A Preliminary Exploration On Mechanisms Of Jieze No.1 Decoction Anti-genital Herpes Virus Infection VK2/E6E7 Cells In Vitro

Part I An optimization model of herpes simplex virus type 2 infection of VK2/E6E7 cells in vitroObjective In order to ensure the stability of the study, an optimization model of herpes simplex virus type 2 infection of human vaginal epithelial cells in vitro was necessary.Methods To culture HSV-2 virus in Vero cells and calculate TCID50 showed as the virus titers by the Reed-Muench methods. The diluted virus and VK2/E6E7 cells were cultured to abtain virus solution again which was diluted respectively 5-fold, 10-fold, 20-fold, 100-fold to coincubate with 12000 VK2/E6E7 cells on 96 well plates for 24 h.Then observed under the microscope and calculated cell viability by MTT assays.Result The TCID50 is 10-6.84, which meant that the virus solution had 107.84 TCID50 per 1 ml.The groups of diluted respectively 5-fold, 10-fold, 20-fold virus solution with VK2/E6E7 cells showed numbers of abnormal cells and intercellular connections disappeared. The results of MTT assays indicated cell viability of 5-fold group was 51.26% ± 0.95%, of 10-fold group was 54.45% ± 1.85%, of 20-fold group was 65.44% ± 1.63%, and of 100-fold group was 78.51% ± 1.75%.Conclusion To ensure the stability of the study, diluting virus 10-fold with 12000 VK2/E6E7 cells was chosed as the model.Part II Effects of prevention and treatment of Jieze No.1 decoction anti HSV-2 in vitroObjective To study how Jieze NO.1 Decoction protect human vaginal epithelial cells form HSV-2,we had to distinguish prevention and treatment of Jieze NO.1 decoction anti HSV-2 in vitro.Methods To set up prevention and treatment group, drugs and VK2/E6E7 cells incubated with HSV-2 for 30 min as prevention groups, while HSV-2 and VK2/E6E7 cells were incubated for 30 min then adding drugs as treatment groups. To observe cell morphology under an inverted microscope and to calculate cell viability by MTT assays.Result Under the inverted microscope, HSV-2 infection group showed a large number of round cells and cell number reduced compared with the normal group. While comparing with HSV-2 infection group, Jieze No.1 prevention group and acyclovir prevention group showed the majority of spindle cells, but berberine(the main ingredient of Jieze No.1) was no better than HSV-2 infection group; While all drugs treatment groups were slightly more cells than HSV-2 infection group and more spindle cells.The number and cell morphology of Jieze No.1 prevention group were significantly more nearly the normal group. MTT results showed that: only the cell viability of Jieze No.1 prevention and treatment groups were statistically significant compared with HSV-2 infection group(P<0.001).Conclusion Jieze No.1 decoction could protect human vaginal epithelial cells against HSV-2. And effects of prevention of Jieze No.1 decoction were better than the treatment in the study.Part III Mechanisms of prevention of Jieze No.1 decoction anti-genital herpes virus in vitroPart A Inactivation of Jieze No.1 decoction on HSV-2 in 30minsObjective To explain mechanisms of prevention of Jieze No.1 decoction, we must know whether Jieze No.1 decoction could inactivate HSV-2 directly.Methods HSV-2 preincubated with Jieze No.1 decoction for 30 mins was added to VK2/E6E7 cells on 96 well plates(experimental group). HSV-2 with Jieze No.1 decoction was added to VK2/E6E7 cells directly as control(prevention goup). To calculate cell viability by MTT assays after 24 hours.Result The difference of cell viability between Jieze No.1 prevention group and HSV-2 infection group was 27.27% ± 1.67%, it of Jieze No.1 experimental group and HSV-2 infection group was 23.87% ± 7.56%. The difference of cell viability between berberine prevention group and HSV-2 infection group was 22.27% ± 4.48%, it of berberine experimental group and HSV-2 infection group was 6.06% ± 2.87%.The difference of cell viability between acyclovir prevention group and HSV-2 infection group was 13.75% ± 2.66%, it of acyclovir group and the experimental HSV-2 infection group was 7.49% ± 1.86%. The result indicated that Jieze No.1 couldn’t inactivate HSV-2 in 30 mins directly, neither could berberine and acyclovir.Conclusion Prevention of Jieze No.1 decoction on HSV-2 was not based on inactivation of Jieze No.1 decoction on HSV-2 in 30 mins.Part B Experiments of human vaginal epithelial cells pretreated by Jieze No.1 decoction blocking HSV-2 infectionObjective To determine whether Jieze No.1 decoction achieved the role of anti-virus by affecting the host cell.Methods To set up preconditional groups that VK2/E6E7 cells pretreated by Jieze No.1 decoction for 24 hours/12 hours cocultured with HSV-2 only for another 24 hours. VK2/E6E7 cells cocultured with HSV-2 and Jieze No.1 decoction simultaneously as control. To calculate cell viability by MTT assays and contrast the preconditional groups with the control group.Result There were no significant differences between the preconditional groups and the control group(P<0.001) and no significant differences between the preconditional groups and HSV-2 infection group.(P>0.05)Conclusion Jieze No.1 decoction may not block receptors of the host cells infected by HSV-2. And we didn’t show a direct proof.Part C Effects of Jieze No.1 decoction on receptor expression of human vaginal epithelial infected by HSV-2Objective To further explore the preventive effects of Jieze No. 1 molecular mechanisms, we assumed that Jieze No.1 decoction may affect receptors of human vaginal epithelial cells infected by HSV-2 to block the membrane fusion of HSV-2 and the host cell.Methods To set up Jieze prevention group, HSV-2 infection group and the normal group. To detect expression of membrane protein receptors- HVEM and nectin-1 on VK2/ E6E7 cells of all groups by Western Blotting and analyze grey of protein bands of HVEM and nectin-1.Result No significant difference existed on HVEM and nectin-1 expression of Jieze No.1 decoction group and HSV-2 infection group.(P>0.05)Conclusion Jieze No.1 decoction had no effects of Jieze No.1 decoction on receptor of human vaginal epithelial infected by HSV-2.Part IV Efficacy decay curve of Jieze No.1 decoction anti-HSV-2Objective Early study found that the efficacy of traditional Chinese medicine decreased with time. To confirm the preliminary hypothesis, we designed this experiment to provide certain references for the period of validity of traditional Chinese medicine.Methods We calculated cell viability of Jieze No.1 decoction(Lot: 20140815) prevention group and HSV-2 infection group on December 2014, January 2015, April 2015, June 2015 and August 2015 and drawed efficacy decay curve.Result Cell viability of HSV-2 infection group on December 2014, January 2015, April 2015, June 2015 and August 2015 was respectively 54.53% ? 4.39%,52.36% ? 1.23%,42.89% ? 0.56%,51.26% ? 0.95%, and cell viability of Jieze prevention group was respectively 84.93% ? 4.04%, 78.58% ? 3.96%,64.37% ? 1.53%, 61.30% ? 1.44%, 56.17% ? 2.51%. The difference of cell viability of two groups was less and less with time.Conclusion The efficacy of Jieze No.1 decoction anti-HSV-2 decreased gradually over one year.