Ribavirin Mediates Anti-hepatitis C Virus With Distinct Regulation Of MAPK And STAT Pathways

Hepatitis C Virus (HCV) is an RNA virus belonging to the family Flaviviridae andinfects nearly175million people worldwide. In China, HCV incidence reaches about3%,with a30million infected population. Most HCV infected individuals develope chronichepatitis C, and some of them even progress into cirrhosis and hepatacellular carcinoma.Interferon-α (IFN-) has been used clinically for the treatment of HCV infection since1986. Later ribavirin was introduced in the therapy and that greatly improved the clearanceof viremia and reduced recurance after treatment. The recently developed HCV proteininhibitor treatment is also applied with ribavirin to enhance the response to treatment.Though ribavirin has shown great significance in clinic, the mechanism of its role remainsevasive.It has been shown that ribavirin alone cannot clear HCV viremia, yet it increases thepatient’s response to IFN-α. The antiviral effect of IFN-α is realized through its mediationof protein phosphorylation involved in the Mitogen-activated protein kinase (MAPK) andSignal transducer and activator of transcription (STAT) signal pathway to induce theexpression of antiviral genes. Hence, we hypothesize that ribavirin exerts biological effectthrough the modulation of signaling pathway in cell. This research aims at discoveringroles of ribavirin in the regulation of MAPK and STAT pathways during the treatment ofHCV.Cell culture derived HCV (HCVcc) was used here as HCV infection model.Combining other techniques including flow cell cytometry, western blot analysis, andreal-time PCR, this research investigates the influence of ribavirin on ERK, p38MARK,and STAR pathway, as well as HCV replication in human hepatoma cells.Influence of ribavirin on MAPK pathwaysTreatment with1-100μg/ml of ribavirin for24hours reduces the phosphorylation ofRaf,MEK,ERK in MARK pathway.Phosphorylation of P38MARK increases withascending concentration of ribavirin. Treatment with20μg/ml of ribavirin for12,48,72,96hours and determine the phosphorylation of kinase in MAPK pathways by Westernblotting.Ribavirin suppress Raf,MEK,ERK phosphorylation within the observation period(24-96hours). p38MAPK phosphorylation was increased at12,48, and96hours afterribavirin treatment yet decrease at72hours. Pretreatment of U0126before ribavirin inhibitribavirin-induced ERK phosphorylation. The inhibition of ERK phosphorylation by U0126 showed specific regulation of the ERK pathways by ribavirin. These results indicate thatribavirin mediates ERK and p38MAPK in distinct ways.Influence of ribavirin on STAT pathwaysThen we concentrate on the influence of ribavirin on STAT pathways.Including theup-stream kinase Tyk2,Jak1and down-stream kinase STAT1,STAT3.Treatment with5-100μg/ml of ribavirin for24hours reduces Tyk2phorsphorylation in STAT pathway,10and20μg/ml of ribavirin treatment enhance Jak1phosphorylation. Therefore, ribavirindown-regulates the phosphorylation of STAT1but up-regulate STAT3. Kinase cascadereaction kinetics showed that Tyk2phosphorylation declined in48,72,96hours after20μg/ml of ribavirin treatment. Jak1phosphorylation was increased after the treatment for12hours.STAT3phosphorylation increases at12,48, and96hours after ribavirin treatmentyet decreases at72hours. STAT1phosphorylation decreases at12,48,96hours afterribavirin treatment but not at72hours. Pretreatment of Jak Inhibitor I before ribavirininhibit ribavirin-induced STAT3phosphorylation. The inhibition of STAT3phosphorylation by Jak Inhibitor I showed specific regulation of the STAT3pathways byribavirin.Interference of IFN-with ribavirin-induced MAPK and STAT pathwaysSince ribavirin has significant curing effect combined with interferon, this researchdiscussed the impact of ribavirin on MAPK and STAT applied in combination withinterferon. The joint effect of ribavirin and interferon is to down-regulate ERKphosphorylation and up-regulate p38MAPK at48hours. The joint effect of ribavirin andinterferon is to down-regulate STAT3and p38MAPK phosphorylation at72hours.Contraryto the inhibiting effect of ribavirin on STAT1phosphorylation, IFN-enhances itsphosphorylation and expression. Therefore, IFN-and ribavirin co-regulate ERK,p38MAPK, and STAT3, yet they have separate effects on STAT1.Down-regulation of ribavirin-induced MAPK and STAT pathways by HCVccIn clinic practices, HCV infection might influence efficacy of ribavirin-interferontreatment and its regulation on MAPK and STAT pathway. Here HCVcc model is used tosimulate HCV infection to test how this situation affects ribavirin induced signalingpathway. The result is that HCV infection has negative effects on ribavirin induced MAPKand STATS pathways. The assumed mechanism is that HCVcc damages the pathwaysinduced by ribavirin. HCVcc up-regulates the phosphorylation of ERK and p38MAPK,reduced ribavirin dose dependent ERK, p38MAPK and Tyk2phosphorylation, mildly down-regulates STAT1phosphorylation, and up-regulates ribavirin dose dependentP-STAT3.Inhibition of HCV replication and induction of antiviral genes by ribavirinOur data show that ribavirin inhibits HCV RNA replication and HCV structuralprotein E2and non-structure protein NS3expression in dose dependent manner. At higherdose up to80μg/ml, ribavirin had maximum eliminating effect on HCV RNA levels andNS3,E2protein expression.In HCV infected cells, ribavirin up-regulates the expression ofIRF9and ISG15, the optimized dosage is80μg/ml, which was consistent with thesignificant decreased HCV RNA levels upon80μg/ml ribavirin treatment. Henceforce，ribavirin inhibits HCV RNA replication and induces antiviral genes.Inhibition of HCV replication and induction of antiviral genes by the combinedtreatmentRibavirin at1μg/ml and5μg/ml significantly decreased HCV RNA levels (P <0.05),and that5U/ml,10U/ml, and20U/ml IFN-in combination with the ribavirin resulted ina greater inhibition of the virus replication than that of ribavirin at the dose of1μg/ml and5μg/ml (P <0.05), Similarly, the inhibition of HCV NS3and E2protein expression wasalso observed following the treatment of ribavirin in combination with IFN-.suggestingthat ribavirin and IFN-exert a synergetic inhibitory effect on HCV replication.However,IFN-α could better induce IRF9and ISG15than ribavirin and they do not show synergicaction when applied together in HCVcc infection. In summary, Ribavirin mediatesanti-hepatitis C virus with distinct regulation of MAPK and STAT pathways.