| Objective:To explore the mechanism of the external Chinese herbal prescription JieZe-1(JZ-1)and its effective ingredient of the monarch in the treatment of genital herpes based on autophagy and apoptosis.Methods:In vivo,female Balb/c mice were infected with HSV-2,then the virus infection of the vulva,vagina,uterus and spinal nerve of mice were detected at different time points.Different concentration drug groups of JZ-1 gel were set up to explore the efficacy and optimal concentration of JZ-1 in the treatment of genital herpes.Then,CQ was used as an autophagic flux inhibitor to detect the regulatory effects of JZ-1 on autophagy and apoptosis in various tissues of GH mice.In addition,the possible upstream mechanism of JZ-1 regulating autophagy and apoptosis was explored by detecting Akt/mTOR pathway.In vitro,VK2/E6E7 cells were infected with HSV-2,and Baf A1 was selected as an autophagic flux inhibitor to further validate the role of JZ-1 against HSV-2 infection by regulating autophagy and apoptosis.Subsequently,the antiviral effect and mechanism of monarch’s composition were explored in VK2/E6E7 cells.The main active components of the monarchs of JZ-1,phellodendrine,ginkgolide A and ginkgolide C,were selected as the research objects.Baf A1 was applied to detect the autophagic flux,and the autophagy inhibitor 3-MA was applied to clarify whether the drugs could exert anti-HSV-2 infection effects by regulating autophagy and apoptosis.Results:The peak of vulvar infection in GH mice was on day 9 after modeling.The peak of vaginal pathological changes was also on day 9,but the peak of viral protein gD expression was on day 5.The peak of uterine and spinal nerve infection was on day 14 and 5,respectively.High doses of JZ-1(JH)were the most effective,significantly improving vulvar symptoms and weight loss,and reducing gD expression,inflammatory cell infiltration and pro-inflammatory cytokine expression at all sites of GH mice(P<0.01).After CQ application,HSV-2+CQ+JH administration significantly increased the expression of LC3B-Ⅱ and SQSTM1/p62 compared to HSV-2+CQ group(P<0.05).Expression level of Atg5 was consistently decreased in HSV-2 group and was consistently increased in JZ-1 treated group(P<0.01).Moreover,the application of CQ increased the percentage of apoptotic cells and the expression of apoptotic proteins cleaved Caspase-3 and Bax,decreased the expression of anti-apoptotic protein Bcl-2,and attenuated the inhibitory effect of JH on viral protein gD(P<0.01),aggravated the vulval symptoms and pathological changes in mice.In addition,JH significantly inhibited the phosphorylation of Akt and mTOR caused by HSV-2 infection(P<0.01).In vaginal and uterine tissues,CQ similarly significantly attenuated the effects of JZ-1 in decreasing the expression of the apoptotic proteins cleaved Caspase-3,Bax and viral protein gD and increasing the expression of the anti-apoptotic protein Bcl-2(P<0.05).In the spinal nerve,inhibition of autophagy similarly affected the inhibitory effect of JZ-1 on gD(P<0.01).The results of in vitro experiments were the same as in vivo.In the exploration of the active ingredients of monarchs of JZ-1,1μM(H1)and 0.1 μM(H0.1)of phellodendrine significantly increased the survival of HSV2 infected cells(P<0.01),while ginkgolide A and ginkgolide C had no such effect.After application of Baf Al,LC3B-Ⅱ protein expression was significantly increased in the Baf A1+H0.1 and Baf A1+H1 groups compared with the H0.1 and H1 groups,respectively(P<0.001).By applying 3-MA,phellodendrine no longer exerted the effect of reducing the expression of apoptotic protein cleaved Caspase-3 and Bax protein and increasing the expression of anti-apoptotic protein Bcl-2(P<0.01).At the same time,phellodendrine inhibited the phosphorylation levels of Akt and mTOR(P<0.01).Conclusion:In genital herpes mice,HSV-2 first enters the vaginal epithelium,then travels up to the spinal nerve and then down to the vulva,while infecting the endometrium,and the whole process occurs continuously.JZ-1 can treat genital herpes caused by HSV-2 infection by inducing autophagy and inhibiting apoptosis,and its potential mechanism may be related to the inhibition of Akt/mTOR pathway.Ginkgolide A and ginkgolide C,the main components of ginkgo semen,have no anti-HSV-2 infection effect,while phellodendrine,the main component of Phellodendri Chinensis Cortex,exerts significant anti-HSV-2 infection effect,and this effect is exerted by inducing autophagic flux and inhibiting apoptosis.Part Ⅰ:Changes of infections in different parts of genital herpes mice with time pointsObjective:To explore the changes in viral replication and histopathological changes in various parts of the genital tract(including vulva,vagina,and uterus)and spinal nerves in genital herpes mice over the time of HSV-2 infection,so as to explore the pathological process of GH caused by HSV-2 infection in animal model.Methods:8-week-old female Balb/c mice were infected with HSV-2 transvaginal,then vulvar symptom scores and body weight were recorded daily.Specimens were collected at the corresponding time points(day 1,3,5,7,9,12 and 14 after modeling),including vulva,vagina,uterus and spinal nerve.Then the degree of infection was assessed by qRT-PCR and Western Blot for viral protein expression,and HE staining for histopathological changes.Results:The results of vulvar symptom scores and weight changes showed that on the 9th day after modeling,the mice had the most severe vulvar symptoms and the most obvious weight changes.And the results of HE staining showed that the vulvar tissues of normal uninfected mice had clear structures and normal stratification,the vulvar structures of mice on the 1st and 3rd days after modeling were like the normal group,with no obvious abnormalities.On the 5th day,the vulvar structures began to appear discontinuous and a large number of lymphocytes infiltrated,and the above changes were more obvious on the 7th day,with tissue hyperplasia appeared.On day 9,the hyperplasia was most obvious,and there was a large number of lymphocytes infiltrating from the epidermis to the subcutaneous tissue layer.Day 14,there were still some hyperplasia and lymphocyte infiltration.The pathological changes in the vaginal tissues started to appear on day 1 after infection.The vaginal epithelium was still continuous on days 1 and 3 after viral infection,but there was partial lymphocytic infiltration.On day 5,discontinuity of the epithelial layer and a little cellular vacuolation started to appear,with the peak of symptoms on day 9 after modeling,which showed massive cellular vacuolation necrosis and massive lymphocytic infiltration.In contrast to the vagina,the adjacent endometrium showed later pathological changes,with endothelial epithelial disruption starting on day 5 and vacuolar degeneration on day 7,which gradually worsened,and some degree of endothelial hyperplasia by day 14.qRT-PCR and Western Blot showed that viral protein gD expression in vulvar tissues did not appear until day 5 post-modeling,with peak expression on day 9.In vaginal tissues,gD expression peaked on day 5,while in uterine tissues,gD expression started on day 5 and gradually increased to the highest level on day 14.Viral proteins were detectable in the spinal nerve starting on day 3 post-modeling,with a rapid peak of replication on day 5.The peak time point of vulval symptoms,weight change,vulval pathological changes and viral protein replication were all on day 9 after modeling,thus day 9 after modeling was chosen as the experimental endpoint in the subsequent experiments.Conclusions:After vaginal infection,HSV-2 first infects the vaginal epithelium,and then the virus travels up the nerve to the spinal nerve and then down to the vulva while also infecting the endometrium,and the whole process occurs simultaneously.Part Ⅱ:Treatment of genital herpes through induction of autophagy and inhibition of apoptosis by JieZe-1 in vitro and in vivoObjective:To investigate the efficacy and mechanism of the external Chinese herbal prescription JZ-1 in the treatment of genital herpes from the perspective of autophagy and apoptosis.Methods:In the in vivo part of the experiment,8-week-old female balb/c mice were infected with HSV-2,and different concentration drug groups of JZ-1 gel and acyclovir gel positive control groups were set up to explore the optimal drug concentration.After determining the optimal drug concentration,CQ was then used as an autophagic flux inhibitor to explore the regulatory effects of JZ-1 on autophagy and apoptosis.The body weight and symptom scores of mice in each group were recorded daily during the experiment.According to the results of the part I,day 9 after modeling was selected as the final time point of the experiment.Specimen were collected from vulva,vagina,uterus and spinal nerve as experimental subjects,then the pathology of each site was detected by HE staining,viral protein expression was detected by qRT-PCR and Western Blot,inflammatory response was assessed by IHC and qRT-PCR,autophagic flux was detected by TEM,IHC and Western Blot,and apoptotic expression explored by TUNEL staining,IHC and Western Blot.In addition,the Akt/mTOR pathway was detected by Western Blot to explore the possible upstream mechanisms of JZ-1 regulation of autophagy and apoptosis.In part of the in vitro experiments,HSV-2 infected VK2/E6E7 cells were used as the experimental model,Baf A1 was selected as an autophagic flux inhibitor,and Western Blot was used to detect key proteins of autophagy and apoptosis to further validate the role of JZ-1 against HSV-2 infection by regulating autophagy and apoptosis in VK2/E6E7 cells.Results:Based on vulvar symptom scores,body weight changes,expression of viral protein gD in the vulva and remission of inflammation,it is clear that high doses of JZ-1 gel(JH)are the most effective,significantly reducing vulvar symptom scores,improving body weight loss,reducing viral protein gD expression in various sites,inflammatory cells(CD3+,CD4+,CD68+and MPO+)infiltration and expression of the pro-inflammatory cytokines IL-1β,IL-6,TNF-α,and CCL2 at various sites(vulva,uterus,and spinal nerve)(P<0.05).As for apoptosis,JH treatment significantly reduced the proportion of apoptotic cells in the vulva of model mice,decreased the expression of apoptotic proteins cleaved Caspase-3 and Bax,and increased the expression of anti-apoptotic protein Bcl-2(P<0.05).Meanwhile,TEM and molecular biology experiments showed that HSV-2 infection inhibited autophagy vesicle generation and autophagy key protein expression,while JH reversed the above phenomena(P<0.01).The results after applying CQ showed that inhibition of autophagy increased the proportion of apoptotic cells and the expression of apoptotic proteins cleaved Caspase-3 and Bax,decreased the expression of anti-apoptotic protein Bcl-2,and attenuated the inhibitory effect of JH on viral protein gD(P<0.01),and increased viral replication and aggravated vulvar symptoms and pathological changes in mice.In addition,JH significantly decreased the phosphorylation of Akt and mTOR caused by HSV-2 infection(P<0.01),suggesting that the promotion of autophagy by JZ-1 may be mediated by the Akt/mTOR signaling pathway.In vaginal and uterine tissues,the autophagy inhibitor CQ similarly significantly attenuated the effects of JZ-1 in reducing the expression of the cleaved Caspase-3 and Bax,increasing the expression of Bcl-2,and inhibiting the expression of the viral protein gD(P<0.05).In the spinal nerve,viral protein gD expression was significantly increased in the spinal nerve of mice after application of CQ to inhibit autophagy compared with the JH treatment group(P<0.001).In addition,the results of in vitro experiments showed that,as in vivo,the autophagy inhibitor Baf A1 similarly attenuated the effects of JZ-1 in reducing cleaved Caspase-3 and Bax expression,increasing Bcl-2 expression,and inhibiting the expression level of viral protein gD in VK2/E6E7 cells(P<0.05).Conclusions:JZ-1 can treat GH due to HSV-2 infection by inducing autophagy to inhibit apoptosis,and the underlying mechanism may be related to the inhibition of the Akt/mTOR pathway.Part Ⅲ:In vitro experimental study on the induction of autophagy thus inhibition of apoptosis against HSV-2 infection by active ingredients of the monarchs of JZ-1Objective:To explore the effect and mechanism of the active ingredient of JZ-1 monarchs against HSV-2 infection based on the regulation of autophagy and apoptosis.Methods:Phellodendrine,ginkgolide A and ginkgolide C,the main active components of the JZ-1 monarchs Phellodendri Chinensis Cortex and Ginkgo Semen,were selected for the study.The drug toxicity of phellodendrine,ginkgolide A and ginkgolide C in VK2/E6E7 cells was examined by CCK-8,and the non-toxic concentrations were screened before acting on HSV-2-infected VK2/E6E7 cells to determine the drugs and concentrations with anti-HSV-2 effects.Subsequently,the expression of autophagy-related proteins was detected by immunofluorescence and Western Blot,and autophagic flux inhibitor Baf A1 was applied to clarify the effect of drugs on autophagic flux in HSV-2-infected cells.Meanwhile,apoptosis and the expression level of viral protein gD in cells were detected by apoptosis flow assay,immunofluorescence,qRT-PCR,and Western Blot.With the application of autophagy inhibitor 3-MA,apoptosis and viral replication were then detected to clarify whether the drugs could exert anti-HSV-2 infection effects by regulating autophagy and apoptosis.In addition,Western Blot was performed to detect the Akt/mTOR pathway to explore the possible upstream mechanisms of drug regulation of autophagy and apoptosis.Results:CCK-8 cytotoxicity results showed that 10 μM and higher concentrations of phellodendrine and ginkgolide A were cytotoxic,and 1000 μM and higher concentrations of ginkgolide C were cytotoxic in VK2/E6E7 cells.Efficacy results showed that 1 μM(H1)and 0.1 μM(H0.1)phellodendrine had anti-HSV-2 infection effects(P<0.01),while there was no difference in cell survival rates of each concentration of ginkgolide A and ginkgolide C compared with the model group.Therefore,1 μM and 0.1 μM of phellodendrine were selected for exploring the anti-HSV-2 effect,while JZ-1 and penciclovir control groups were set up as positive control groups.The results of bright-field of cells showed that compared with the model group,the number of cells and cell status of the 1 μM and 0.1 μM phellodendrine group recovered significantly,and which of the high-dose phellodendrine group were close to those of JZ-1 and penciclovir groups.mRNA and protein expression levels of HSV-2 viral protein gD,pro-inflammatory cytokines IL-1β,IL-6,TNF-α and chemokine CCL2 showed that both 1 μM and 0.1 μM of phellodendrine had anti-HSV-2 infection effect(P<0.01),and the high-dose group had better effect than the low-dose group,which was close to the JZ-1 and penciclovir groups.The results of autophagy assay showed that the protein expression levels of LC3B fluorescence and LC3B-Ⅱ protein expression were significantly lower and the protein expression of SQSTM1/p62 and Atg5 were significantly higher in both concentration groups of phellodendrine compared with the HSV2 infection group(P<0.05).After the application of the autophagic flux inhibitor Baf A1,LC3B-Ⅱ protein expression was significantly increased in the Baf A1+H0.1 and Baf Al+H1 groups compared with the H0.1 and H1 groups,respectively,indicating that both H0.1 and H1 could increase autophagic flux in VK2/E6E7 cells(P<0.001).And the results of apoptosis assay showed that HSV-2 infection significantly increased the fluorescence expression of apoptotic proteins cleaved Caspase-3 and Bax protein,while significantly decreased the fluorescence expression of anti-apoptotic protein Bcl-2,while H0.1 and H1 reversed this phenomenon(P<0.01).The ability of phellodendrine to improve apoptosis was diminished after the application of 3-MA.The mRNA and protein expression of gD in the 3-MA+H0.1 and 3-MA+H1 groups were significantly increased compared with the H0.1 and H1 groups(P<0.01),indicating that 3-MA could effectively block the anti-HSV-2 effect of phellodendrine.In addition,the phosphorylation levels of Akt and mTOR were detected and found to be significantly increased in HSV-2-infected VK2/E6E7 cells compared with the normal group(P<0.01),while which returned to normal levels in the phellodendrine treatment group.Conclusions:Ginkgolide A and ginkgolide C,the main components of Ginkgo Semen,have no anti-HSV-2 infection effect,while the main component of Phellodendri Chinensis Cortex,phellodendrine,exerts significant anti-HSV-2 infection effect,and this effect is exerted by inducing autophagic flux and inhibiting apoptosis. |
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