The Study On The Anti-HSV-2 Effects Of Styela Plicata In Vitro And In Vivo

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.ObjectiveBecause 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,Methods1. Anti-HSV-2 activiy of the ethanol extracts of Styela plicataThe 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 vitroThe 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 vaginaMice 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 vivoMice 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 plicataThe 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 analysisAll 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.Results1. The cytotoxic effect of alcohol extracts of Styela plicata on Vero cellMTT 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 plicataThe 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-2MTT 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-2MTT 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-2MTT 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 plicataMTT 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 replicationHSV-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 herpesAfter 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 miceAfter 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 miceAfter 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 miceAfter 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 cellMTT 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 plicataMTT 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 plicataMTT 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 plicataMTT 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-2MTT 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-2MTT 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-2MTT 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. Conclusions1. 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.