Fatty Acids Inhibits HBV Replication And The Secretions Of HBsAg Via Induction Of Endoplasmic Reticulum Stress Of Hepatocytes In Vitro

Objective: Hepatitis B virus(HBV)infection is a serious public health problem. Patients with chronic hepatitis B and nonalcoholic fatty liver disease have became more and more common. However, the effects of fatty liver on HBV replication, HBV related viral antigen secretion and the involved mechanism are not currently understood. Endoplasmic reticulum stress(ER stress) is a common pathological and physiological phenomenon in various liver diseases. The aims of this study are to establish a cell model of fatty liver with HBV replication, and to explore the possible effects and mechanism of fatty liver on HBV replication.Methods: Hep G2.2.15 cell with HBV replication was used in this study. Stearic acid(SA) and oleic acid(OA)were used to induce Hep G2.2.15 cell steatosis. The viability of Hep G2.2.15 cells was detected by MTS method. The lipid droplets within the cells were stained by oil red O-hematoxylin staining method. Intracellular triglyceride(TG) level was deteced by GPO-PAP method. The expressions of glucose regulated protein 78(GRP 78), eukaryotic initiation factor 2α(e IF2α), PKR-like ER kinase(PERK), sterol regulatory element binding protein 1c(SREBP1c)were detected by the method of western blotting(WB). HBV DNA in the supernatant was detected by real-time reverse transcriptase polymerase chain reaction(RT-PCR) method, HBs Ag levels in the supernatant were determinted by the method of enzyme-linked immunosorbent assay(ELISA). We also observed the effects of SA on HBV replication after using4-phenylbutyric acid(PBA)and GRP78 small interfering RNA(GRP78 si RNA) to alleviate ER stress and inhibit GRP78 expression, repectively.Results:1、 Fatty acids induced stestosis in Hep G2.2.15 cells. 100μM SA and 0.2m M OA significantly increased intracellular lipid droplets and TG level in Hep G2.2.15 cells.2、 Fatty acids induced ER stress in Hep G2.2.15 cells. SA at the concentrations of 50μM, 100μM, 200μM and OA at the concentrations of 0.1m M, 0.2m M, 0.4m M dose dependently increased GRP 78 expression in Hep G2.2.15 cells. 100μM SA and 0.2m M OA also significantly induced GRP 78 and SREBP1 c expression and the phosphorylation of PERK,e IF2α from 24 to 72 h.3、 Fatty acids inhibits the secretions of HBs Ag. 50μM, 100μM, 200μM SA and 0.1m M, 0.2m M, 0.4m M OA significantly inhibited HBs Ag secretion in Hep G2.2.15 cells at 48h. 200μM SA also significantly inhibited HBe Ag secretion. 100μM SA and 0.2m M OA significantly inhibited HBs Ag secretion in Hep G2.2.15 cells from 24 to 72 h. HBe Ag secretion was also inhibited by 100μM SA or 0.2m M OA from 24 to 72 h, but their inhibitory effects were not significantly.4、 Fatty acids inhibits HBV DNA replication. 50μM, 100μM, 200μM SA and 0.1m M, 0.2m M, 0.4m M OA significantly decreased HBV DNA leves in Hep G2.2.15 cells at 48h. 100μM SA and 0.2m M OA also significantly inhibited HBV DNA replication in Hep G2.2.15 cells from 24 to 72 h.5、 Fatty acids inhibits HBV replication and the secretions of HBs Ag via induction of ER stress. PBA partly alleviated ER stress induced by SA in Hep G2.2.15 cells. HBV replication and HBs Ag secretion inhibited by SA can also be significatly restored by PBA. These results indicated that fatty acids inhibited HBV replication and the secretions of HBs Ag via induction of ER stress.6、 GRP78 si RNA inhibited SA induced GRP78 expression, however, we observed that after inhibiting GRP78 indcued by GRP78 si RNA, the HBV DNA and HBs Ag were still inhibited in Hep G2.2.15 cells by SA. These results indicated that the inhibitory effects of SA on HBV DNA replication and HBs Ag secretion were not depended on induction of GRP78 expression.Conclusion: Fatty acids inhibits HBV replication and the secretions of HBs Ag via induction of ER stress of hepatocytes. This inhibitory effects are not depended on induction of GRP78 expression.