Effects Of Hepatic Endoplasmic Reticulum Stress On HBV Replication And The Antiviral Activity Of Entecavir

Objective: Endoplasmic reticulum stress(ERS)is a common pathological phenomenon in many liver diseases,which can be induced by factors such as hepatitis B virus(HBV).In previous studies,we found that hepatocyte ERS significantly reduced the level of extracellular HBV DNA and related antigens.However,the impact of ERS on intracellular HBV DNA replication and antigen synthesis is still unclear.The purpose of this project is to explore the effect of ERS on HBV replication and the antiviral activity of entecavir(ETV).Methods: In this study,HBV transfected cells HepG2.2.15 was employed to explore the effects of thapsigargin(TG)-induced ER stress on HBV replication and the antiviral activity of ETV.The cell apoptosis rate was detected by flow cytometry.The expression of glucose regulated protein 78(GRP 78),phosphorylated eukaryotic translation initiation factor 2α(p-eIF2α)and ER degradation-enhancing α-mannosidase-like protein(EDEM)were detected by the method of western blotting(WB).The transcription level of GRP78,inositol requiring enzyme-1(IRE1),activating transcription factor-6(ATF6)and protein kinase R-like ER kinase(PERK)were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR).HBV DNA,HBsAg and HBeAg in supernatant were determined by the methods of real-time fluorescent polymerase chain reaction(PCR),enzyme-linked immunosorbent assay(ELISA)and immunoassay of chemiluminescence particles.Intracellular HBV DNA,HBsAg and their related mRNA were measured by PCR,WB,qRT-PCR to investigate the effect of TG-induced ERS on HBV replication.Transcription factors,such as farnesoid X receptor(FXR),hepatocyte nuclear factor 1(HNF1),nuclear factor kappa B(NF-κB),et al,which can stimulate or suppress HBV genome transcription and replication,were detected by qRT-PCR.Subsequently,ETV was used to intervene TG-induced ERS andto observe the effect of TG-induced ERS on the antiviral effect of ETV.Results:1.Flow cytometry showed that 0.5 M TG did not induce apoptosis in HepG2.2.15 cells for 24 h,48 h and 72 h.2.qRT-PCR results showed that the mRNAs of GRP78,PERK,ATF6 and IRE1 were significantly up-regulated after the treatment of 0.5μM TG on HepG2.2.15 cells.WB results showed that GRP78,EDEM and p-eIF2α were also increased after TG treatmen.The above results confirmed the TG significantly induced ERS in HepG2.2.15 cells and activated ER-associated degradation pathway(ERAD).3.HBsAg and HBeAg in supernatant were significantly reduced following TG treatment.On the contrary,the intracellular HBV mRNAs,HBV DNA,HBsAg and HBcAg were significantly increased following TG treatment.These data strongly suggested that ERS of hepatocyte induced by TG promoted intracellular HBV DNA and HBsAg accumulation,which may be associated with enhanced HBV replication and impaired HBV DNA and HBsAg secretion.4.After we treated HepG2.2.15 cells for 48 h,we found that only FXR was up-regulated among 11 transcription factors,while the others were down-regulated to different degrees.Transcription factor NF-κB,which has been reported inhibiting HBV replication,was significantly downregulated.5.10μM ETV was used for 4 days to observe the effect of TG induced ERS on the antiviral effect of ETV.The results showed that the ETV significantly inhibited HBV DNA,HBeAg and HBsAg in the supernatant,as well as the intracellular HBV DNA,3.5kb mRNA,HBV-s mRNA,HBsAg and HBcAg protein in no TG treated HepG2.2.15 cells.In TG treated HepG2.2.15 cells,ETV also significantly inhibited the intracellular and extracellular HBV DNA and HBV-related proteins.However,intracellular HBV DNA,3.5kb mRNA,HBV-s mRNA,HBsAg and HBcAg protein were significantly higher in TG treated HepG2.2.15 cells than that of no TG treated HepG2.2.15 cells after ETV treatment.6.After inhibiting HBV DNA and decreasing HBV-related antigen by ETV for 4days,GRP78,PERK,ATF6 and IRE1 mRNA were significantly down-regulated,as were GRP78 protein expression and the eIF2α phosphorylation in TG treated HepG2.2.15 cells.Conclusion: 1.ERS induced by TG has a complex effect on HBV life cycle.On the one hand,it promotes HBV transcription;on the other hand,it inhibits HBV DNA,HBsAg and HBeAg secretion,so as to accumulate HBV DNA and HBsAg in cells.2.The enhancement of HBV replication may be related to the increased promotion of FXR and the decreased inhition of NF-κB on HBV replication.3.TG-induced HepG2.2.15 cells ERS prolonged the time for ETV to clear intracellular HBV.4.After ETV antivial therapy,ERS can be partially alleviated in TG treated HepG2.2.15 cells.