| Chronic hepatitis B is a serious public health risk for human health problem. China is a high HBV endemic area, and the rate of hepatitis B surface antigen (HBsAg)-positive is about 9%, and the HBV carriers are about 110-120 million, some of which got varying degrees of liver cell damage and may develop into cirrhosis of the liver and liver cancer.Currently, clinical anti-HBV drugs are: interferons, nucleoside/nucleoside analogues and immunomodulators. IFN is a cytokine secreted by lymphocytes after infected by virus or bacteria, and has a variety of functions such as anti-virus, anti-tumor and immune regulation. In accordance with immunogenicity the different molecular structures, IFNs can be divided intoα,β,γcategories, and the antiviral effect of IFN-αis strongest.Interferons (IFNs) are a family of soluble glycoproteins produced by different cells and can be divided intoα,β,γcategories. IFN-αis one of anti-HBV drugs commonly used in clinic, and exhibits antiviral and immunomodulation functions.Conventional IFN-αhas been used for the treatment of CHB for many decades. However, it also has some shortcomings, such as short half-life (4~6h), which makes patients have to accept frequent injections for at least half a year. In order to overcome these shortcomings, rHSA-IFNα-2b was developed, whose half-life is much longer than that of IFNα-2b, and even longer than that of PEG-IFNα. The reduction of injection frequency would greatly facilitate patient who need long period treatment. At the same time, long-acting interferon provided a more stable plasma concentration, which will also improve efficacy and reduce side effects.Animal experiments and clinical trials have proved rHSA-IFN to be an effective anti-HCV drug. However, whether it can reach the same anti-HBV effect, will be further research. In this study, we are trying to investigate its anti-HBV effect and some mechanisms in vitro and in vivo, and to provide information for the clinical research of rHSA-IFNα-2b in CHB therapy.IFN-αtriggers a series of signal transduction events induced by the binding of IFN-αto its cell surface receptors, and a series of enzymes are actived and produce a group of anti-viral protein, including 2'-5'-oligoadenylate synthetase (OAS), phosphodiesterase (PDE) and protein kinase. OAS can prevent virus reproduction in host cells by catalyzing oligonucleotide synthesis, activating endogenous endonuclease, and inhibiting the information transmission of virus mRNA. PDE could end virus tRNA degradation; and protein kinase can inhibit protein synthesis and viral replication. At the same time, in order to explore its anti-HBV mechanism, this study was to examine the effects of rHSA-IFNα-2b on antiviral protein OAS, and on the relationship of Jak-Stat pathway and p38-MAPK pathway.(A) Pharmacodynamics in vitroHepG2 2.2.15 cells were selected as the model of hepatitis B in vitro. Firstly, we study the toxicity of rHSA-IFNα-2b by MTT method. The cells were cultured for 9 days in different concentrations of rHSA-IFNα-2b (500, 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9 nmol/L), then MTT was added, and then DMSO. The absorbance (A) was measured by absorbance microplate reader to examine the degree of cell damage. The results showed that, 2.2.15 cells were slightly damaged by rHSA-IFNα-2b, but damage rate had no significant direct relationship with changes of morphology, and TC50>500 nmol/L, well above its effective dose.Subsequently, the 2.2.15 cells were cultured in three different concentrations of rHSA-IFNα-2b (0.075, 0.3 and 1.2 nmol/L) for 9 days, and the culture medium was replaced every three days. HBsAg and HBeAg in cell supernatants were detected using ELISA kits, and the copies of HBV DNA were detected by real-time fluorescence quantitative PCR. The experimental results showed that, rHSA-IFNα-2b significantly inhibited secretion of HBsAg and HBeAg, and there was obvious dose-dependent relationship between the secretion of HBsAg with rHSA-IFNα-2b at concentrations from 0.075 to 1.2 nmol/L, but not with HBeAg. And the effect of rHSA-IFNα-2b was no significant difference with that of IFNα-2b. Three days after administration of rHSA-IFNα-2b (0.075,0.3,1.2 nmol/L), the cells were collected and total RNA was extracted by Trizol.The expressions of signal transducers and transactivator 1 (STAT1), IFN-stimulated gene factor 3 (ISGF3) and 2'-5'-Oligoadenylate synthetase 1 (OAS1) in HepG2 2.2.15 cells were detected by RT-PCR. And then the changes of HBsAg, HBV DNA and 2'-5'-OAS were detected after the intervention of p38 inhibitor or JAK inhibitor.The results showed that, three days after the treatment of rHSA-IFNα-2b, the expression of OAS1 in HepG2 2.2.15 cells was increased in a dose-dependent manner; STAT1 and ISGF3 expression also increased, but no dose-dependent manner.rHSA-IFNα-2b could inhibit the secretion of HBsAg and HBV-DNA, increase the expression of 2'-5'-OAS protein. p38 inhibitor had a minor impact on the effects of rHSA-IFNα-2b, JAK inhibitors had great influence, the combination of two inhibitors could produce a more significant impact, but did not block the rHSA-IFNα-2b role completely. This indicated that rHSA-IFNα-2b produces a marked effect primarily through the Jak-Stat pathways, and partly through p38-MAPK pathway.(B) The efficacy study in vivoThe DHBV-infected Anhui ducks were selected as the animal model in vivo. Firstly, the DHBV-positive ducklings were judged by PCR, then rHSA-IFNα-2b (0.25, 0.5, 1.0 nmol/kg) were injected subcutaneously, once a week for 4 weeks. IFNα-2b was the positive control, 0.2 nmol/kg, 3 times a week for 4 weeks. The animals were sacrificed one week after cessation of the treatment, and the livers were obtained immediately after collecting blood from the vena cruralis. The Changes of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum total bilirubin (TBIL) and DHBV DNA were explored, and the duck livers were examined by pathological method; and the expression of liver 2'-5'-OAS was detected by Western Blotting.The results showed that, four weeks after treatment with rHSA-IFNα-2b, the major liver enzymes (ALT, AST and TBIL) were reduced significantly, and ALT was more significantly in a dose-dependent manner. IFNα-2b can significantly reduce the levels of ALT, AST, TBIL, and the ALT level had a rebound at day 35, but not obviously in rHSA-IFNα-2b-treated groups.rHSA-IFNα-2b could markedly decrease the concentrations of DHBV DNA in the duckling model, and was dose-dependent after 4 weeks` treatment. All animals in the IFNα-2b-treated group showed a reduction in DHBV DNA levels over the course of treatment, with less potency than those treated with rHSA-IFNα-2b.Histopathological profiles of the liver from the control group ducks revealed necrosis, inflammatory cell infiltration and massive ballooning degeneration of the hepatic cytoplasm. Administrations of rHSA-IFNα-2b to the experimental animals showed a dose-dependent improvement of the hepatocellular architecture over the control group. rHSA-IFNα-2b resulted in more obvious improvements than that IFNα-2b did.rHSA-IFNα-2b could increase the expression of 2'-5'-OAS protein in liver cell remarkably, and showed significant dose-dependent manner.In Conclusion, rHSA-IFNα-2b can effectively inhibit the proliferation of HBV in vitro and in vivo, and it is mainly through the Jak-Stat pathways, and partly through p38-MAPK pathway, to increase the secretion of OAS. |
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