An Exploration On Part Of The Mechanisms Of JieZe No.1 Decoction Against Herpes Simplex Virus Type 2 Infection In Vitro

Part 1 The supplement of some related parameters of the vitro model about the herpes simplex virus type 2 infect VK₂/E6E7?Objective?Early,the team members have been build a relatively stableVitro model of the herpes simplex virus type 2 infect VK2/E6E7,but in the later experiments,some shortcomings have been found and interfer with the progress of the eperiment.So,doing some suplements of the related parameters of the model is necessary.?Methods?The HSV-2 virus was amplificated by Vero cell,its titer was showed by the TCID50 calculated by the Reed-Muench method.And,the TCID50 was mesured once a week on average for nine weeks?the HSV-2 virus were stored in 4?refrigerator?.The HSV-2 virus were incubated in Vero cells again for the HSV-2virus concentration solution,then were observed under the microscope and havested for the final concentrated HSV-2 virus solution which were diluted by 5,10,20,fold.The diluted HSV-2 virus were respectively incubated in VK₂/E6E7 cells for 24h,then the vability was calculated by MTT method.The good state VK₂/E6E7 cells which were spindle,like Paving stones,abundant cytoplasm,large volume,clear cell menbrane and cell nucleus were selected for the coincubation with the diluted HSV-2virus solution for 48h to 96h,and coincubate twice continuously.The titer of the later HSV-2 virus was mesured by MTT method.In additional,the poor stste VK₂/E6E7cells which were round,small volum,dim cell membrane,etc,were also choosed to repeate the above experiment.?Result?The TCID50 of the cultivated HSV-2 virus solution was 10^-7.69,its titer decline at the speed of 10^-0.250.25 per week on average from the first week to the forth week,but was at 10^-5.05.0 stablely for the next three weeks,finaly,decline again at the same speed of the first four weeks from the seventh week to the nineth week.The diluted 5 and 10 fold respectively of the concentrated HSV-2 virus solution showed reduced numbers of cells,numbers of round cells,stronger cell refraction.MTT assay results of respective group indicated that the vability of 5-fold,10-fold,20-fold group is 43.5%±1.4%?45.1%±1.1%?48.2%±1.8%.The titer of HSV-2 virus solution incubated in good sate VK₂/E6E7 cells was calculated by MTT method,and the result was that the vability of the diluted 5,10,20-fold respectively was 44.6%±3.9%?53.3%±3.4%?62.7%±5.1%;but,it was found that a great number of VK₂/E6E7cells turned to be spindle after coincubation with HSV-2 virus solution for 24h,and the vability was 85.7%±4.1%??Conclusion?When the HSV-2virus was stored in 4?refrigerator,its titer was also declined by the time at the speed of TCID50=10^-0.25/W on average.Based on the previous'experimental resultsof Qiao Tingting,the vability of the virus infection group which could meet for the successful vitro model's standard was set for about50%,the titer of concentrated HSV-2 virus solution cultured in Vero cells could reach the standard and the rusult was quite stable.The HSV-2 virus could not be rejuvenated in poor state VK₂/E6E7 cells stablely,so it could be infered that the state of VK₂/E6E7 cells and the titer of HSV-2 virus could also infect the replication and assembly of HSV-2 virus.Part 2 The comparsion of the different preparation processes of Jie Ze No.1 Decoction against HSV-2 infection?Objective?The differences are compared between the JieZe No.1 decoction used in clinical with the precise process prepared Jie Ze No.1,providing references for the later experiments.?Methods?The experimental groups were designed,namely,the treatment group which HSV-2 was coincubated with VK2/E6E7 cells for 30 min,then Jie Ze No.1 was added for 24h;the preventative group,Jie Ze No.1 and VK2/E6E7 cells were incubated together for 1h,then,the supernatant was removed and HSV-2 was added for 24h;the interventional group,HSV-2?VK2/E6E7 cells and Jie Ze No.1 were incubated together for 24 h.Looking at the CPE of the VK2/E6E7 cells,combined with the MTT method,the viability was calculated to assess the difference of Jie Ze No.1 against HSV-2 in two prepared process.?Result?The VK2/E6E7 cells of the virus infection group were round,stronger cell refraction and reduced cell number compared with the nomarl group;Compared with the virus infection group,the VK2/E6E7 cells of the treatment group and the interventional group of both different processed Jie Ze No.1 decoction were mostly spindle,a greater number of cells than the virus infection group,additionally,the percent of spindle cells in the interventional group was high than the treatment group.But,the state and number of VK2/E6E7 cells in the preventative group of both processed Jie Ze No.1 decoction was almostly like the virus infection group.The result of MTT assay was as the following,the viability of the interventional group in he precise process prepared Jie Ze No.1,the treatment group and the preventative group respecttively was 98.2% ± 4.6%?82.2% ± 2.7%?42.5% ± 3.6%,correspondingly,in the Jie Ze No.1 decoction used in clinical,was 62.1% ± 2.4% ? 57.0% ± 2.1% ?42.6% ± 2.2%.After statistics analysis,the viability of the treatment group and the interventional group in both different prepared Jie Ze No.1 showed great difference with corresponding the virus infection group?P?0.05?,but,those preventative groups turned to be nearly with the virus infection group;the treatment group and intervetional group in the precise process prepared Jie Ze No.1 indicated significantly difference with the Jie Ze No.1 in clinical use?P?0.01?.?Conclusion?The two different process prepared JieZe No.1 were both aginst HSV-2 virus infection,and the effect of the interventional group was better than the treatment group,but the preventative group showed no anti-HSV-2 virus infection,it indicated that Jie Ze No.1 had no preventation.The precise process prepared Jie Ze No.1 against HSV-2 virus infection was superior to the Jie Ze No.1 decoction used in clinical.Part 3 The time exploration of Jie Ze No.1 anti-HSV-2 virus infection?Objective ?To uncover the part of mechanism of Jie Ze No.1 decoction against HSV-2 virus infection,the incubation time of HSV-2,VK2/E6E7 cell and Jie Ze No.1needed to be resolved,subsequently,it was better to explore whether Jie Ze No.1decoction influence HSV-2 virus adhesion and penetration into VK2 / E6E7 cells.? Method ? HSV-2 virus and VK2/E6E7 cell incubated for 5min,10 min,20min,30 min,40min,50 min,60min,then,removed the supernatant and added culture medium for 24 h.The viability was calculated by MTT method to confirm the time of HSV-2 virus into VK2/E6E7 cell.VK2/E6E7 cells were pre-chilling in 4? for 30 min,Setting up the experiment groups: the virus infection 24 h group?the V24h?that HSV-2 incubated with VK2/E6E7 in 4?for 20 min and then in incubator for 24 h,the virus infection 20 min group?the V20min?which was that HSV-2 pre-coincubate with VK2/E6E7 in 4 ? for 20 min,removed the supernatant and added the Cn-TPR medium in incubator for 24h;the drug intervention 24 h group?The VJ24 h group?that was HSV-2 virus and VK2/E6E7 cell incubated together in 4?for 20 min and then removed the supernatant and added the JZNO.1 in incubator for 24h;the drug intervention 20 min group?The JV20 min group?that was HSV-2 virus,JZNo.1 and VK2/E6E7 cell incubated 4?for 20 min then removed the supernatant and added the Cn-TPR medium in incubator for 24h;the viability was counted by MTT method.?Result?5min,10 min,20min,30 min,40min,50 min,60min group were statistically significant compared with the nomal group?P?0.05?,but each time group showed no difference between each other.The V24 h group showed great dfifference compared with the V20 min group?P?0.05?,which indicated that when the temperature declined to 4?,it could reduced HSV-2 into VK2/E6E7.The JV24 h group and the JV20 min group had respectively statistical distinction compared with the V20 min group?P?0.05?.?Conclusion?HSV-2 virus could penetrate into VK2/E6E7 cell in 5 min.Based on the use of different temperature 4? and 37?,it allowed to be infered that Jie Ze No.1 may affect the HSV-2 virus adhesion and penetration into VK2/E6E7 cell.In addition,the Jie Ze No.1 played an important role in anti-HSV-2 virus in the early phase of infection.Part 4 Effect of Jie Ze No.1 on the expression of HSV-2 envelope protein g D? Objective ? Jie Ze No.1 may affect HSV-2virus adhesion and penetration into VK2/E6E7 cell,HSV-2 envelope protein g D play an important role in meditate the process,so,whether Jie Ze No.1 affected the expression of HSV-2 envelope protein g D was needed to be explored.?Method?To set up the experiment groups,the HSV-2 virus infection group was HSV-2 virus and VK2/E6E7 cells incubated for 30 min,6h,12 h,24h;the Jie Ze No.1 group was that HSV-2 virus,VK2/E6E7 cells,Jie Ze No.1 incubated together for30 min,6h,12 h,24h,VK2/E6E7 incubated with Cn-TPR medium for the same time as control.The expression of HSV-2 envelope protein g D could be observed by immunofluorescence method.?Result?The HSV-2 virus infection group for 30 min and 6h turned to be no the expression of HSV-2 envelope protein g D,but in another time points,12 h and 24 h,it could be observerd and g D protein expressing quantity of the 24 h was more than the12h;the Jie Ze No.1 group showed no the expression of specificity fluorescent protein g D in the whole time.?Conclusion ? The expression quantity of HSV-2 envelope protein g D increased gradually by time;The Jie Ze No.1 could reduce the g D expression.Part 5 Effect of Jie Ze No.1 on cytokines production of of VK2/E6E7 cell infected by HSV-2 virus in vitro?Objective?To detect the change of the level of IL1-??IL-6?IL-8?IL-10 and TNF-? cytokines in VK2/E6E7 infected by HSV-2 virus in vitro,it could be betterly understood how the above cytokines influenced HSV-2 virus infection.? Methods ? Setting up experimental group,that were the HSV-2 virus infection group which HSV-2 virus and VK2/E6E7 cell incubated for 2h,4h,6h,12 h,18h,24h;the Jie Ze No.1 group which was that HSV-2 virus,VK2/E6E7 cell,Jie Ze No.1incubate together for 2h,4h,6h,12 h,18h,24 h.Collect the supernatant of each group and repackaging supernatant.The amount of IL1-?,IL-6,IL-8,IL-10 and TNF-?were detected by CBA method?Cytometric Beads Array?.?Result?The production of IL-1??TNF-??IL-10 and IL-12 were low in the control group,even infected by HSV-2 virus;and treated by JZNO.1,the above cytokines were higher than the HSV-2 virus infection group?P?0.05?;the level of IL-6 in the HSV-2 virus infection group was statistically higher than the control?P?0.05?,and,it also was slightly higher than JZNO.1 group?P ? 0.05?.The production of IL-8 in HSV-2 virus infection was higher than JZNO.1 group in post-infection 6h?P?0.05?,but,after 6h,it was lower than JZNO.1 group?P?0.05?.?Conclusion?JZNO.1 could promote the production of IL-1??TNF-??IL-10?IL-12 and IL-8 and enhance the immune response,which played an significant role in the removal of HSV-2 virus in body.