Study On The Mechanism Of The Anti-HSV-2 Effect Of Chinese Herbal Prescription Jieze-1 Based On Natural Immune Toll Like Receptor Signaling Pathway

Part ?: The Variation of Toll-like Receptor Signaling Pathways After Different Time-points of VK2 Cells Infected by HSV-2Purpose: In order to establish a stable model of HSV-2 infection of VK2/E6E7 cells in vitro and satisfy the demands of HSV-2 virus of the progressive completement of follow-up experiments,a stablized,convenient and fast amplified method that can meet the demands of virulence of HSV-2 viruses needs to be explored.We base on stable model to investigate the changes of crucial proteins and terminal immune factors in Toll-like receptor signaling pathway at different time points after HSV-2 infection of VK2 cells,so as to establish foundation to further explore the mechanism of Jieze-1 against HSV-2 infection based on natural immune Toll-like receptor signaling pathway.Methods: Firstly,the routine method in Vero cells,continuous rejuvenation method in VK2/E6E7 cells and concentration method in Vero cells are used to cultivate HSV-2 viruses.The TCID50 of HSV-2 amplified by each method is detected by Reed-Muench method and the cell viability of VK2/E6E7 cells infected by HSV-2 which amplified by three methods is detected through MTT method.Thus aim to evaluate the virulence of the HSV-2 viruses and determine whether the virulence meet the standard of successful modeling.After 0,2,4,6,12,18,and 24 hours of infection by HSV-2,the cell viability is measured by MTT assay and the apoptotic rate is measured by flow cytometry respectively.The protein expression at different time-points of TLR2,TLR3,TLR4,TLR7,TLR9,HVEM,My D88,TRIF,I?B?,P-I?B?,IRF3,P-IRF3,IRF7,and P-IRF7 are detected by Western Blotting.The fluorescent expression of g D and NF-KB are detected by immunofluorescence.The nucleoprotein and plasma proteins are separated by a nuclear protein extraction kit.Then the expressed levels at above different time points of NF-KB in nuclear proteins and plasma proteins are detected by Western blotting respectively.The concentrations of IL-6,IL-8,IL-1?,TNF-?,IFN-? and IFN-? at different time-points are detected by Elisa method.Result: The TCID50 of the viruses amplified by the conventional method in Vero cells,the continuous rejuvenation method in VK2/E6E7 cells and the concentration method in Vero cells are 10-7.16,10-9.45,and 10-12.5,respectively.The cell viability in VK2 cells infected by diluted 5-fold viruses respectively are 75.74% ± 9.66%,49.7% ± 2.51%,and 28.31% ± 2.58%.The cell survival rate gradually decreased and the apoptosis rate increased gradually after 0,2,4,6,12,18 and 24 h in VK2 cells infected by HSV-2.The protein expression of TLR3,TLR4,TLR7,TLR9,g D,TRIF,P-I?B?,P-IRF3 and nuclear NF-KB is dramatically increased at 12 h,18h,and 24 h after infection.While TLR2,P-IRF7 cytoplasmic NF-KB protein expression is significantly decreased at 6 h,12 h,18 h,and 24 h after infection.The protein levels of My D88 increased significantly at 6 h,12 h,and 18 h after l infection,and there is no alteration in total protein levels of NF-KB,I?B?,IRF3,IRF7 and HVEM at different time points after infection.The concentration of IL-6 peaked at 4h,the concentration of IL-8 peaked at 18 h and the concentration of IL-1? peaked at 6 h after infection.The concentration of TNF-? and IFN-? peaked at 24 h,while the concentration of IFN-? is gradually decreased reaching the lowest peak at 24 h after infection.Immunofluorescence results show that g D virus protein is expressed in cell membrane,and its fluorescence expression could be seen from 12 h,and gradually increased until 24 h,while NF-KB is expressed in cytoplasm at 2,4,and 6h after infection and the expression of fluorescence transfer into the nuclear from 12 h to 24 h.Conclusion: Among the three methods,the virulence of the virus amplified by concentration method in Vero cells is the highest,and the expression levels of important proteins and effector molecules in the Toll-like receptor signaling pathway are different at different time-points after HSV-2 infection.Each protein and fator has their corresponding crucial time-poiints.Part ?: Exploration the mechanism of Jieze-1 recipe anti-HSV-2 in vitro based on Toll like receptor signaling pathwayPurpose: Based on the variable characteristics of each pivotal protein and effector factor of different time-points in the Toll-like receptor signaling pathways after HSV-2 infection,we select the best time-point of observation and disquisition to investigate the influence of of Jieze-1 recipe on TLRs signaling pathyway and the involved mechanism of Jieze-1 anti-HSV-2 infection in vitro.We attempt to partly clarify the effective mechanism of Jieze-1 anti-HSV-2 infection to provide theoretical foundation to rich the scientifical connotation of traditional Chinese medicine anti herpes virus.Method: Firstly,the cell viability of aciclovir,ganciclovir,and penciclovir are measured by MTT assay to determine their respective minimum cytotoxic concentration.The acyclovir,ganciclovir and penciclovir of seven drug concentrations are co-incubated with virus for 24 hours respectively.The morphological changes of the cells are observed under the microscope.The cell viability is determined by MTT assay to obtain the optimal anti-HSV-2 concentration.The minimum cytotoxic and the best anti-HSV-2 concentration of berberine and Jieze-1 could also be determined by similar methods as above.Flow cytometry is used to detect the apoptosis rate of infected VK2 cells when treated by penciclovir,berberine and Jieze-1.Protein levels of TLR2,TLR3,TLR4,TLR7,TLR9,g D,HVEM,My D88,TRIF,I?B?,P-I?B?,IRF3,P-IRF3,IRF7,P-IRF7 and NF-?B in total protein,nuclear protein and cytoplasm protein are detected by Western Bloting after intervention of the three drugs.The fluorescent expression of g D virus protein and the distribution of NF-?B of cytoplasm and nucleus are detected by immunofluorescence assay.Elisa assay is used to detect the concentration of IL-6,IL-8,IL-1?,TNF-?,IFN-? and IFN-? which are secreted by infected VK2 cells when treated by penciclovir,berberine and Jieze-1.Result: There is no significant cytotoxic effect from concentration of 1.25 mg/ml among acyclovir,ganciclovir and penciclovir.The best antiviral effect concentration of acyclovir,ganciclovir,and penciclovir are 0.078125 mg/ml,0.15625 mg/ml and 0.078125 mg/ml,respectively.The highest cell viability is 62.07%±1.17%,76.63% ± 5.39%,87.46% ± 5.49%,respectively.After treatment with Jieze-1,berberine and penciclovir,the apoptosis rate of the infected VK2 cells is significantly decreased compared with HSV-2 infected group,but the apoptosis rate of berberine group is still high.The levels of IL-6,IL-1?,TNF-?,and IFN-? decreased significantly after the treated with the three drugs,and the concentration of IL-8 is significantly decreased after penciclovir and Jieze-1 treatment.While the levels of IFN-? increased significantly after berberine and Jieze-1 treatment.After penciclovir and Jieze-1 treatment,there is no apparent nuclear fluorescence expression of NF-KB,and the fluorescence expression of g D is significantly decreased,and the condition of Jieze-1 is close to normal.Although the nuclear fluorescence expression of NF-KB is reduced after berberine treatment,but the fluorescence expression of g D is still strong.After intervention of the three drugs,the protein levels of TLR3,TLR4,TLR7,TLR9,g D,My D88,TRIF,P-I?B?,P-IRF3 and nuclear NF-KB are significantly reduced.The protein expression of TLR2,P-IRF7 and cytoplasmic NF-KB are significantly increased.There are no significant changes in the expression of total proteins of NF-KB,I?B?,IRF3 and IRF7.Conclusion: Penciclovir is the best positive control drug among acyclovir,ganciclovir,and penciclovir.Jieze-1 exerts the best effect of anti-HSV-2 virus.The involved mechanism may be related to the regulation of Toll-like receptor signaling pathway.Jieze-1 achieves the antiviral effect probably by promoting the secretion of interferon and down-regulating the inflammatory factors.Part ?: Construction of HSV-2 virus infection model in mousePurpose: In order to establish the foundation of investigating the effect and the possible mechanism of Jieze-1 of anti-HSV-2 virus infection in vivo,we re-establish and optimize a stable HSV-2 infection model in mouse,then establish a model evaluation system.Method: All mice are intramuscularly injected with progesterone for 5 days.Then the mice are divided into vaginal physical friction groups and free-physical stimulation groups.Subsequently the mice of physical friction or free-physical stimulation are inoculated HSV-2 virus at six virus titer by direct injection,submucosal injection and sponge gel filling methods respectively.The six virus titers are 10-10,10-9,10-8,10-7,10-6 and 10-5 which using TCID50 as unit.After the model was established,the vaginal symptoms of mice are observed daily to score based on the grading standard for symptom scores.The survival or the death condition of each mose was recorded daily to calculate the success rate of the modeling and mortality.The mice are weighed and the virus titers of vaginal secretions of each mouse is calculated on 1d,3d,7d,10 d,14d,17 d and 21 day after successful modeling.The blood and vaginal tissues are collected on these corresponding days.Result: When the vagina of each mouse was not physical stimulated,none of the three inoculated methods at the six virus titers could be successfully modeled.When the vagina of mice is physically rubbed,the mice which inoculated HSV-2 virus with the three methods are all successful at the titer of 10-10,10-9,10-8.But the success rate of modeling and the death rate is different between the nine groups.When the virus titer of TCID50 is 10-10,the success rate of modeling and the death rate is supernal.When the virus titer of TCID50 is 10-8,the mortality rate of the three inoculation methods all is low,and the success rate of direct injection method is higher than the other two.When the direct injection method is selected to establish the model,the success rate of the modeling is high and the mortality rate is low at the virus titer of TCID50 of 10-8 among the six virus titers of 10-5 to 10-10.When the body weight of mice is from18 g to 30 g,both the success rate and the mortality rate is decreased followed with the increased body weight.When the volume of the virus fluid is 10 ul,15ul,and 20 ul,the success rate of modeling is decreased with reduced volume of virus fluid.After successful modeling,the symptom score and the virus titer of vaginal secretion rises highest from 7d to 12 d after modeling.While the survival rate drops highest and the weight of mice is lowest from 7d to 12 d after modeling.Conclusion: The most key factor which determines the outcome of modeling is the mouse vaginal whether physically rubbed.Simultaneously,the virus titer,the method of inoculation,the volume of the virus fluid and the weight of each mouse are related with the outcome of modeling.The symptoms of HSV-2 infection in mice progresse worst during the period from 7d to 12 d.Part ?: Preliminary investigation on the efficacy of Jieze-1 gel anti-HSV-2 virus infection in mousePurpose: On the basis of the stable HSV-2 infection model in mouse established,the curative effect of low,middle,high and extra high dose of nosocomial(clinical medication)and extracted(for vitro experiments)Jieze-1 gel is explored.We initially investigate whether there is an efficacy of Jieze-1 on anti-HSV-2 infection in vivo and compare.the difference of efficacy between the two pharmaceutical techqiques of Jieze-1.Method: First,the nosocomial and the extracted Jieze-1 soup is made into Jieze-1gel at four concentrations of 2 g/l(extra high dose),1.5 g/l(high dose),1 g/l(medium dose),and 0.5 g/l(low dose).Acyclovir solution is made into a 3% acyclovir gel,penciclovir liquid is made into 1% penciclovir gel,berberine is made into 0.03% berberine gel,and the blank gel is made from double distilled water.After 5 days of intramuscular injection of progesterone,the vaginas of all mice are physically rubbed,then 20 ul HSV-2 virus fluid is inoculated.Afterwards,the model group is added with 20 ul of blank gel by lavage needle,and other groups of mice are added with 20 ul of the corresponding concentration of drug gel.After modeling and treatment with drugs,the mortality and vulval symptoms of each mouse is observed daily,and the symptoms are scored.The mice are weighed and the virus titer of vaginal secretion is measured every 3 days.The mice are sacrificed on the 21 st day after modeling,and blood and vaginal tissues are collected.Result: When the model was successfully established,the survival rate,the virus titer of the vaginal secretion and the body weight of the mice in the model group is decreased significantly compared with the control group.While the symptom score of mice in model group is significantly increased.The survival rate of extra high dose,high dose of nosocomial Jieze-1 and high-dose of extract Jieze-1 groups is statistically increased from that of the model group.The virus titer of vaginal secretions of extra high,high,and middle dose groups of nosocomial Jieze-1 and extracted Jieze-1 groups,as well as the penciclovir and berberine groups,is significantly decreased compared with the model group.The body weights of mice of the extra high,high and medium dose of nosocomial Jieze-1 and the extra high,high dose of extracted Jieze-1groups,as well as the penciclovir and berberine groups,is statistically increased compared with HSV-2 infected group.The symptom score of mice in the nosocomial and extrated Jieze-1 groups is decreased with the increased dose.The symptom score of berberine groups is lower than that of penciclovir and acyclovir groups.The lowest symptom score is the extra high dose of nosocomial Jieze-1.Conclusion: In the drug anti-HSV-2 virus infection experiment in vivo,the effectiveness of the nosocomial Jieze-1 anti-HSV-2 virus infection is better than extracted Jieze-1,and the efficacy of penciclovir and berberine is better than acyclovir.Compared the effect of all drugs on anti-HSV-2 virus infection,the extra-high dose of nosocomial Jieze-1 is the best.