| Hepatitis B virus （HBV） is a hepatotropic, non-cytopathic DNA virus （3.2kb partially double-stranded DNA） that causes acute and chronic hepatitis. Patients with a persistent infection of HBV are at a high risk of developing chronic hepatitis, cirrhosis, and/or hepatocellular carcinoma. The interactions between HBV replication and immune responses against HBV infection play an important role in determining the outcome of virus infection. It has been shown that cytokines are likely to be involved in both the regulation of the immune responses and the direct inhibition of HBV replication. Several cytokines effectively suppress HBV replication in a noncytopathic manner in a cell culture system and in HBV transgenic mice. Interleukin-6effectively suppresses HBV replication and prevents the accumulation of HBV covalently closed circular DNA by preventing the formation of genome-containing nucleocapsids in a human hepatoma cell line. Interleukin-12, inteiieukin-18and intrahepatic induction of alpha/beta interferon （IFN-α/β） effectively inhibit HBV replication in the liver of transgenic mice. Furthermore, interleukin-4and transforming growth factor beta-1（TGF-β1） suppress HBV replication in hepatoma cells through the transcriptional regulation of HBV RNA. These studies suggest that inflammatory cytokines play an important role in the antiviral response against HBV infection. T helper17（Th17） cells arc a newly identified subset of T helper cells and participate in the disease progression and pathogenesis of liver injury in hBV infected patients. The functions oF th17cells are mediated via the production of several cytokines including interleukin-17A （IL-17A） and interleukin-22（IL-22）. Th17cells were reported to have a rapid increase with the entecavir-induced suppression of HBV replication and plasm IL-17A levels and Th17frequency negatively correlated with plasma HBV DNA load in patients with chronic HBV infection.We therefore hypothesized that IL-17A and IL-22might have a potent suppressive effect on HBV replication. In our present study, we analyzed the suppressive effect of IL-17A and IL-22on HBV replication in the hepatocellular carcinoma cell line HepG2.2.15.Part I Suppressive Effect of IL17on the Replication of HBV in HepG2.2.15CellsObjectives:To study the suppressive effect of IL17on the replication of hepatitis B virus in HepG2.2.15.Materials and method:HepG2.2.15cells were separately incubated with culture medium containing different concentration of IL17or anti-IL17R antibody （Ab）. Cell proliferation was determined with the Cell Counting Kit-8（CCK-8） assay. The amount of HBsAg and HBeAg in the culture medium at day3and day5was measured using the electrochemiluminescence assay. The amount ofintracellular and extracellular HBV DNA was measured by real-time PCR.Results:1. The proliferation of HepG2.2.15cells was not significantly inhibited or enhanced by IL17or anti-IL17R Ab （Tab.1-2, P>0.05）.2. In the IL-17-treated cells, the levels of intracellular HBV DNA decreased than control （6.79±0.15,7.14±0.13, P<0.05）. At5d, the levels of HBsAg in culture medium decreased in the IL-17-treated cells than control （7.56±0.60,9.19±1.20, P<0.05）. At3d and5d, the levels of HBeAg in culture medium decreased in the IL-17-treated cells than control （34.90±3.68,43.10±5.46, P<0.05;24.92±6.18,43.95±6.64, P<0.05）.3.In the anti-IL-17R Ab-treated cells, the levels of intracellular HBV DNA increased than control （7.47±0.16,7.14±0.13, P<0.05）. At3d and5d, the levels of extracellular HBV DNA in culture medium increased in the anti-IL-17R Ab-treated cells than control （5.00±0.21,4.47±0.34,P<0.05;5.10±0.21,4.57±0.38, P<0.05）. At3d, the levels of HBsAg in culture medium increased in the anti-IL-17R Ab-treated cells than control （12.78±0.96,10.72±0.95, P<0.05）.4. The levels of all indexes did not differ significantly among various dose groups of IL17（Tab1-6, P>0.05）. At day5, IL-17decreased the level of HBsAg, but at day3, IL-17did not significantly suppress HBsAg compared with HBsAg in the control. Additionally, IL-17suppressed HBeAg43.31%at day5, but only19.04%at day3.Conclusions:IL-17could effectively inhibit HBV replication in a noncytopathic manner. The suppressive effect of IL-17is not dose-dependent within the concentration range of250pg/ml to4ng/ml, but the suppressive effect of IL-17was time-dependent. Our findings highlight the utility of IL-17as a novel and potentially effective drug for viral suppression.Part Ⅱ Suppressive Effect of IL22on the Replication of HBV in HepG2.2.15CellsObjectives:To study the suppressive effect of IL22on the replication of hepatitis B virus in HepG2.2.15.Materials and method:HepG2.2.15cells were separately incubated with culture medium containing different concentration of IL22or anti-IL22R Ab. Cell proliferation was determined with CCK-8assay. The amount of HBsAg and HBeAg in the culture medium at day3and day5was measured using the electrochemiluminescence assay. The amount of intracellular and extracellular HBV DNA was measured by real-time PCR.Results:1. The proliferation of HepG2.2.15cells was not significantly inhibited or enhanced by IL22or anti-IL22R antibody （Tab.2-1,P>0.05）.2. The levels of intracellular HBV DNA did not significantly differ between the IL-22-treated group and the control group （7.28±0.26,7.14±0.13,P>0.05） or between the anti-IL-22R Ab-treated group and the control group （7.24±0.17,7.14±0.13, P>0.05）. 3. At3d and5d, the levels of extracellular HBV DNA in culture medium did not significantly differ between the IL-22-treated group and the control group （4.87±0.63,4.47±0.34, P>0.05;4.68±0.70,4.57±0.38, P>0.05） or between the anti-IL-22R Ab-treated group and the control group （4.70±0.50,4.47±0.34, P>0.05;4.77±0.33,4.57±0.38, P>0.05）.4. At3d and5d, the levels of HBsAg in culture medium did not significantly differ between the IL-22-treated group and the control group （11.07±1.15,10.72±0.95, P>0.05;8.99±0.42,9.19±1.20, P>0.05） or between the anti-IL-22R Ab-treated group and the control group （9.80±1.50,10.72±0.95, P>0.05;10.27±1.24,9.19±1.20, P>0.05）.5. At3d and5d, the levels of HBeAg in culture medium did not significantly differ between the IL-22-treated group （45.42±6.80,43.10±5.46, P>0.05;37.80±7.55,43.95±6.64,P>0.05） and the control group or between the anti-IL-22R Ab-treated group and the control group （40.06±4.29,43.10±5.46, P>0.05;39.45±6.99,43.95±6.64, P>0.05）.The levels of intracellular HBV DNA, extracellular HBV DNA, HBsAg and HBeAg in culture medium all did not significantly differ between the IL-22-treated group and the control group or between the anti-IL-22R Ab-treated group and the control group.Conclusions:IL22has no directly suppressive effect on replication of HBV.Part Ⅲ Suppressive effect of the antiviral protein related with IL17on replication of HBV.Objectives:To observe the effect of IL17on the transcription of some genes and its relationship with Ill7’s anti-HBV activity in HepG2.2.15Cells.Materials and Methods:With or without IL17treatment, the mRNA levels of myxovirus resistance AMxA, IFN-stimulated gene factor3（ISGF3）, oligoadenylate synthetase （OAS）, signal transducer and activator of transcription1（STAT1）, and signal transducer and activator of transcription2（STAT2） were quantified by real-time RT-PCR using the LightCycler480. The effect of MxA and OAS gene being silenced by siRNA or not were assessed by quantifying intracellular HBV DNA by realtime PCR.Results:1. Ratio of MxA and OAS mRNA levels of IL-17-treated group to control were16.56（P<0.05） and19.88（P<0.05）. Ratio of STAT1, STAT2and ISGF3, mRNA levels of IL-17-treated group to control were1.67（P>0.05）,1.98（P>0.05） and1.93（P>0.05）. The expression of MxA and OAS increased in the IL-17A-treated group than the control group. The expression of ISGF3, STAT1and STAT2did not significantly differ between the IL-17A-treated group and the control group.2. MxA siRNA can effectively silence MxA gene transcription with a ratio up to82.63%. The amount of intracellular HBV DNA increased in the MxA-targeted siRNA group than the control （7.09±0.21,6.76±0.28,P<0.05）.3. OAS siRNA can effectively silence OAS gene transcription with a ratio up to81.07%. The amount of intracellular HBV DNA increased in the OAS-targeted siRNA group than the control （7.19±0.13,6.76±0.28, P<0.05）.Conclusions:MxA-targeted siRNA and OAS-targeted siRNA abolished the suppressive effect of IL-17on IIBV replication. MxA and OAS are two important antiviral proteins and may be involved in the suppression of HBV replication by IL-17A. |
PDF Full Text Request