| Part ?: Study on the characteristics of adhesion and penetration of HSV-2 into VK2/E6E7 cellsAim of study: In order to study the effect and mechanism of Jie Ze No.1(JZ-1)against HSV-2 in vitro focusing on adhesion and penetration stage,we explored the time required for HSV-2 to infect VK2/E6E7 cells and the characteristics of host cells under normal temperature and temperature control technology based on the stable model of HSV-2 infection of VK2/E6E7 cells in vitro.Then we established an in vitro model of HSV-2 adhesion and penetration into VK2/E6E7 cells.Methods: The virus with stable virulence and meet the standard of modeling was obtained by amplifying HSV-2 in Vero cells,and the survival rate of infected VK2/E6E7 was 40-60% of control group 24 h post-infection.The exact time required for adhesion and penetration of HSV-2 into VK2/E6E7 among 1 s,30 s,1 mins,2 mins,3 mins,4 mins,5 mins,10 mins and 30 mins at room temperature and among 1 s,10 mins,20 mins,30 mins,40 mins,50 mins and 60 mins under temperature control techniques was measured,and the effect of HSV-2 adhesion and penetration of VK2/E6E7 on survival rate of host cells,expression location of viral glycoprotein D(g D),expression level of viral proteins g D,g B,VP16,ICP5,and ICP4,expression level of cell membrane receptor proteins Nectin-1,Nectin-2,and HVEM,and ultrapathological changes of host cells were explored,to provide time basis and theoretical basis for establishing the in vitro model of HSV-2 adhesion and penetration into VK2/E6E7 cells by temperature control technology.Results: HSV-2 can fully adhered and penetrated into host cells within 5 mins of contact between HSV-2 and VK2/E6E7 at room temperature while it took 60 mins under temperature control techniques,which provides the possibility for exploring the antiviral stage of JZ-1.The survival rate of VK2/E6E7 in virus adhesion group and virus penetration group was only about 50% of the control group,which proved that there were enough virons to infect host cells and make the survival rate reaches the standard of modeling under temperature control technology.Under the temperature control technology,the expression of g D in the virus adhesion group was mainly detected on the membrane of the host cells,and the expression of viral structural proteins g D,VP16,and ICP5 increased significantly,while the expression of viral replication protein ICP4 cannot be detected.A large number of virons attached to the cell membrane could be observed under scanning electron microscope(SEM),which confirmed that the virons could fully adhere to the cell membrane without penetrating under temperature control technology.Under the temperature control technology,the expression of g D in the virus penetration group was mainly detected in the cytoplasm of the host cells,and the expression of viral structural proteins g D,VP16,and ICP5 increased significantly,while the expression of viral replication protein ICP4 cannot be detected,making sure that the virons could fully penetrate into the cell membrane without entering the nucleus under temperature control technology.The above results provide the possibility for exploring the antiviral stage of JZ-1.Conclusion: The stable model of HSV-2 adhesion and penetration into VK2/E6E7 cells in vitro can be established by temperature control technology,which lays a foundation for exploring the mechanism of JZ-1 against HSV-2 adhesion and penetration into VK2/E6E7 cells in the follow-up experiments.Part ?: Study on the effect and mechanism of Chinese Herbal Prescription Jie Ze-1 against the adhesion and penetration of HSV-2 into VK2/E6E7 cellsAim of study: On the basis of a stable model of HSV-2 adhesion and penetration of VK2/E6E7 cells in vitro,the effect and mechanism of JZ-1 against adhesion and penetration of HSV-2 into VK2/E6E7 cells were studied.Methods: On the basis of a stable model of HSV-2 infecting VK2/E6E7 cells in vitro,cell morphology,ultrastructural pathology,cell viability and expression of viral glycoprotein D were assessed after 6 h,12 h,18 h,and 24 h of JZ-1 treatment in order to explore its anti-HSV-2 effect in vitro.After which we treated VK2/E6E7 with JZ-1,penciclovir,or berberine at the adhesion and penetration stages,and explored the mechanism of JZ-1 in blocking HSV-2 adhesion and penetration into host cells by assessing the host cell ultrastructural pathology,viability,viral(g B,g D,VP16,ICP5,and ICP4)and host cell(HVEM,Nectin-1,and Nectin-2)proteins.Results: JZ-1 and penciclovir showed significant anti-HSV-2 effects,with improved host cell morphologies and increased host cell viabilities observed after treatment for 24 h compared with negative controls.The antiviral effect of JZ-1 was superior to that of penciclovir while berberine,the main component of monarch drug of JZ-1,showed no significant anti-HSV-2 effect in vitro.The anti-HSV-2 effect of JZ-1 can be detected after treatment for 6 h while the anti-HSV-2 effect of penciclovir was not obvious until treatment for 12 h.JZ-1 showed distinct effects on HSV-2 adhesion and penetration into VK2/E6E7 by significantly reducing the expression of viral(g B,g D,VP16,ICP5,and ICP4)proteins,improving cell morphology and increasing cell viability.However,these effects were apparently not exerted via downregulated expression of membrane fusion-related proteins such as HVEM,Nectin-1 or Nectin-2.The specific anti-HSV-2 mechanisms of JZ-1 need to be further explored.Neither penciclovir nor berberine could significantly inhibit HSV-2 adhesion and penetration into host cells.Conclusion: The anti-HSV-2 effect of JZ-1 was superior to that of penciclovir and berberine,and was mainly mediated by enhancing host cell defense and blocking adhesion and penetration of HSV-2.Part ?: Construction and optimization of genital herpes model in miceAim of study: In order to further verify the anti-HSV-2 effect of JZ-1 in vivo and explore the mechanism,it is necessary to establish a stable model of genital herpes in mice.On the basis of the previous research of our research group,the parameters such as week age and body weight of mice,modeling method,inoculation quantity and virulence of HSV-2,and symptom scoring standard were refined to further optimize the in vivo model of genital herpes.The changes of indexes at different time points of genital herpes mice model were detected to provide time basis for follow-up experiments.Methods: The source,strain,and feeding conditions of experimental mouse were controled,the week age and weight of mouse were defined,the modeling operation method were unified,the symptom scoring standard and successful model evaluation standard of genital herpes mice model were improved.Combining TCID50 and PFU methods to detect the titer of HSV-2,and comparing the successful modeling rate and mortality rate of mice with different titer of HSV-2.Choosing the HSV-2 solution with appropriate titer to make sure the successful modeling rate is between 80% and 100%,and the mortality rate is between 0% and 20%.The changes of body weight,vulvar symptom score,the degree of vaginal lesion,the infiltration of T cells,NK cells,and DC cells in vaginal tissue were measured,and the expression of tissue membrane fusion-related proteins(g D,VP16,ICP5 and Nectin-1,Nectin-2,HVEM)were detected at 12 hours,1,3,5,7,9,12 and 14 days after the establishment of genital herpes model.Results: Controlling the source,feeding conditions,and modeling operation methods,the SPF BALB/c female mice of 20±2g were adaptively feeding for one week.The mice were injected with progesterone intramuscularly for 5 days.On the 6th day,the mice were anesthetized with pentobarbital sodium and rubbed repeatedly against the vagina for 50 times.Infecting the mice with 20 ?l HSV-2 solution with the titer between 1×106 TCID50/0.1ML~1×108 TCID50/0.1ML,or 2×104 PFU/ML~2×106 PFU/ML to make sure the peak vulva symptom score of mice is between 1.5 and 4 points,and the weight loss of mice is more than 1g after 5~8 days of infection.The successful modeling rate is between 80% and 100%,and the mortality rate is between 0% and 20%,so the mice model of genital herpes was established stably.12 h after modeling,the expression of virus proteins g D,VP16,ICP5 and cell membrane fusion receptor proteins HVEM,Nectin-1,and Nectin-2 reached the peak in vaginal tissue of mice,and the infiltration of inflammatory cells increased significantly.So the 12 h after modeling was chosen as the time point to explore the effect of JZ-1 against membrane fusion of HSV-2 in vivo.On the 9th day after modeling,the symptoms of mice reached the peak,the body weight of mice decreased to the lowest point,the score of vulvar symptoms increased to the highest point,and the pathological performance of vaginal mucosa reached the peak.The infiltration of inflammatory cells in vaginal tissue also reached the peak.Therefore,the 9th day after modeling was chosen as the time point to explore the anti-HSV-2 effect of JZ-1 in vivo.Conclusion: By controlling the source,strain,week age,body weight,feeding conditions,titer and quantity of the HSV-2 solution,unifying the modeling method,improving the symptom score standard and successful model evaluation standard,a stable mice model of genital herpes is established and the time basis for exploring the anti-HSV-2 effect of JZ-1 in vivo is provided.Part ?: Study on the anti-virus effect and mechanism of Chinese Herbal Prescription Jie Ze-1 on genital herpes in miceAim of study: On the basis of a stable model of genital herpes in mice,exploring the anti-virus effect and mechanism of JZ-1 based on membrane fusion of HSV-2.Providing theoretical basis for the development of new anti-HSV-2 drugs.Methods: Under the premise of controlling the quality of medicinal materials and the technology of gel preparation,JZ-1 gel with stable properties was prepared.Comparing the effects of different concentrations of JZ-1 on the score of vulvar symptoms and body weight of mice with genital herpes to explore the effective concentration of JZ-1 against HSV-2 in vivo.The optimum concentration gradient of JZ-1 gel was selected.The body weight,vulvar symptom,spleen index,viral load of spinal ganglion,pathological changes of vagina and vulva,virus shedding of vaginal secretions,infiltration of T cells,NK cells,and DC cells in vaginal and vulvar tissues,expression level of virus proteins g D,VP16,ICP5 and cell membrane fusion receptor proteins HVEM,Nectin-1,Nectin-2 in vaginal and vulvar tissue were measured,the ultrapathological changes of vaginal and vulvar tissues were detected to explore the anti-HSV-2 effect and mechanism of JZ-1 in mice.Results: 2.5g/ml,2g/ml,1.5g/ml,1g/ml and 0.5g/ml JZ-1 could alleviate the weight loss of genital herpes mice,and 2.5g/ml,2g/ml,1.5g/ml and 1g/ml JZ-1 could alleviate the vulva symptoms of genital herpes mice.Therefore,three concentration gradients of 2.5g/ml,1.5g/ml and 0.5g/ml were selected for follow-up experiments to explore the effect and mechanism of JZ-1 against genital herpes in vivo.In the treatment of mice with genital herpes,JZ-1 can reduce the infected virons in mice vagina and vulva tissue by down-regulating the expression of virus proteins g D,VP16,ICP5 and cell membrane fusion receptor proteins HVEM and Nectin-2.JZ-1 can also protect organelles and maintain normal cell morphology and function,reduce the infiltration of tissue T cells,NK cells,and DC cells,relieve local and systemic inflammation,and promote tissue keratosis repair,so as to reduce vulvar symptoms and weight loss of genital herpes mice.By down-regulating the load of spinal ganglion virus,JZ-1 can control the recurrence of genital herpes to some extent.By reducing the amount of vaginal virus shedding,JZ-1 can also be used to prevent cross-infection.JZ-1 of 2.5g/ml showed the best curative effect.The anti-HSV-2 effect of JZ-1 in vivo is similar to that of acyclovir,which is commonly used in clinic,while berberine showed no such effect.Conclusion: JZ-1 showed clear anti-HSV-2 effect in vivo,which can play a role by intervening the membrane fusion of virus at both early stage of infection and the stage of symptoms peak in mice.It can also control the recurrence of genital herpes as well as prevent cross-infection.The antiviral effect of JZ-1 in vivo is similar to that of acyclovir,but use alone of berberine showed no such effect. |
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