| Objective：Hepatitis B virus (HBV) infection has been a global publichealth problem. One million HBV-infected patients are dying fromHBV-associated liver disease every year. It is well known that active HBVreplication is the key factor of leading to liver injury and disease progression;therefore, the aim of treatment for chronic hepatitis B (CHB) is to achievesustained suppression of HBV replication and remission of liver disease. Atpresent, two types of therapy are available for the treatment of CHB:(i)nucleoside analogues (ii) interferon alpha (IFN-α). IFN-α has a dual functionwith both antiviral and immunomodulatory effects. Because the advantages ofIFN-α therapy include a limited treatment course and a lack of resistance,INF-α treatment has been one of the main antiviral therapies with chronichepatitis B. However, currently IFN-α therapy can achieve only30–40%hepatitis B e antigen (HBeAg) seroconversion, and over60%of patients stillcontinue to suffer from chronic active hepatitis B even with IFN-α therapy.There are many factors related to IFN-α treatment failure, the latest researchhot spot is the interferon-stimulated genes in the interferon antiviral path.Recently, ubiquitin-specific peptidase18(USP18/UBP43) has been describedas the important one of these genes. There are some reports that USP18provides a negative-feed back loop that restricts IFN-α signals about hepatitisC treatment. But it is rarely reported that USP18inhibits IFN-inducedJAK/STAT signal transduction about chronic hepatitis B therapy. In this study,our purpose was to investigate the mechanism of action and clinicalsignificance of USP18in the IFN-α treatment with patients of chronic HBVinfection.Methods: All of70chronic HBV infection patients were in immuneclearance phase. All patients underwent liver biopsy. Both inflammation and fibrosis attained G2/S2or above it. After24-week course of IFN-α treatment,32patients achieved complete virological response (CVR),18patients hadnon virological response (NVR), and20patients had partial virologicalresponse (PVR).10healthy persons were in control group.1The incubation of PBMCs: The PBMCs were isolated by densitygradient centrifugation separation from individual peripheral blood, culturedby serum-free suspension. All were divided into two groups. One group addednothing as blank controller, another group added IFN-α and cultured for4h.2The peripheral blood was obtained at0w,1w,12w and24w duringIFN-α treatment.3Real-time PCR was used to detect the quantity of mRNA transcribedfrom genes of USP18and STAT-1in PBMCs.4Western blot was applied to detect the expression levels of USP18,STAT-1and P-STAT-1in PBMCs.Results:1The expression of USP18wasn’t detected in healthy control group2The changes of USP18mRNA expressions in PBMCs during follow-upobservationA total of70patients with CHB received USP18mRNA expressions inPBMCs detection during IFN-α treatment. The relative quantitative express-ions of GAPDH mRNA in PBMCs were set to1.00. Before IFN-α treatmentthe expression of USP18mRNA in PBMCs was higher in NVR group(0.073±0.026) than that in CVR（0.043±0.023）and PVR（0.044±0.022）groups(P<0.001).At the same time point, USP18mRNA expression were higher inNVR group than the other two (P<0.01). At the different time point, USP18mRNA expression in NVR, CVR and PVR groups were all lower at0w thanother time points (P<0.01).3The changes of USP18protein expressions in PBMCs during follow-upobservationThe relative quantitative expressions of β-actin protein in PBMCs wereset to1.00. Before IFN-α treatment the expression of USP18protein in PBMCs was higher in NVR group (0.308±0.046) than that inCVR(0.250±0.044) and PVR（0.248±0.048）groups (P<0.001). At the sametime point, USP18protein expressions were higher in NVR group than othertwo groups (P<0.01). At the different time point, USP18protein expressionsin NVR, CVR and PVR groups were all lower at0w than other time points(P<0.01).4The morphologic observation of PBMCs:The PBMCs were adherent after2hours. The majority of suspensioncells were B cells and peripheral blood DC. After they were incubated for4h,both their morphology and amount were unchanged5Variation of USP18, STAT-1mRNA/protein expressions in PBMCs in vitroAfter IFN-stimulated, USP18mRNA/protein expressions in PBMCswere up-regulated considerably. There was significant difference betweenblank control group and IFN-α group (P<0.01). There was no significantdifference on STAT-1mRNA/protein expressions in PBMCs among eachgroup.6The correlation between USP18and P-STAT-1protein expressionsAfter IFN-stimulated, P-STAT-1protein expressions were lower innon-responder group than the other two (P<0.01). There was negativecorrelation between USP18and P-STAT-1protein expressions. Pearsoncorrelation was-0.753. USP18protein expression was the highest in NVRgroup, yet P-STAT-1protein expression was the lowest.7Correlation of different influencing factors and IFN-α therapeutic effectThere was correlation between HBV-DNA baseline level, ALT level, thephasing of liver tissue inflammation, USP18expression level and IFN-αtherapeutic effect. Through the elimination process step by step, the threevariables in HBV-DNA baseline level, the phasing of liver tissueinflammation and USP18expression level went into regression equation,y=0.588+20.552x1-39.299x2+19.641x3(χ2=35.682, P<0.01).Conclusions:1The expression level of USP18was up-regulated considerably by IFN-α stimulated. These results imply that USP18is induced by IFN, LPS andviral infections.2There was negative correlation between the expression level of USP18and P-STAT-1. It is demonstrated that USP18inhibits phosphorylated activeforms of STAT1by binding to the IFNAR2subunit of the IFN-α.3There was obvious difference about the expression of USP18betweenCVR, PVR and NVR groups in a period of time from0w to24w. It is exactlyillustrated that USP18has been described as negative regulator in TypeⅠIFNsignaling.4USP18may become a new target of treating non-responders as negativepredictor. |
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