The Chinese Herbal Prescription JZ-1 Induces Autophagy To Protect Against Herpes Simplex Virus-2 In Human Vaginal Epithelial Cells By Inhibiting The PI3K/Akt/mTOR Pathway

Part Ⅰ Changes in expression of key autophagy protein LC3B-II at different time points in HSV-2 infected VK2/E6E7 cells[Objective] In order to explore the changes of autophagy with infection time in VK2/E6E7 cells infected with HSV-2,we measure the expression of key autophagic proteins at different time points,to lay the foundation for exploring the relationship between autophagy and anti-HSV-2 infection.[Methods] Vero cells were used as a host to cultivate HSV-2,and the virulence of the harvested HSV-2 was measured by median tissue culture infective dose(TCID50),and the harvested HSV-2 is used to infect VK2/E6E7 cells.The success criterion of the model is that the cell survival rate of VK2/E6E7 cells infected with HSV-2 for 24 h by MTT assay is between 40% and 60%.VK2/E6E7 cells are harvested at 0,6,12,18,24,and 48 hours after HSV-2 infection,respectively.Western blot is used to detect the expression level of key autophagy protein LC3B-II.[Results] The virus harvested 60-72 h post-infection is determined to have a TCID50 of 106-107 by the Reed-Muench method.Infecting VK2/E6E7 cells with this virulence HSV-2 for 24 hours allowed the cell survival rate is between 40% and 60%,which means that the infectious model is successfully created.HSV-2 infected VK2/E6E7 cells at different times.After harvesting the cells(at 0,6,12,18,24,and 48 h postinfection),western blot results show that compared with the control group,the expression of endogenous LC3B-II in HSV-2 infected cells significant increased at each corresponding time point,with the most increase at 24 h of infection.[Conclusion] According to the previous research of our team,the method of amplifying HSV-2 virus using Vero cells as a host can successfully model.In addition,compared with the control group,LC3B-II increased the most 24 h after HSV-2 infection of VK2/E6E7 cells,so 24 h post-infection is selected as the time point for subsequent experiments.Part Ⅱ Changes of autophagy flux in HSV-2 infected VK2/E6E7 cells[Objective] To study the changes of autophagy flux in VK2/E6E7 cells infected by HSV-2,and to explore the effect of HSV-2 infection on autophagy flux,which is the basis for the subsequent research on the relationship between autophagy and antiviral infection.[Method] According to the part I,the modeling method was to infect VK2/E6E7 cells with HSV-2 for 24 h.After applying autophagolysosomal inhibitor Bafilomycin A1(Baf A1),the number of autophagosomes was observed by immunofluorescence,and the expression of autophagy proteins LC3B-II,SQSTM1/p62,and Atg5 was measured by western blot to evaluate the autophagy flux.[Results] Immunofluorescence and western blot show that when treated with the autophagolysosomal inhibitor Baf A1 alone,the LC3 B light spots,and LC3B-II and SQSTM1/p62 protein expression levels were reduced in the Baf A1+HSV-2 group.At the same time,the protein expression of autophagy protein Atg5 is reduced in HSV-2 infected cells.[Conclusion] In VK2/E6E7 cells infected with HSV-2,the expression of autophagy protein LC3 B increased,and the expression of SQSTM1/p62 decreased.However,compared to Baf A1 treated alone,the expression of autophagy proteins LC3B-II,SQSTM1/p62 don’t increase in the Baf A1+HSV-2 group,which indicating that autophagy flux in HSV-2 infected VK2/E6E7 cells is blocked.Reduced Atg5 expression also confirms this.Part Ⅲ Chinese herbal prescription JZ-1 induces autophagy against herpes simplex virus-2 in VK2/E6E7 cells[Objective] The above experiments showed that autophagy flux was inhibited in HSV-2 infected VK2/E6E7 cells.By studying the effect of JZ-1 on the autophagy flux in HSV-2 infected VK2/E6E7 cells,the relationship between JZ-1 anti-HSV-2 infection and its regulation of autophagy flux was explored.[Method](1)We performed MTT assays to exam the cytotoxicity and antiviral activity of JZ-1 on VK2/E6E7 cells,various concentrations(0.625,1.25,2.5,5,10,20mg/m L)of JZ-1 were added to VK2/E6E7 cells for 24 h in the presence or absence of HSV-2.(2)After m RFP-GFP-LC3 B lentivirus was transfected into VK2/E6E7 cells,the autophagy structure was observed with a confocal microscope.Since the GFP signal is p H sensitive,this method can be used to quantify the number of autophagosomes and autolysosomes.The yellow spots represent overlapping red and green spots,indicating autophagosomes that have not fused with lysosomes,while the red spots correspond to autolysosomes.At the same time,TEM was used to observe the changes of autophagy structure in cells after different treatments.(3)The autophagy flux was detected by Baf A1 as described in the part II,and the expression of key autophagy proteins and HSV-2 virus protein g D and ICP5 were detected by immunofluorescence and Western blot to evaluate the autophagy flux and the antiviral effect of JZ-1 in different situations.(4)After using Rapamycin(Rapa)or 3-MA to induce or inhibit autophagy,the expression of autophagy structure / protein and viral protein in the HSV-2,JZ-1+HSV-2 group was detected.[Results](1)JZ-1 was cytotoxic at concentrations > 5 mg/m L and 1.25,2.5,5,10,20mg/m L JZ-1 all exerted an antiviral activity to HSV-2 infection.Consider the above results,we chose 5mg/m L as JZ-1’s final concentration used in this study.Western blot showed that the expression of ICP5 and g D were increased in HSV-2 infected cells,indicating enhanced viral replication.However,the expression levels of ICP5 and g D were significantly reduced in the JZ-1+HSV-2 treated group.(2)The m RFP-GFP-LC3 B lentivirus is transfected to VK2/E6E7 cells to evaluate the autophagy structure by confocal microscopy.Twenty-four hours after infection,we found a marked increase in the number and proportion of yellow puncta in the cells infected with HSV-2 compared with the number and proportion in the nontreated cells,which indicates autophagosomes are not fused to lysosomes.Taken together,these results indicate that HSV-2 blocks autophagic flux in VK2/E6E7 cells.And a significant increase in the number of autolysosomes in the JZ-1+HSV-2 group compared with those in the group treated with only HSV-2.In addition,TEM observation showed that autophagy-related structures were reduced in cells 24 hours after HSV-2 infection,and increased autophagy structure in JZ-1 treated HSV-2 infected cells.(3)The immunofluorescence and Western blot results showed that compared with the Baf A1+HSV-2 treatment group,the LC3 B points and LC3B-II and SQSTM1/p62 protein expression levels of the JZ-1+Baf A1+HSV-2 treatment group increased.This indicates that JZ-1 induces autophagy flux.The increase of Atg5 in Western blot further confirms the above conclusion.(4)The results of m RFP-GFP-LC3 B lentivirus showed that the number of autolysosomes increased after Rapa treatment in the HSV-2 group or the JZ-1+HSV-2 group,but decreased significantly after 3-MA treatment.Consistently,immunofluorescence and western blot showed that the numbers of LC3 B puncta and the LC3B-II and Atg5 proteins expression levels were both increased in the Rapa+HSV-2 group while decreased in 3-MA+HSV-2 group.Virus proteins ICP5 and g D were determined to evaluate the anti-HSV-2 effect,the results showed that in Rapa+HSV-2 group,the expression of ICP5 and g D was decreased compared with the HSV-2 group,while in 3-MA+HSV-2 group,the expression was further increased compared with that of HSV-2 group.Moreover,the ICP5 and g D proteins expression levels of the JZ-1+3-MA+HSV-2 treated group were more than the JZ-1+HSV-2 group.[Conclusion] Increased expression of ICP5 and g D in HSV-2 infected cells indicate enhanced virus replication.However,the expression levels of ICP5 and g D were significantly reduced in the JZ-1+HSV-2 group,indicating that JZ-1 has an anti-HSV-2 effect.Changes in the expression levels of autophagosomes and autophagy proteins after applying Baf A1,Rapa and 3-MA indicate that JZ-1 induced autophagy flux.The detection of ICP5 and g D expression levels showed that the activation of autophagy can prevent HSV-2 infection,and the anti-HSV-2 effect of JZ-1 can be mediated by inducing its autophagic flux.Part Ⅳ JZ-1 induces autophagy via inhibiting PI3K/Akt/mTOR pathway[Objective] The PI3K/Akt/mTOR pathway is one of the main pathways of mammalian autophagy mutations.Based on the PI3 K / Akt / mTOR pathway,we explored the mechanism of JZ-1 regulating autophagy in HSV-2 infected VK2 / E6E7 cells.[Methods] qRT-PCR and western blot were used to detect the expression of key molecules PI3 K,p-Akt,Akt,p-mTOR,and mTOR in the PI3K/Akt/mTOR pathway of different treatment groups.In addition,the PI3 K inhibitor LY294002 was used to inhibit the pathway to detect the above indicators and autophagy-related indicators.The relationship between the pathway and JZ-1 enhanced autophagic influx was explored from the positive and negative directions.[Result] Compared with the control group,qRT-PCR analysis and Western blot results showed that PI3 K expression level,phosphorylation levels of the Akt and mTOR(pAkt and p-mTOR)were significantly increased in HSV-2 infected VK2 cells,but significantly reduced in the JZ-1+HSV-2 group.After LY294002 was used to inhibit PI3 K,western blot and qRT-PCR analysis results showed that the expression of key proteins in the PI3K/Akt/mTOR pathway were inhibited.There was no difference in expression of key autophagy proteins LC3B-II and Atg5 between the LY294002 treatment group and LY294002+JZ-1 group.[Conclusion] JZ-1 can indeed inhibit the expression of PI3K/Akt/mTOR pathway,and JZ-1 can no longer enhance autophagy after applying LY294002.JZ-1 can indeed enhance the autophagy effect by inhibiting the PI3K/Akt/mTOR pathway judging from the positive and negative directions.