2致细胞病变抑制效应
MTT法测定皱瘤海鞘水部位对HSV-2致细胞病变抑制效果。结果显示,2、1及0.5 mg/ml浓度组吸光度值与病毒对照组比较均具有统计学意义(P<0.01,<0.05),2,1 mg/ml瘤海鞘水部位对HSV-2病毒抑制率分别达到91.43%,77.14%,其EC50为0.67mg/ml, TI值大于14.93,结果表明,皱瘤海鞘水部位对HSV-2具有一定抑制作用。
17.皱瘤海鞘水部位对HSV-2增殖的影响
MTT法测定皱瘤海鞘水部位对HSV-2增殖的影响。实验结果显示,皱瘤海鞘水部分各浓组吸光度值与病毒对照组比较均无统计学意义(P>0.05),皱瘤海鞘水部位2、1和0.5mg/ml时,其病毒抑制率分别达82.98,65.96和63.93%,EC50=0.49mg/ml, T>20.41。结果表明,皱瘤海鞘水部位对HSV-2增殖具有一定抑制作用。
18.皱瘤海鞘水部位对HSV-2直接杀伤作用
MTT法测定皱瘤海鞘水部位对HSV-2直接杀伤作用,结果显示,皱瘤海鞘水部位各浓度组吸光度值与病毒对照组比较均无计学意义(P>0.05),皱瘤海鞘水部位在2和1mg/ml时,其病毒抑制率分别达74.29%,60.00%, EC50=0.80mg/ml,T>12.50。结果表明,皱瘤海鞘水部位对病毒具有一定的杀伤活性。19.皱瘤海鞘水部位对HSV-2吸附的阻断作用
MTT法测定皱瘤海鞘水部位对HSV-2吸附的阻断作用。实验结果显示,皱瘤海鞘水部位各组吸光度值与病毒对照组比较均无统计学意义(P>0.05),在2mg/ml时,其病毒抑制率只有19.23%,没有达到50%。实验结果表明皱瘤海鞘水部位对HSV-2病毒吸附没有阻断作用,
研究结论
1.运用细胞模型证实皱瘤海鞘对HSV-2复制具有抑制作用。
2.运用细胞模型证实皱瘤海鞘对HSV-2具直接杀伤作用,对HSV-2生物合成具有抑制作用,而对HSV-2吸附侵入细胞没有阻断作用。
3.运用细胞模型证实皱瘤海鞘对HSV-2 DNA链复制延长具有抑制作用,其作用靶点之一是抑制HSV-2 DNA聚合酶基因表达。
4.皱瘤海鞘能够对阴道感染HSV-2小鼠生殖器疱疹具有治疗作用。
5.皱瘤海鞘能够提高阴道感染HSV-2小鼠外周血液CD4+和CD8+T细胞亚群百分比,调节小鼠T细胞亚群的平衡；能提高阴道感染HSV-2小鼠血液Th细胞因子水平,从而调节和改善小鼠的免疫功能。这些可能是其对生殖器疱疹具有治疗作用一个有效机制。
6.皱瘤海鞘提取物抗HSV-2的有效活性部位是水溶性成分,而不是正丁醇和三氯甲烷等有机溶剂部位,其最可能的成分是小分子多肽等物质。
Styela plicata (lesueur) is an ascidian which belongs to class Ascidiacea, subphylum urochordata. They mainly distribute over the tropical zone and subtropical zone maritime space. The scabbard contains many physiological activity compounds, and most of them have strong biological activity, such as anti-tumor, anti-virus, antibiosis, etc. There are abundant ascidian resources in the South China Sea. So, study on the medicinal value of Styela plicata for development and utilization of the resources of our country is of great practical significance.
Objective
Because we finds the Styela plicata can inhibit hepatitis b virus in vitro and in vivo before. So, for expanding and understanding the Styela plicata of antiviral spectra, this issue is to study on the effects of Styela plicata on herpes simplex virus 2(HSV-2)and its mechanism of action in vitro and in vivo. The research will provide scientific theory for development of new anti-HSV-2 drugs and for further development and utilization of Styela plicata,
Methods
1. Anti-HSV-2 activiy of the ethanol extracts of Styela plicata
The Vero cell infected with HSV-2 was used as experimental model. CPE assay and MTT assay were applied to investigate anti-HSV-2 activiy of the ethanol extracts of Styela plicata in vitro. Survival rate of cell and CC50 value were applied to evaluate cytotoxicity of medicine; the viral inhibition ratio and TI value were used to evaluate anti-HSV-2 activity of alcohol extracts of Styela plicata.
2. The study on the mechanism of the anti-HSV-2 action of the ethanol extracts of Styela plicata in vitro
The Vero cell infected with HSV-2 was used as experimental model. CPE assay and MTT assay were applied. The viral inhibition ratio and TI value were used to evalueate the effect of the ethanol extracts of Styela plicata on HSV-2. Fluorescence detection method of PCR assay was applied to investigate the effect of the ethanol extracts of Styela plicata on the replication of HSV-2 DNA chain. RT-PCR assay was applied to investigate the effect of the ethanol extracts of Styela plicata on the expression of HSV-2 DNA pol gene.
3. Protective effect of Styela plicata on the mice infected with HSV-2 in vagina
Mice infected with HSV-2 in vagina were applied to investigated the effect of Styela plicata on genital herpes. The mice cunnus pathological changes extent value and histopathology were used to eveluate the effect of Styela plicata on genital herpes. The contrast of different group mice cunnus pathological changes extent were investigated by rank-sum test.
4. The study on the mechanism of the anti-HSV-2 action of the ethanol extracts of Styela plicata in vivo
Mice infected with HSV-2 in vagina was applied. After treatment was completed, the mice were killed. The peripheral blood of the mice were collected and anticoagulated. Flow cytometry assay was applied to determined the percentage of CD4+T cells and CD8+T cells, respectively. One-Way ANOVA assay was applied to analyze the difference of T cell subgroups in peripheral blood of all class mice. The effect of Styela plicata on immunologic function was evaluated.
Mice infected with HSV-2 in vagina were applied. Aafter treatment was completed, the mice were killed, the peripheral blood of the mice was collected. The sera were collected. ELISA assay was applied to determined the concentation of IL-2, IFN-y, IL-4 and IL-10 in the sera. One-Way ANOVA assay was applied to analyze the difference of concentation of IL-2, IFN-y, IL-4 and IL-10 in the sera among all class mice. The effect of Styela plicata on immunologic function was evaluated.
5. The screening of effective fraction for anti-Herpes simplex virus 2 of Styela plicata
The Vero cell infected with HSV-2 was used as experimental model. CPE assay and MTT assay were applied to investigate the effect of the different fraction of Styela plicata on HSV-2 in vitro. Survival rate of cell and CC50 value were applied to evaluate cytotoxicity of the different fraction of Styela plicata; the viral inhibition ratio and TI value were used to evaluate anti-HSV-2 activity of the different fraction of Styela plicata.
6. Statistical analysis
All measurement data in this paper were analyzed by One-way ANOVA test. Homoscedasticity was analyzed, ANOVA test was used to analyze the data while homoscedasticity, Welch test was used to analyze the data while homoscedasticity of variance.The ranked data were analyzed by K Independent Samples Tests. P value of less than 0.05 was considered to be statistically significant.
Results
1. The cytotoxic effect of alcohol extracts of Styela plicata on Vero cell
MTT assay was applied to investigate the cytotoxic effect of the ethanol extracts from Styela plicata on Vero cell. The results displayed the absorbance values of the ethanol extracts of Styela plicata groups had significant difference compared to that of vero cell control group (P<0.05); the absorbance values of ACV(not more than lmg/ml) groups were not significantly difference compared to that of vero cell control group (P>0.05). The vero cell survival rate was 74.88% at concentration of 10mg/ml the ethanol extracts of Styela plicata. The CC50 value of the ethanol extracts of Styela plicata on Vero cell was more than lOmg/ml, which indicated the cytotoxic effect of alcohol extracts of Styela plicata on Vero cell was low. The CC50 value of ACV was 1.57mg/ml.
2. Anti-HSV-2 activiy of the ethanol extracts of Styela plicata
The results displayed the absorbance values of the ethanol extracts of Styela plicata groups had significant difference compared to that of HSV-2 control group (P<0.01); the absorbance values of ACV(0.04～0.01 mg/ml) groups had significant difference compared to that of vero cell control group (P<0.01). The EC50 value of the ethanol extracts of Styela plicata on HSV-2 is 0.40 mg/ml, TI value is more than 25, which indicated the ethanol extracts of Styela plicata had good protective effect on Vero cell infected with HSV-2. The EC50 value of ACV on HSV-2 is 0.0067mg/ml, TI value was 224, which indicated ACV had good protective effect on Vero cell infected with HSV-2.
3. The direct lethal effect of the ethanol extracts of Styela plicata on HSV-2
MTT assay was applied to investigate the direct lethal effect of the ethanol extracts of Styela plicata on HSV-2. The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was calculated. The results displayed the absorbance values of alcohol extracts of Styela plicata group(1 mg/ml) had significant difference compared to that of HSV-2 control group (P<0.01). The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was 74.19% at 2mg/ml concentration. The EC50 was 0.63mg/ml, TI value was 15.87, which indicated the ethanol extracts of Styela plicata had good direct lethal effect on HSV-2.
4. The effect of the ethanol extracts of Styela plicata on multiplication of HSV-2
MTT assay was applied to investigate the effect the ethanol extracts of Styela plicata on multiplication of HSV-2. The results displayed the absorbance values of the ethanol extracts of Styela plicata groups(2.0 and 1.0mg/ml) had significant difference compared to that HSV-2 control group (P<0.05). The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was calculated. The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was 82.98% at 2mg/ml concentration, the inhibition ratio was 29.79% at 0.25mg/ml concentration. The EC50 was 0.66mg/ml, TI value was 15.15, which indicated the ethanol extracts of Styela plicata could inhibit the multiplication of HSV-2.
5. The effect of the ethanol extracts of Styela plicata on adsorbtion HSV-2
MTT assay was applied to investigate the effect of the ethanol extracts of Styela plicata on HSV-2 adsorbtion. The inhibition ratio the ethanol extracts of Styela plicata on HSV-2 was calculated. The results displayed the absorbance values of the ethanol extracts of Styela plicata groups had not significant difference compared to that HSV-2 control group (P>0.05). The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was 23.08% at 2mg/ml concentration, it was not more than 50%, which indicated the ethanol extracts of Styela plicata could not inhibit HSV-2 invased into host cell.
6. Effect of addition time on the antiviral activity of the ethanol extracts of Styela plicata
MTT assay was applied to investigate time-of-addition effect of the ethanol extracts of Styela plicata. The results displayed the absorbance values the ethanol extracts of Styela plicata groups had significant difference compared to that of HSV-2 control group but 10 hour post infection group(P<0.01). The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was calculated. The experimental results indicated the ethanol extracts of Styela plicata could well inhibit HSV-2 replicate when the extracts were added at 0,2,4,6h post infection. At 0,2 hour post infection, the inhibition ratios of the ethanol extracts of Styela plicata on HSV-2 were more than 65%, At 4,6h, they were 41.27% and 34.92%, respectively. At 10 hours post infection, the ethanol extracts of Styela plicata could not inhibit HSV-2 replication.
7. The effect of the ethanol extracts from Styela plicata on HSV-2 DNA chain replication
HSV-2 DNA copy in the ethanol extracts of Styela plicata group (at 2,1,0.5 ml/mg) had statistical significance (P<0.01) compared with the virus control, the experimental results prompted the ethanol extracts of Styela plicata could interrupt HSV-2 DNA chain replicated.
8. The effect of the extracts of Styela plicata on HSV-2 DNA pol gene (UL30) expression.
After 40 cycle period RT-PCR amplification, all sample could be amplificated specificβ-actin frags. HSV-2 DNA polymerases mRNA of the virus control, the Styela plicata (2mg/ml) group and ACV group could be detected, respectively. Compared with virus control, HSV-2 DNA polymerases mRNA of the Styela plicata (2mg/ml) group and ACV group reduced significantly. The experimental results prompted the ethanol extracts of Styela plicata could interrupt HSV-2 DNA pol gene (UL30) expressed.
9. The therapeutical effect of Styela plicata on genital herpes
After treatment completed, the mice were killed. The lesion of mice cunnus and genital were investigate to evalueate the therapeutical effect of Styela plicata on genital herpes. The ethanol extracts of Styela plicata could relieve engorgement of mice cunnus. Compared with virus the control group, the score of the cunnus pathological changes of ACV group mice and the ethanol extracts of Styela plicata groups mice (5.0,2.5g/kg) could be relieved significantly(P<0.05). The histopathological experimental results indicated, the normal control group mice vaginal mucosa were integrity, there were not phlegmasia cells. The vaginal mucosa of virus control group mice were severerly injuryed, there were many bleeding points and many phlegmasia cells. The vaginal mucosa of ACV group mice and the ethanol extracts of Styela plicata groups mice(5.0,2.5g/kg) were lightly injuryed, there were not bleeding point, and there were small amounts phlegmasia cells. The experimental results prompted the ethanol extracts of Styela plicata had good therapeutical effect on genital herpes.
10. The effect of Styela plicata on the subsets of T cell in peripheral blood of the mice
After treatment completed, the mice were killed. The percentages of CD4+T cell and CD8+T cell in the peripheral blood of the mice were investigated. The percentages of CD4+Tcell and CD8+T cell in the peripheral blood of the model group mice significantly decreased compared to those of the blank control group mice(P<0.01). The percentages of CD4+Tcell and CD8+T cell in the peripheral blood of the mice administrated the ehanol extracts of Styela plicata and ACV significantly increased compared to those of the model control group mice(P< 0.01). The relative value of CD4+T cell/CD8+T cell of the model control group mice significantly increased compared to that of the blank goup mice(P<0.01). The relative value of CD4+Tcell/CD8+Tcell of the mice administrated the ethanol extracts of Styela plicata and ACV significantly decreased compared to that of the model goup mice(P<0.01, P<0.05). There was not significant deviation between the relative value of CD4+Tcell/CD8+Tcell of the mice administrated the ethanol extracts of Styela plicata and that of the blank group mice(P>0.05). There was significant deviation between the relative value of CD4+Tcell/CD8+Tcell of the mice administrated ACV and that of the blank group mice(P<0.01).
11. The effect of Styela plicata on the levels of IL-2 and IFN-y of the mice
After treatment completed, the mice were killed. The levels of IL-2 and IFN-y in the peripheral blood of the mice were investigated. The levels of IL-2 in the peripheral blood of the model control group mice significantly decreased compared to that of the blank control group mice(P<0.01); the levels of IL-2 in the peripheral blood of the mice administrated ethanol extracts of Styela plicata significantly increased compared to that of the model control group mice(P<0.01); the level of IL-2 in the peripheral blood of the mice administrated ACV did not significantly increase compared to that of the model control group mice(P>0.05); the levels of IFN-y in the peripheral blood of any other group mice did not significantly increas compared to that of the model control group mice(P>0.05).
12. The effect of Styela plicata on the levels of IL-4 and IL-10 of the mice
After treatment completed, the mice were killed. The concentrations of IL-4 and IL-10 in the peripheral blood of the model control group mice were not significant difference compared to that of the blank control group mice(P>0.05); the levels of IL-4 and IL-10 in the peripheral blood of the ACV control group mice was not significant difference compared to that of the model control group mice(P>0.05); the levels IL-4 and IL-10 in the peripheral blood of the mice administrated the ethanol extracts of Styela plicata significantly elevated compared to those of the model control group mice(P<0.01, P<0.05).
13. The cytotoxicity of different fraction of Styela plicata on Vero cell
MTT assay was applied to investigate the cytotoxicity of different fraction of Styela plicata on Vero cell. The results displayed the absorbance values of the chloroform frction, n-butanol fraction and water fraction of Styela plicata groups(0.08～10mg/ml) had significant difference compared to that of vero cell control group (P<0.01, P<0.05). The results displayed that the CC50 value of the chloroform frction of Styela plicata was 4.62mg/ml, the CC50 value of the n-butanol fraction of Styela plicata was more than 10mg/ml, and the CC50 value of the water fraction of Styela plicata was more than 10mg/ml.
14. Anti-HSV-2 activiy of the chloroform frction of Styela plicata
MTT assay was applied to investigate anti-HSV-2 activiy of the chloroform frction of Styela plicata. The results displayed the absorbance values of chloroform frction of Styela plicata groups had not significant difference compared to that of HSV-2 control group (P>0.05). The results displayed that the inhibition ratio of the chloroform frction of Styela plicata on HSV-2 was 19.23% at 2mg/ml concentraion. The results indicated the chloroform frction of Styela plicata could not inhibit HSV-2.
15. Anti-HSV-2 activiy of the n-butanol frction of Styela plicata
MTT assay was applied to investigate anti-HSV-2 activiy of the n-butanol frction of Styela plicata. The results displayed the absorbance values of the n-butanol frction of Styela plicata groups had not significant difference compared to that of HSV-2 control group (P>0.05). The inhibition ratio of the n-butanol frction of Styela plicata on HSV-2 was 11.54% at 2mg/ml concentraion. The results indicated the n-butanol frction of Styela plicata could not inhibit HSV-2.
16. Anti-HSV-2 activiy of the water frction of Styela plicata
MTT assay was applied to investigate anti-HSV-2 activiy of the water frction of Styela plicata. The results displayed the absorbance values of the water frction of Styela plicata groups had significant difference compared to that of HSV-2 control group (P<0.01, P<0.05). The inhibition ratio of the water frction of Styela plicata on HSV-2 were 91.43% and 77.14% at 2 and 1mg/ml concentraion, respectively, it's value of EC50 was 0.67mg/ml, The value of TI was 14.93. The results indicated the water frction of the Styela plicata could inhibit HSV-2.
17. The effect of the water fraction of Styela plicata on multiplication of HSV-2
MTT assay was applied to investigate the effect of the water fraction of Styela plicata on multiplication of HSV-2. The results displayed the absorbance values of the water frction of Styela plicata groups had not significant difference compared to that of HSV-2 the control group (P>0.05). The inhibition ratio of the water fraction of Styela plicata on HSV-2 was calculated. The inhibition ratio of the water fraction of Styela plicata on HSV-2 was 82.98% at 2mg/ml concentraion, EC50=0.49mg/ml, TI>20.41, which indicated the water fraction of Styela plicata had good effect on multiplication of HSV-2.
18. The direct lethal effect of the water fraction of Styela plicata on HSV-2
MTT assay was applied to investigate the direct lethal effect of the water fraction of Styela plicata on HSV-2. The inhibition ratio of the water fraction of Styela plicata on HSV-2 was calculated. The results displayed the absorbance values of the water frction of Styela plicata groups were not significantly difference compared to that of HSV-2 control group (P>0.05). The inhibition ratio of the water fraction of Styela plicata on HSV-2 were 74.29% and 60.00% at 2 and 1 mg/ml concentraion, respectively, EC50=0.80mg/ml, TI> 12.50, which indicated the water frction of Styela plicata had good direct lethal effect on HSV-2.
19. The effect of the water fraction of Styela plicata on adsorbtion of HSV-2
MTT assay was applied to investigate the effect of the water fraction of Styela plicata on adsorbtion of HSV-2. The inhibition ratio of the water fraction of Styela plicata on HSV-2 was calculated. The results displayed the absorbance values of the water frction of Styela plicata groups had not significant difference compared to that of HSV-2 control group (P>0.05). The inhibition ratio of the water fraction of Styela plicata on HSV-2 was 19.23% at 2mg/ml concentraion, which indicated the water fraction of Styela plicata could not inhibit HSV-2 invase into host cell.
Conclusions
1. Styela plicata can inhibit the replication of HSV-2 in vitro.
2. Styela plicata can directly kill HSV-2 and can ininhibit the biosynthesis of HSV-2, but can not inhibit HSV-2 invade into host cell.
3. Styela plicata can interrupt HSV-2 DNA chain replicate. One of its target is to interrupt HSV-2 DNA polymerases gene (UL30) expression.
4. Styela plicata has good therapeutical effect on genital herpes.
5. Styela plicata can increase the percentage of T cell subgroup in the blood of mice infected with HSV-2 in vagina, and can regulate the balance of T cell subgroups. Styela plicata can elevate the concentration of Th cell cytokine of the mice infectec with HSV-2 in vagina, can regulate and improve immunological function of the mice. These may be other mechanisms of action of Styela plicata on genital herpes.
6. The water fraction of Styela plicata can inhibit HSV-2, but n-butanol fraction and chloroform fraction can not inhibit HSV-2.
研究目的
课题组前期研究发现皱瘤海鞘在体内外对乙型肝炎病毒(HBV)都有一定的抑制作用,显示出良好的研究和开发前景。因此,本课题拟对皱瘤海鞘对同是具有包膜的DNA病毒单纯疱疹病毒Ⅱ型(HSV-2)药效活性和作用机制进行研究。为进一步了解皱瘤海鞘的抗病毒谱,为新型抗病毒药物的研究和开发提供理论依据,为皱瘤海鞘的进一步深入研究和开发利用提供科学的理论基础。
研究方法1.皱瘤海鞘体外抗HSV-2药效学研究
运用HSV-2在非州绿猴肾细胞(Vero细胞)上的增殖模型,通过观察细胞病变(CPE法)和四甲基偶氮唑盐比色法(MTT比色法)检测细胞活性,以病毒抑制率和治疗指数(TI)为指标对皱瘤海鞘体外抗HSV-2的药效活性作用进行评价。
2.皱瘤海鞘体外抗HSV-2病毒作用机制研究
运用HSV-2在Vero细胞上的增殖模型,在HSV-2复制的不同阶段用药物进行干预,运用CPE法结合MTT比色法检测细胞存活率,以病毒抑制率和TI为指标考察了药物在体外对HSV-2病毒直接杀伤作用、对HSV-2吸附的阻断作用、对HSV-2病毒生物合成的抑制作用和在不同时间点加入药物对HSV-2复制的抑制作用；运用荧光定量PCR方法,从分子水平上探讨了皱瘤海鞘对HSV-2DNA双链复制的影响；运用RT-PCR方法进一步研究皱瘤海鞘对HSV-2DNA聚合酶基因表达的影响。
3.皱瘤海鞘对阴道感染HSV-2小鼠的保护作用
运用小鼠阴道感染HSV-2的致病模型,研究了皱瘤海鞘对小鼠生殖器疱疹的治疗作用。以小鼠外阴病变程度评分和小鼠阴道粘膜组织病理学检查为指标评价皱瘤海鞘对生殖器疱疹的治疗作用。4.皱瘤海鞘体内抗HSV-2作用机制研究
运用小鼠阴道感染HSV-2的致病模型,研究了皱瘤海鞘对小鼠免疫细胞的的调节作用。在治疗周期结束后,小鼠处死,取小鼠外周血抗凝,流式细胞术测定CD4, CD8阳性T细胞百分比,比较用药后各组小鼠外周血T细胞亚群的差异,评价皱瘤海鞘对小鼠的免疫功能的调节作用。
运用小鼠阴道感染HSV-2的致病模型,研究了皱瘤海鞘对小鼠Th细胞因子的调节作用。在治疗周期结束后,小鼠处死,取小鼠外周血,收集血清,用酶联免疫吸附分析法测定血清中IL-2, IFN-y, IL-4, IL-10水平。比较用药后各组小鼠外周血IL-2, IFN-y, IL-4和IL-10的差异,评价皱瘤海鞘对小鼠的免疫功能的调节作用。
5.皱瘤海鞘抗HSV-2活性有药效部位的筛选
运用HSV-2在Vero细胞上的增殖模型,以CPE法和MTT比色法检测细胞活性。以细胞存活率和半数细胞毒性浓度(CC50)为指标考察药物的细胞毒性；以病毒抑制率为指标,对皱瘤海鞘氯仿部位、正丁醇部位,水部位体外抗HSV-2的药效活性作用进行评价；以病毒抑制率为指标考察了皱瘤海鞘水部位在体外对HSV-2病毒直接杀伤作用、对HSV-2吸附的阻断作用以及对HSV-2病毒生物合成的抑制作用。
6.统计学处理
本文所有计量资料数据均采用单因素方差分析方法进行,先进行方差齐性检验,若方差齐,则应用One-way ANOVA方法进行整体检验,用LSD方法进行组间多重比较,若方差不齐,则应用Welch方法进行整体检验,用DunnettT3方法进行组间多重比较。等级资料数据应用多个独立样本非参数检验进行分析。P<0.05,可认为组间比较具有统计学意义,数据用SPSS13.0软件处理。
研究结果
1.药物对Vero细胞毒性
实验结果显示,与细胞对照组比较,皱瘤海鞘各组吸光度值均无统计学意义(P>0.05), ACV组在1 mg/ml浓度以下各组均无统计学意义(P>0.05)。皱瘤海鞘对Vero细胞的毒性作用小,在10mg/ml浓度下细胞存活率仍有74.88%,其对Vero细胞的CC50值大于10mg/ml,阳性对照药ACV的CC50为1.57mg/ml。
2.药物对HSV-2致细胞病变的抑制作用
实验结果显示,皱瘤海鞘各组吸光度值与病毒对照组比较均具有统计学意义(P<0.01),阳性对照药阿昔洛韦(acylovir, ACV) 0.01～0.04 mg/ml组吸光度与病毒对照组比较均具有统计学意义(P<0.01)。皱瘤海鞘对HSV-2的半数有效浓度(EC50)为0.40 mg/ml, TI>25,对HSV-2感染Vero细胞显示出较强的保护作用；ACV50%病毒抑制率药物浓度EC50=0.0067mg/ml,治疗指数TI=224,对HSV-2显示出较强的抑制作用。
3.皱瘤海鞘对HSV-2直接杀伤活性
应用MTT法测定细胞活性,计算皱瘤海鞘对HSV-2病毒的抑制率。结果显示,皱瘤海鞘在1 mg/ml时吸光度与病毒对照组比较具有统计学意义(P<0.01),在2和1 mg/ml时,其对病毒抑制率分别达74.19,64.52%,EC50为0.63mg/ml, TI值大于15.87。结果表明,皱瘤海鞘对病毒具有一定的直接杀伤活性。
4.皱瘤海鞘对HSV-2增殖的影响
运用MTT法测定细胞活性,计算皱瘤海鞘对HSV-2病毒的抑制率。实验结果显示,皱瘤海鞘2和1mg/ml组吸光度与病毒对照组比较均具有统计学意义(P<0.05),在2和1mg/ml时,其对病毒的抑制率分别达82.98%和65.96%,在0.25mg/ml时,其对病毒抑制率也达29.79%。其ECso值为0.66mg/ml, TI值大于15.15,实验结果提示,皱瘤海鞘对病毒复制具有一定的抑制作用。
5.皱瘤海鞘对HSV-2吸附的阻断作用
运用MTT法测定细胞活性,计算皱瘤海鞘对HSV-2病毒的抑制率。实验测得皱瘤海鞘各组吸光度值与病毒对照比较均无统计学意义(P>0.05),经计算求得,其在2mg/ml时,其对病毒抑制率达只有23.08%,没有超过50%。实验结果提示皱瘤海鞘对病毒的吸附没有阻断作用。
6.在病毒感染前后不同时间点加入皱瘤海鞘对HSV-2病毒的影响
运用MTT法测定在不同时间点加入皱瘤海鞘对病毒复制的抑制作用,计算皱瘤海鞘对HSV-2病毒的抑制率。结果显示,在病毒感染前2小时和感染后0,2,4,6小时加入药物,最后测得的吸光度值与病毒对照比较均具有统计学意义(P<0.01)；病毒在感染后0,2小时加入药物对病毒抑制率达65%以上,对病毒显示出明显的抑制作用。在感染后4,6小时加入药物,对病毒抑制率分别是41.27%,34.92%,在感染后10小时加入药物,对病毒复制没有抑制作用。
7.荧光定量PCR方法测定皱瘤海鞘对HSV-2病毒DNA复制的影响
实验结果显示,与病毒对照组比较,皱瘤海鞘2,1,0.5 mg/ml组能使HSV-2DNA拷贝数下降2-3个数量级,差异具有统计学意义(P<0.01)。实验结果提示,皱瘤海鞘对HSV-2 DNA链复制有一定抑制作用。
8. RT-PCR法检测皱瘤海鞘对HSV-2 DNA聚合酶UL30基因表达的影响
RT-PCR扩增40个循环后,所有样品均能扩增出特异性的β-actin片段,在皱瘤海鞘(2mg/ml)组和ACV组的均能检测到DNA聚合酶UL30 mRNA的表达,但是与病毒对照组相比,表达量显著降低,提示皱瘤海鞘对HSV-2DNA聚合酶的基因表达具有一定的干扰作用。
9.皱瘤海鞘对小鼠生殖器疱疹的治疗作用
病毒攻击后,经过连续7天治疗。实验结果显示,海鞘可明显减轻阴道感染HSV-2小鼠外阴肿胀,缩短病程。与模型组比较,阳性药ACV组,海鞘高、低剂量组小鼠外阴病变评分都较小,差异具有统计学意义(P<0.05)。小鼠阴道组织病理学检查,光镜下,正常组的阴道粘膜完整,没有炎细胞浸润,没有损伤表现。模型组小鼠阴道粘膜损伤严重,有大量的出血点,有大量炎细胞浸润；ACV组,皱瘤海鞘高、低剂组阴道粘膜损伤较轻,只有少量炎细胞浸润,没有出血点。结果表明,皱瘤海鞘对小鼠生殖器疱疹具有一定治疗作用。
10.皱瘤海鞘对感染HSV-2小鼠外周血T细胞亚群的影响
病毒攻击后,连续给药治疗7天。实验结果显示,与空白对照组相比较,模型对照组外周血中CD4+T细胞,CD8+T细胞百分比均具有显著差异(P<0.01),可认为造模后两者均显著减少,免疫功能受到一定抑制。ACV治疗组、海鞘治疗组和模型对照组相比,小鼠外周血中CD4+T细胞,CD8+T细胞百分比均有显著差异(P<0.01),可认为经治疗后,CD4+T细胞和CD8+T细胞数量有了显著提高。与空白对照组相比较,模型组外周血CD4+T细胞/CD8+细胞比值有显著差异(P<0.01),可认为模型组CD4+T细胞和CD8+细胞平衡失调。ACV治疗组、皱瘤海鞘治疗组和模型对照组相比,外周血CD4+T细胞/CD8+细胞比值均具有显著性差异(P<0.01,P<0.05),可认为经治疗后,小鼠T细胞平衡失调得到明显改善；与正常组比较,皱瘤海鞘治疗组CD4+T细胞/CD8+细胞比值无显著差异(P>0.05),ACV治疗组CD4+T细胞/CD8+细胞比值有显著差异(P<0.01)。
11.皱瘤海鞘对小鼠IL-2, IFN-γ细胞因子水平的影响
病毒攻击后,连续给药治疗7天。实验结果显示,与空白对照组相比较,模型对照组小鼠血清IL-2水平显著降低(P<0.01)；与模型组比较,皱瘤海鞘高、低剂量组小鼠血清IL-2水平均有显著增加(P<0.01),可认为皱瘤海鞘能提高小鼠IL-2的表达水平；与模型组比较,阳性药(ACV)组小鼠血清IL-2水平有升高趋势,但无统计学意义(P>0.05)；与模型组比较,各组小鼠血清IFN-y水平均无显著性差异(P>0.05)。
12.皱瘤海鞘对小鼠IL-4,IL-10细胞因子水平的影响
病毒攻击后,连续给药治疗7天。实验结果显示,与空白对照组相比较,模型对照组小鼠血清IL-4, IL-10水平无显著性差异(P>0.05)；与模型组比较,阳性药(ACV)组小鼠血清IL-4,IL-10水平均无显著性差异(P>0.05)；皱瘤海鞘高、低剂量组与模型组比较,小鼠血清IL-4, IL-10水平均具有显著性差异(P<0.01,P<0.05),可认为皱瘤海鞘能够提高致病小鼠IL-4,IL-10水平。
13.皱瘤海鞘不同部位对Vero细胞毒性
MTT法测定皱瘤海鞘不同部位对Vero细胞毒性。实验测得皱瘤海鞘氯仿部位、正丁醇部位和水部位0.08～10 mg/ml组吸光度值与细胞对照组比较均具有统计学意义(P<0.01,P<0.05)；皱瘤海鞘氯仿部位对Vero细胞的CC50为4.62mg/ml,皱瘤海鞘正丁醇部位对Vero细胞的CCso大于10mg/ml,皱瘤海鞘水部位对Vero细胞的CC50大于10mg/ml。
14.皱瘤海鞘氯仿部位对HSV-2致细胞病变抑制效应
MTT法测定皱瘤海鞘氯仿对HSV-2致细胞病变抑制效果,结果显示,各浓度组吸光度值与病毒对照组比较均无统计学意义(P>0.05),2mg/ml皱瘤海鞘氯仿部位对HSV-2抑制率为19.23%,没有达到50%。实验结果表明,皱瘤海鞘氯仿部位对HSV-2无抑制作用。
15.皱瘤海鞘正丁醇部位对HSV-2致细胞病变抑制效应
MTT法测定皱瘤海鞘正丁醇部位对HSV-2致细胞病变抑制效果,结果显示,各浓度组吸光度值与病毒对照组比较均无统计学意义(P>0.05),2mg/ml皱瘤海鞘正丁醇部位HSV-2抑制率为11.54%。实验结果表明,皱瘤海鞘正丁醇部位对HSV-2无抑制作用。
16.皱瘤海鞘水部位对HSV-2致细胞病变抑制效应
MTT法测定皱瘤海鞘水部位对HSV-2致细胞病变抑制效果。结果显示,2、1及0.5 mg/ml浓度组吸光度值与病毒对照组比较均具有统计学意义(P<0.01,<0.05),2,1 mg/ml瘤海鞘水部位对HSV-2病毒抑制率分别达到91.43%,77.14%,其EC50为0.67mg/ml, TI值大于14.93,结果表明,皱瘤海鞘水部位对HSV-2具有一定抑制作用。
17.皱瘤海鞘水部位对HSV-2增殖的影响
MTT法测定皱瘤海鞘水部位对HSV-2增殖的影响。实验结果显示,皱瘤海鞘水部分各浓组吸光度值与病毒对照组比较均无统计学意义(P>0.05),皱瘤海鞘水部位2、1和0.5mg/ml时,其病毒抑制率分别达82.98,65.96和63.93%,EC50=0.49mg/ml, T>20.41。结果表明,皱瘤海鞘水部位对HSV-2增殖具有一定抑制作用。
18.皱瘤海鞘水部位对HSV-2直接杀伤作用
MTT法测定皱瘤海鞘水部位对HSV-2直接杀伤作用,结果显示,皱瘤海鞘水部位各浓度组吸光度值与病毒对照组比较均无计学意义(P>0.05),皱瘤海鞘水部位在2和1mg/ml时,其病毒抑制率分别达74.29%,60.00%, EC50=0.80mg/ml,T>12.50。结果表明,皱瘤海鞘水部位对病毒具有一定的杀伤活性。19.皱瘤海鞘水部位对HSV-2吸附的阻断作用
MTT法测定皱瘤海鞘水部位对HSV-2吸附的阻断作用。实验结果显示,皱瘤海鞘水部位各组吸光度值与病毒对照组比较均无统计学意义(P>0.05),在2mg/ml时,其病毒抑制率只有19.23%,没有达到50%。实验结果表明皱瘤海鞘水部位对HSV-2病毒吸附没有阻断作用,
研究结论
1.运用细胞模型证实皱瘤海鞘对HSV-2复制具有抑制作用。
2.运用细胞模型证实皱瘤海鞘对HSV-2具直接杀伤作用,对HSV-2生物合成具有抑制作用,而对HSV-2吸附侵入细胞没有阻断作用。
3.运用细胞模型证实皱瘤海鞘对HSV-2 DNA链复制延长具有抑制作用,其作用靶点之一是抑制HSV-2 DNA聚合酶基因表达。
4.皱瘤海鞘能够对阴道感染HSV-2小鼠生殖器疱疹具有治疗作用。
5.皱瘤海鞘能够提高阴道感染HSV-2小鼠外周血液CD4+和CD8+T细胞亚群百分比,调节小鼠T细胞亚群的平衡；能提高阴道感染HSV-2小鼠血液Th细胞因子水平,从而调节和改善小鼠的免疫功能。这些可能是其对生殖器疱疹具有治疗作用一个有效机制。
6.皱瘤海鞘提取物抗HSV-2的有效活性部位是水溶性成分,而不是正丁醇和三氯甲烷等有机溶剂部位,其最可能的成分是小分子多肽等物质。
Styela plicata (lesueur) is an ascidian which belongs to class Ascidiacea, subphylum urochordata. They mainly distribute over the tropical zone and subtropical zone maritime space. The scabbard contains many physiological activity compounds, and most of them have strong biological activity, such as anti-tumor, anti-virus, antibiosis, etc. There are abundant ascidian resources in the South China Sea. So, study on the medicinal value of Styela plicata for development and utilization of the resources of our country is of great practical significance.
Objective
Because we finds the Styela plicata can inhibit hepatitis b virus in vitro and in vivo before. So, for expanding and understanding the Styela plicata of antiviral spectra, this issue is to study on the effects of Styela plicata on herpes simplex virus 2(HSV-2)and its mechanism of action in vitro and in vivo. The research will provide scientific theory for development of new anti-HSV-2 drugs and for further development and utilization of Styela plicata,
Methods
1. Anti-HSV-2 activiy of the ethanol extracts of Styela plicata
The Vero cell infected with HSV-2 was used as experimental model. CPE assay and MTT assay were applied to investigate anti-HSV-2 activiy of the ethanol extracts of Styela plicata in vitro. Survival rate of cell and CC50 value were applied to evaluate cytotoxicity of medicine; the viral inhibition ratio and TI value were used to evaluate anti-HSV-2 activity of alcohol extracts of Styela plicata.
2. The study on the mechanism of the anti-HSV-2 action of the ethanol extracts of Styela plicata in vitro
The Vero cell infected with HSV-2 was used as experimental model. CPE assay and MTT assay were applied. The viral inhibition ratio and TI value were used to evalueate the effect of the ethanol extracts of Styela plicata on HSV-2. Fluorescence detection method of PCR assay was applied to investigate the effect of the ethanol extracts of Styela plicata on the replication of HSV-2 DNA chain. RT-PCR assay was applied to investigate the effect of the ethanol extracts of Styela plicata on the expression of HSV-2 DNA pol gene.
3. Protective effect of Styela plicata on the mice infected with HSV-2 in vagina
Mice infected with HSV-2 in vagina were applied to investigated the effect of Styela plicata on genital herpes. The mice cunnus pathological changes extent value and histopathology were used to eveluate the effect of Styela plicata on genital herpes. The contrast of different group mice cunnus pathological changes extent were investigated by rank-sum test.
4. The study on the mechanism of the anti-HSV-2 action of the ethanol extracts of Styela plicata in vivo
Mice infected with HSV-2 in vagina was applied. After treatment was completed, the mice were killed. The peripheral blood of the mice were collected and anticoagulated. Flow cytometry assay was applied to determined the percentage of CD4+T cells and CD8+T cells, respectively. One-Way ANOVA assay was applied to analyze the difference of T cell subgroups in peripheral blood of all class mice. The effect of Styela plicata on immunologic function was evaluated.
Mice infected with HSV-2 in vagina were applied. Aafter treatment was completed, the mice were killed, the peripheral blood of the mice was collected. The sera were collected. ELISA assay was applied to determined the concentation of IL-2, IFN-y, IL-4 and IL-10 in the sera. One-Way ANOVA assay was applied to analyze the difference of concentation of IL-2, IFN-y, IL-4 and IL-10 in the sera among all class mice. The effect of Styela plicata on immunologic function was evaluated.
5. The screening of effective fraction for anti-Herpes simplex virus 2 of Styela plicata
The Vero cell infected with HSV-2 was used as experimental model. CPE assay and MTT assay were applied to investigate the effect of the different fraction of Styela plicata on HSV-2 in vitro. Survival rate of cell and CC50 value were applied to evaluate cytotoxicity of the different fraction of Styela plicata; the viral inhibition ratio and TI value were used to evaluate anti-HSV-2 activity of the different fraction of Styela plicata.
6. Statistical analysis
All measurement data in this paper were analyzed by One-way ANOVA test. Homoscedasticity was analyzed, ANOVA test was used to analyze the data while homoscedasticity, Welch test was used to analyze the data while homoscedasticity of variance.The ranked data were analyzed by K Independent Samples Tests. P value of less than 0.05 was considered to be statistically significant.
Results
1. The cytotoxic effect of alcohol extracts of Styela plicata on Vero cell
MTT assay was applied to investigate the cytotoxic effect of the ethanol extracts from Styela plicata on Vero cell. The results displayed the absorbance values of the ethanol extracts of Styela plicata groups had significant difference compared to that of vero cell control group (P<0.05); the absorbance values of ACV(not more than lmg/ml) groups were not significantly difference compared to that of vero cell control group (P>0.05). The vero cell survival rate was 74.88% at concentration of 10mg/ml the ethanol extracts of Styela plicata. The CC50 value of the ethanol extracts of Styela plicata on Vero cell was more than lOmg/ml, which indicated the cytotoxic effect of alcohol extracts of Styela plicata on Vero cell was low. The CC50 value of ACV was 1.57mg/ml.
2. Anti-HSV-2 activiy of the ethanol extracts of Styela plicata
The results displayed the absorbance values of the ethanol extracts of Styela plicata groups had significant difference compared to that of HSV-2 control group (P<0.01); the absorbance values of ACV(0.04～0.01 mg/ml) groups had significant difference compared to that of vero cell control group (P<0.01). The EC50 value of the ethanol extracts of Styela plicata on HSV-2 is 0.40 mg/ml, TI value is more than 25, which indicated the ethanol extracts of Styela plicata had good protective effect on Vero cell infected with HSV-2. The EC50 value of ACV on HSV-2 is 0.0067mg/ml, TI value was 224, which indicated ACV had good protective effect on Vero cell infected with HSV-2.
3. The direct lethal effect of the ethanol extracts of Styela plicata on HSV-2
MTT assay was applied to investigate the direct lethal effect of the ethanol extracts of Styela plicata on HSV-2. The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was calculated. The results displayed the absorbance values of alcohol extracts of Styela plicata group(1 mg/ml) had significant difference compared to that of HSV-2 control group (P<0.01). The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was 74.19% at 2mg/ml concentration. The EC50 was 0.63mg/ml, TI value was 15.87, which indicated the ethanol extracts of Styela plicata had good direct lethal effect on HSV-2.
4. The effect of the ethanol extracts of Styela plicata on multiplication of HSV-2
MTT assay was applied to investigate the effect the ethanol extracts of Styela plicata on multiplication of HSV-2. The results displayed the absorbance values of the ethanol extracts of Styela plicata groups(2.0 and 1.0mg/ml) had significant difference compared to that HSV-2 control group (P<0.05). The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was calculated. The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was 82.98% at 2mg/ml concentration, the inhibition ratio was 29.79% at 0.25mg/ml concentration. The EC50 was 0.66mg/ml, TI value was 15.15, which indicated the ethanol extracts of Styela plicata could inhibit the multiplication of HSV-2.
5. The effect of the ethanol extracts of Styela plicata on adsorbtion HSV-2
MTT assay was applied to investigate the effect of the ethanol extracts of Styela plicata on HSV-2 adsorbtion. The inhibition ratio the ethanol extracts of Styela plicata on HSV-2 was calculated. The results displayed the absorbance values of the ethanol extracts of Styela plicata groups had not significant difference compared to that HSV-2 control group (P>0.05). The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was 23.08% at 2mg/ml concentration, it was not more than 50%, which indicated the ethanol extracts of Styela plicata could not inhibit HSV-2 invased into host cell.
6. Effect of addition time on the antiviral activity of the ethanol extracts of Styela plicata
MTT assay was applied to investigate time-of-addition effect of the ethanol extracts of Styela plicata. The results displayed the absorbance values the ethanol extracts of Styela plicata groups had significant difference compared to that of HSV-2 control group but 10 hour post infection group(P<0.01). The inhibition ratio of the ethanol extracts of Styela plicata on HSV-2 was calculated. The experimental results indicated the ethanol extracts of Styela plicata could well inhibit HSV-2 replicate when the extracts were added at 0,2,4,6h post infection. At 0,2 hour post infection, the inhibition ratios of the ethanol extracts of Styela plicata on HSV-2 were more than 65%, At 4,6h, they were 41.27% and 34.92%, respectively. At 10 hours post infection, the ethanol extracts of Styela plicata could not inhibit HSV-2 replication.
7. The effect of the ethanol extracts from Styela plicata on HSV-2 DNA chain replication
HSV-2 DNA copy in the ethanol extracts of Styela plicata group (at 2,1,0.5 ml/mg) had statistical significance (P<0.01) compared with the virus control, the experimental results prompted the ethanol extracts of Styela plicata could interrupt HSV-2 DNA chain replicated.
8. The effect of the extracts of Styela plicata on HSV-2 DNA pol gene (UL30) expression.
After 40 cycle period RT-PCR amplification, all sample could be amplificated specificβ-actin frags. HSV-2 DNA polymerases mRNA of the virus control, the Styela plicata (2mg/ml) group and ACV group could be detected, respectively. Compared with virus control, HSV-2 DNA polymerases mRNA of the Styela plicata (2mg/ml) group and ACV group reduced significantly. The experimental results prompted the ethanol extracts of Styela plicata could interrupt HSV-2 DNA pol gene (UL30) expressed.
9. The therapeutical effect of Styela plicata on genital herpes
After treatment completed, the mice were killed. The lesion of mice cunnus and genital were investigate to evalueate the therapeutical effect of Styela plicata on genital herpes. The ethanol extracts of Styela plicata could relieve engorgement of mice cunnus. Compared with virus the control group, the score of the cunnus pathological changes of ACV group mice and the ethanol extracts of Styela plicata groups mice (5.0,2.5g/kg) could be relieved significantly(P<0.05). The histopathological experimental results indicated, the normal control group mice vaginal mucosa were integrity, there were not phlegmasia cells. The vaginal mucosa of virus control group mice were severerly injuryed, there were many bleeding points and many phlegmasia cells. The vaginal mucosa of ACV group mice and the ethanol extracts of Styela plicata groups mice(5.0,2.5g/kg) were lightly injuryed, there were not bleeding point, and there were small amounts phlegmasia cells. The experimental results prompted the ethanol extracts of Styela plicata had good therapeutical effect on genital herpes.
10. The effect of Styela plicata on the subsets of T cell in peripheral blood of the mice
After treatment completed, the mice were killed. The percentages of CD4+T cell and CD8+T cell in the peripheral blood of the mice were investigated. The percentages of CD4+Tcell and CD8+T cell in the peripheral blood of the model group mice significantly decreased compared to those of the blank control group mice(P<0.01). The percentages of CD4+Tcell and CD8+T cell in the peripheral blood of the mice administrated the ehanol extracts of Styela plicata and ACV significantly increased compared to those of the model control group mice(P< 0.01). The relative value of CD4+T cell/CD8+T cell of the model control group mice significantly increased compared to that of the blank goup mice(P<0.01). The relative value of CD4+Tcell/CD8+Tcell of the mice administrated the ethanol extracts of Styela plicata and ACV significantly decreased compared to that of the model goup mice(P<0.01, P<0.05). There was not significant deviation between the relative value of CD4+Tcell/CD8+Tcell of the mice administrated the ethanol extracts of Styela plicata and that of the blank group mice(P>0.05). There was significant deviation between the relative value of CD4+Tcell/CD8+Tcell of the mice administrated ACV and that of the blank group mice(P<0.01).
11. The effect of Styela plicata on the levels of IL-2 and IFN-y of the mice
After treatment completed, the mice were killed. The levels of IL-2 and IFN-y in the peripheral blood of the mice were investigated. The levels of IL-2 in the peripheral blood of the model control group mice significantly decreased compared to that of the blank control group mice(P<0.01); the levels of IL-2 in the peripheral blood of the mice administrated ethanol extracts of Styela plicata significantly increased compared to that of the model control group mice(P<0.01); the level of IL-2 in the peripheral blood of the mice administrated ACV did not significantly increase compared to that of the model control group mice(P>0.05); the levels of IFN-y in the peripheral blood of any other group mice did not significantly increas compared to that of the model control group mice(P>0.05).
12. The effect of Styela plicata on the levels of IL-4 and IL-10 of the mice
After treatment completed, the mice were killed. The concentrations of IL-4 and IL-10 in the peripheral blood of the model control group mice were not significant difference compared to that of the blank control group mice(P>0.05); the levels of IL-4 and IL-10 in the peripheral blood of the ACV control group mice was not significant difference compared to that of the model control group mice(P>0.05); the levels IL-4 and IL-10 in the peripheral blood of the mice administrated the ethanol extracts of Styela plicata significantly elevated compared to those of the model control group mice(P<0.01, P<0.05).
13. The cytotoxicity of different fraction of Styela plicata on Vero cell
MTT assay was applied to investigate the cytotoxicity of different fraction of Styela plicata on Vero cell. The results displayed the absorbance values of the chloroform frction, n-butanol fraction and water fraction of Styela plicata groups(0.08～10mg/ml) had significant difference compared to that of vero cell control group (P<0.01, P<0.05). The results displayed that the CC50 value of the chloroform frction of Styela plicata was 4.62mg/ml, the CC50 value of the n-butanol fraction of Styela plicata was more than 10mg/ml, and the CC50 value of the water fraction of Styela plicata was more than 10mg/ml.
14. Anti-HSV-2 activiy of the chloroform frction of Styela plicata
MTT assay was applied to investigate anti-HSV-2 activiy of the chloroform frction of Styela plicata. The results displayed the absorbance values of chloroform frction of Styela plicata groups had not significant difference compared to that of HSV-2 control group (P>0.05). The results displayed that the inhibition ratio of the chloroform frction of Styela plicata on HSV-2 was 19.23% at 2mg/ml concentraion. The results indicated the chloroform frction of Styela plicata could not inhibit HSV-2.
15. Anti-HSV-2 activiy of the n-butanol frction of Styela plicata
MTT assay was applied to investigate anti-HSV-2 activiy of the n-butanol frction of Styela plicata. The results displayed the absorbance values of the n-butanol frction of Styela plicata groups had not significant difference compared to that of HSV-2 control group (P>0.05). The inhibition ratio of the n-butanol frction of Styela plicata on HSV-2 was 11.54% at 2mg/ml concentraion. The results indicated the n-butanol frction of Styela plicata could not inhibit HSV-2.
16. Anti-HSV-2 activiy of the water frction of Styela plicata
MTT assay was applied to investigate anti-HSV-2 activiy of the water frction of Styela plicata. The results displayed the absorbance values of the water frction of Styela plicata groups had significant difference compared to that of HSV-2 control group (P<0.01, P<0.05). The inhibition ratio of the water frction of Styela plicata on HSV-2 were 91.43% and 77.14% at 2 and 1mg/ml concentraion, respectively, it's value of EC50 was 0.67mg/ml, The value of TI was 14.93. The results indicated the water frction of the Styela plicata could inhibit HSV-2.
17. The effect of the water fraction of Styela plicata on multiplication of HSV-2
MTT assay was applied to investigate the effect of the water fraction of Styela plicata on multiplication of HSV-2. The results displayed the absorbance values of the water frction of Styela plicata groups had not significant difference compared to that of HSV-2 the control group (P>0.05). The inhibition ratio of the water fraction of Styela plicata on HSV-2 was calculated. The inhibition ratio of the water fraction of Styela plicata on HSV-2 was 82.98% at 2mg/ml concentraion, EC50=0.49mg/ml, TI>20.41, which indicated the water fraction of Styela plicata had good effect on multiplication of HSV-2.
18. The direct lethal effect of the water fraction of Styela plicata on HSV-2
MTT assay was applied to investigate the direct lethal effect of the water fraction of Styela plicata on HSV-2. The inhibition ratio of the water fraction of Styela plicata on HSV-2 was calculated. The results displayed the absorbance values of the water frction of Styela plicata groups were not significantly difference compared to that of HSV-2 control group (P>0.05). The inhibition ratio of the water fraction of Styela plicata on HSV-2 were 74.29% and 60.00% at 2 and 1 mg/ml concentraion, respectively, EC50=0.80mg/ml, TI> 12.50, which indicated the water frction of Styela plicata had good direct lethal effect on HSV-2.
19. The effect of the water fraction of Styela plicata on adsorbtion of HSV-2
MTT assay was applied to investigate the effect of the water fraction of Styela plicata on adsorbtion of HSV-2. The inhibition ratio of the water fraction of Styela plicata on HSV-2 was calculated. The results displayed the absorbance values of the water frction of Styela plicata groups had not significant difference compared to that of HSV-2 control group (P>0.05). The inhibition ratio of the water fraction of Styela plicata on HSV-2 was 19.23% at 2mg/ml concentraion, which indicated the water fraction of Styela plicata could not inhibit HSV-2 invase into host cell.
Conclusions
1. Styela plicata can inhibit the replication of HSV-2 in vitro.
2. Styela plicata can directly kill HSV-2 and can ininhibit the biosynthesis of HSV-2, but can not inhibit HSV-2 invade into host cell.
3. Styela plicata can interrupt HSV-2 DNA chain replicate. One of its target is to interrupt HSV-2 DNA polymerases gene (UL30) expression.
4. Styela plicata has good therapeutical effect on genital herpes.
5. Styela plicata can increase the percentage of T cell subgroup in the blood of mice infected with HSV-2 in vagina, and can regulate the balance of T cell subgroups. Styela plicata can elevate the concentration of Th cell cytokine of the mice infectec with HSV-2 in vagina, can regulate and improve immunological function of the mice. These may be other mechanisms of action of Styela plicata on genital herpes.
6. The water fraction of Styela plicata can inhibit HSV-2, but n-butanol fraction and chloroform fraction can not inhibit HSV-2.
引文
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