Inhibitory Effects Of Picrosides On Hepatitis B Virus Covalently Closed Circular DNA In Vitro

Covalently closed circular DNA(cccDNA),serving as the original template of hepatitis B virus(HBV) replication,is the first replicative intermediate emerging during the life cycle of hepadnavirus,which indicates establishment of viral infection and origination of the replication cycle.A stable pool of cccDNA,has been found in each nucleus of persistently-infected hepatocyte,and is thought to be one of the main factors responsible for reoccurrence of chronic hepatitis B after withdrawal of antiviral agents. Therefore,determination of HBV cccDNA does not only play an vital role in the development and evaluation of aritiviral agents,but also may provide a useful guide to the efficacy of antiviral treatment and the likelihood of long-term response in patients with hepatitis B,and may aid in the decision of when to cease therapy.However,poor efficacy has been achieved aiming at clearance or inhibition of HBV cccDNA with current anti-HBV therapies,and few visible progresses were made in drug development and efficacy monitoring work against HBV cccDNA.In this research,we aimed to confirm the effectiveness and characteristics of picrosides inhibiting cccDNA in HepG2.2.15 cells,based on the specific and sensitive strategy developed earlier by our laboratory to determine HBV cccDNA.This research was composed of three parts:â… .Comparative study on the effectiveness and characteristics in inhibiting HBV cccDNA in HepG2.2.15 cells: picrosides versus adefovir dipivoxil.â…¡.Effect of picrosides on the supercoil configuration of HBV cccDNA molecules in HepG2.2.15 cells.â…¢.Effect of picrosides on the transcription of OAS1,STAT2,ISGF3γ,MyD88 and MxA genes in HepG2.2.15 cellsPartâ… Inhibitory Effect of Pierosides on Hepatitis B Virus Covalently Closed Circular DNA in HepG2.2.15 CellsObjectives To observe the effect of picrosides on HBV cccDNA in HepG2.2.15 cells.Materials and Methods HepG2.2.15 cells were separately incubated with culture medium containing 50μg/ml picrosides,100μg/ml picrosides or 5μg/ml adefovir dipivoxil for 2 and 5 days.HBV DNA in supernatant,intracellular cccDNA, intracellular relaxed circular DNA(rcDNA) and pregenomic RNA(pgRNA) were quantified by specific real-time PCR or RT-PCR.Resultsâ‘ Treatment with 50μg/ml picrosides for 2 and 5 days could reduce the production of HBV in the cell line,as shown by the decline of HBV DNA in the supernatant(49.74%and 79.48%). Intracellular synthesis of cccDNA had also been markedly lowered by 43.55%and 56.43%,and intracellular rcDNA decreased by 43.39%and 63.86%,as well as intracellular pgRNA decreased by 54.72%and 56.08%.â‘¡Treatment with 100μg/ml picrosides for 2 and 5 days resulted in decline of HBV DNA(51.11%and 82.07%) in the supernatant,as well as intracellular cccDNA(41.13%and 57.59%),rcDNA(45.09% and 67.31%) and pgRNA(52.68%and 52.47%).â‘¢Comparative treatment with adeforvir dipivoxil for 2 and 5 days resulted in decline of HBV DNA(25.56%and 92.44%) in the supernatant,cccDNA(18.54%and 47.19%),rcDNA(21.20%and 71.47%) and pgRNA(11.14%and 37.61%) in HepG2.2.15 cells.Conclusions Our research indicated that picrosides can interfere with the replication cycle of HBV, including the formation of cccDNA in HepG2.2.15 cells.The mechanism of picrosides on cccDNA may differ from adefovir dipivoxil's in its earlier inhibition phase.Partâ…¡Impact of Picrosides on the Distribution of Supercoils inside Hepatitis B Virus cccDNA in HepG2.2.15 CellsObjectives To observe the distribution of superhelixes inside HBV cccDNA molecules in HepG2.2.15 cells and the effect of picrosides or adefovir dipivoxil on such distribution.Materials and Methods HepG2.2.15 cells were divided into 3 groups: Cells of normal control group were incubated with fresh complete culture medium, while the other two groups were incubated with culture medium containing 50μg/ml picrosides or 5μg/ml adefovir dipivoxil respectively for 2 and 5 days.HBV cccDNA molecules were extracted from HepG2.2.15 cells by methods based on Hirt-fractionation and then divided into different groups according to the distinct number of innate superhelixes inside each cccDNA molecule through special two-dimensional gel electrophoresis process.Heterogeneous groups of cccDNA with different superhelix number were detected with ³²P-dCTP labeled cccDNA probe after transfer from gels to nitrocellulose blotting membranes by the Southern procedure. Integral gray values of different groups,named as A(containing 0-5 supercoils),B (containing 6-10 supercoils),C(containing 11-15 supercoils) and D(containing 16-20 supercoils) were converted into percentage value and then analyzed with nonparametric test.Resultsâ‘ The median percentage values of A to D distribution group in picrosides group on day 2 were 14.94%,33.32%,35.96%and 14.05%,respectively; while in adefovir dipivoxil group they were 13.00%,20.04%,25.55%and 39.04%and 12.36%,21.04%,23.22%and 40.77%in normal control group.The distribution of HBV cccDNA superhelixes in picrosides group were significantly different from that in adefovir dipivoxil group(Nemenyi Test,x_1,2²=8.28,P＜0.01) and normal control group(x_1,3²=12.86,P＜0.01),while no significant difference was found between adefovir dipivoxil group and normal control group(x_2,3²=2.58,P＞0.25).â‘¡The median percentage values of A to D in picrosides group on day 5 were 15.40%,33.48%, 34.47%and 15.07%,respectivel;while in adefovir dipivoxil group they were 13.30%, 20.52%,23.22%and 42.12%and 13.02%,21.24%,22.91%and 40.61%in normal control group.The distribution of HBV cccDNA superhelixes in picrosides group were significantly different from those of adefovir dipivoxil group(Nemenyi Test,x_1,2²=9.58, P＜0.01) and normal control group(x_1,3²=11.61,P＜0.01),but no significant difference was found between adefovir dipivoxil group and normal control group(x_2,3²=0.86, P＞0.75).â‘¢The distribution of superhelixes inside cccDNA molecules in HepG2.2.15 cells was found as a heterogeneous population,with group D showing the maximum percentage(about 40%),group B and C a less percentage(but both above 20%) and group A the least(less than 15%).No significant difference was found between data on day 2 and day 5(Mann-Whitney U test,U=0.321,P=0.748).Conclusions The distribution of superhelixes numbers inside cccDNA molecules in HepG2.2.15 cells exists as a heterogeneous population,treatment with adefovir dipivoxil for 2 days and 5 days imposed no significant effect on the distribution pattern.Treatment with picrosides reduced the number of innate supercoils inside HBV cccDNA in HepG2.2.15 cells. Such alteration may cause subsequent reduction of cccDNA molecular stability which facilitates the clearance of cccDNA in nucleus.Partâ…¢Effect of picrosides on the transcription of OAS1,STAT2,ISGF3γ, MyD88 and MxA genes in HepG2.2.15 cellsObjectives To observe the effect of picrosides on the transcription of some host genes and its relationship with picrosides' anti-HBV activity in HepG2.2.15 Cells. Materials and Methodsâ‘ A group of host genes including 2',5'-oligoadenylate synthetase 1(OAS1),interferon-stimulated gene factor 3-γ(ISGF3γ),signal transducer and activator of transcription-2(STAT-2),myeloid differentiation primary response gene 88(MYD88) and myxovirus resistance protein A(MxA) were selected as candidate genes according to their reported activity of decreasing pgRNA.Their mRNA levels with or without picrosides treatment were quantitated by realtime reverse transcriptase PCR(RT-PCR) withβ-actin mRNA as internal reference.â‘¡HepG2.2.15 cells were treated with picrosides,and effect of MyD88 gene being silenced by siRNA or not were assessed by quantifying intracellular HBV pgRNA and MyD88 mRNA level as well as total HBV DNA in supernatant by realtime PCR.â‘¢MyD88 gene being silenced by siRNA or not,intracellular HBV cccDNA and MyD88 mRNA level were quantitated by realtime PCR,while the distribution of superhelixes inside HBV cccDNA was evaluated by two-dimensional gel electrophoresis.Resultsâ‘ Ratio of those candidate gene mRNA levels to normal control were 1.17(OAS1),0.87(STAT2),0.93(ISGF3γ), 5.14(MyD88) and 0.83(MxA).The transcription of MyD88 gene was significantly activated by picrosides' treatment(P＜0.01),while no significant difference were observed in the transcription of other candidate genes between groups with or without picrosides treatement(P＞0.05).â‘¡MyD88 siRNA can effectively silence MyD88 gene transcription with a ratio up to 86.35%,while the inhibition rate of HBV DNA in supernatant and intracellular pgRNA by picrosides declined to 5.02%and 6.67% respectively on day 2,indicating the partial block of picrosides' anti-HBV activity by MyD88 siRNA.â‘¢The inhibition rate of intracellular cccDNA by 2 days' picrosides treatment in HepG2.2.15 cells remained as 46.24%in despite of silenced MyD88 gene transcription(with a ratio up to 85.79%),and the inhibition rate of intracellular cccDNA in mock-transfection HepG2.2.15 cells was 52.73%.The A,B,C and D distribution pattern of cccDNA superhelixes in MyD88 siRNA group were 17.08%,33.82%, 34.20%and 14.89%while in normal control group they were 13.82%,20.44%,23.14% and 42.60%,respectively.The distribution of HBV cccDNA superhelixes in MyD88 siRNA group were also significantly different from that of normal control group (Nemenyi Test,x_1,3²=24.74,P＜0.01).Conclusions Treatment with picrosides can activate transcription of MyD88 gene and may subsequently induce the degradation of pgRNA through MyD88 pathway,but MyD88 gene might not participate in the rapid, early inhibition of HBV cccDNA by picrosides.The inhibition effect of picrosides on HBV cccDNA in vitro and the reduction of superhelix number of cccDNA might rely on further activation of other host genes.