| Hepatitis B virus(HBV)is a DNA virus that can cause chronic hepatitis B and seriously endanger human health.In clinical practice,IFNs and nucleotides are the main drugs for the treatment of chronic hepatitis B.However,the treatment is a long-term process,and with the passage of time and the increase in the number of IFN-α,the sensitivity of HBV to IFN-α decreases significantly,which weakens the effects of IFN-α.A large number of clinical studies have also found that hepatitis B patients with long-term IFN-α treatment are easier to lead to drug resistance,which is a huge challenge for the treatment of hepatitis B.IFN-α plays an antiviral activity primarily through the JAK-STAT signaling pathway,and STAT1 plays an important role in it.Studies have shown that STAT1 expression and its modification affect IFN-α effects.Therefore,STAT1 and the expression of its related proteins and genes are of great significance in the study of therapy for HBV with IFN-α.The Hep G2.2.15 cell is a useful tool for in vitro studies of HBV drugs development of reproducing in vitro and secreting HBs Ag,HBe Ag and Dane granules.In this study,we established a model of Hep G2.2.15/IFNα-2b cell line by stimulating the Hep G2.2.15 cells with a low dose(50 IU/ml)of IFNα-2b for a long time(6 months).The sensitivity of drug-resistant cell lines to IFNα-2b was decreased,and the decreased percentage of HBs Ag,HBe Ag and HBV DNA were significantly decreased.This study was aimed to investigate the effect of STAT1 on drug resistance in JAK-STAT signaling pathway and its possible mechanism.Objectives:In this study,we established a model of Hep G2.2.15/IFNα-2b cell line by stimulating the Hep G2.2.15 cells with a low dose(50 IU/ml)of IFNα-2b for a long time(6 month)and aimed to investigate the effect of STAT1 on drug resistance in JAK-STAT signaling pathway and its possible mechanism.Methods:1.The control group,3 months stimulated group and 6 months stimulated group were treated with(0,250,500,1 000 IU / ml)IFNα-2b for 72 h.The protein expression of STAT1,p-STAT1 were detected by Western-blot.2.To investigate the expression of HBs Ag,HBe Ag and HBV DNA in Hep G2.2.15/IFNα-2b cells,Hep G2.2.15 cells as the control group,Hep G2.2.15/IFNα-2b cells as the model group,both were cultured for 24 h with the same number.The supernatant was collected.The levels of HBs Ag and HBe Ag were measured by ELISA and the amount of HBV DNA were detected by PCR-Fluorescence Probing.3.To investigate the antiviral activity of IFNα-2b in Hep G2.2.15/IFNα-2b cells,Hep G2.2.15 cells as the control group,Hep G2.2.15/IFNα-2b cells as the model group,both were treated with 0,IFNα-2b(1 000 IU/ml)for 24 hours with the same number,respectively,comparing with no drug group.The levels of HBs Ag and HBe Ag were measured by ELISA and the amount of HBV DNA was detected by PCR-Fluorescence Probing.The m RNA expression of STAT1,OAS1 and USP18 was detected by q RT-PCR.The protein expression of STAT1,p-STAT1 and OAS1 were detected by Western-blot.4.To investigate the effects of JAK inhibitor and histone deacetylase inhibitor(HDACi)on the antiviral activity of IFNα-2b in Hep G2.2.15/IFNα-2b cells.Hep G2.2.15 cells as the control group,Hep G2.2.15/IFNα-2b cells as the model group,both were treated with 0,IFNα-2b(1 000 IU/ml),IFNα-2b(1 000 IU/ml)+JAK inhibitor(1 μM),IFNα-2b(1 000 IU/ml)+ TSA(30 nmol/ml),IFNα-2b(1 000IU/ml)+ JAK inhibitor(1 μM)+ TSA(30 nmol/ml)for 24 hours with the same number.The levels of HBs Ag and HBe Ag were measured by ELISA and the amount of HBV DNA was detected by PCR-Fluorescence Probing.The m RNA expression of STAT1,OAS1 and USP18 were detected by q RT-PCR.The protein expression of STAT1,p-STAT1 and OAS1 were detected by Western-blot.Results:1.Compared with the control cells,the protein expression of STAT1 was increased in6 months stimulated groups and p-STAT1 was down-regulated.The proportion of p-STAT1 in 6 months stimulated groups was lower than that in the model group.2.The HBs Ag,HBe Ag and HBV DNA in the model group were higher than that in the control group.HBV DNA level was lower than that in the control group.The results were not have statistical significance.3.The expression of STAT1 protein was increased in the model group.The expression of p-STAT1 was down-regulated and the proportion of p-STAT1 in the model group was lower than that in the control group.The m RNA level of STAT1 and USP18 was up-regulated and the level of OAS1 m RNA was down-regulated.4.The results showed that JAK inhibitor and HDACi had different effects on the antiviral activity of IFNα-2b in the control group and the model group.JAK inhibitor and HDACi had a greater effect on the control group than that in the model group.Conclusions:1.Long-time low dose IFNα-2b stimulation of Hep G2.2.15 cells could reduce the sensitivity to IFN-α,which is characterized by the inhibition of HBs Ag,HBe Ag and HBV DNA.2.The down-regulation of STAT1 phosphorylation in JAK-STAT signaling pathway leaded to a decrease in anti-HBV activity of IFN-α,which might be a reason for the resistance of HBV to IFN-α. |
