| Hepatitis B virus (HBV) is a hepatotropic noncytopathic DNA virus, and about5-10%of adult infections and90-95%of neonatal infections lead to persistent infection, which has a high risk of developing chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). HBV infection cause acute and chronic liver disease and is a main health problem worldwide.Although therapeutical nucleos(t)ide analogs, and interferons were used for HBV-infected people, the number of HBV-infected people and HBV-related death continues to increase. Thus, there is a vital need for the development of new therapy agents or candidate sources.Some cationic host defense peptides (HDPs) were characterized from the venomous gland of scorpions by our group recently. These venom peptides were synthesized and tested the activity against HBV. We found that Mucroporin-M1peptide had the most effectively inhibitory activity against HBeAg and HBsAg. Accordingly, Mucroporin-M1peptide was further selectively studied in this study.In the HBV stably transfected cell line, HepG2.2.15, Mucroprin-M1peptide inhibited the expression of HBsAg, HBeAg and HBV DNA with a dose-dependent manner. HBV intracellular DNA, RNA replication intermediates and protein were also potently inhibited by Mucroporin-M1peptide in a concentration-dependent manner. As Mucroporin-M1had an anti-HBV activity in hepatoma cells, we further examined its anti-HBV activity in HBV infection mouse model. Mucroporin-M1peptide inhibited HBV replication in mice hepatocytes and reduced HBV antigens secreting into mice blood.The preceding results suggested that the active target of Mucroporin-M1was at the viral RNA transcription step and not at the HBV DNA polymerase, as with other anti-HBV nucleos(t)ide analogues. So we examine the effect of Mucroporin-M1on HBV promoter activity. The transcriptional activities of HBV promoters were significantly reduced by Mucroporin-M1. Then EMS A assay was used to confirm wether Mucroporin-M1inhibit the binding between host nuclear transcriptional factors and the HBV promoters, while modulate viral promoter activity. We found that the binding between the HNF4a and HBV preCore/Core promoter was significantly decreased.The inhibitory activity of Mucroporin-M1peptide against HBV transcription was explained by the decreasing of the binding between HNF4a and HBV precore/core promoter. As the result of real-time PCR and Western blot, we found that Mucroporin-M1inhibited the expression of HNF4α in HepG2.2.15with a dose-dependent manner. So the decreasing of HNF4α binding to HBV preCore/Core promoter was resulted from the reduction of the HNF4α expression, and further resulted to inhibition of HBV replication.Further analysis of MAPK pathway by Western Blotting, Mucroporin-M1activated the MAPK pathway, and inhibited the expression of HNF4α, leading to suppress HBV replication. While the upstream inhibitors of the MAPK pathway could inhibit the activation of MAPKs by Mucroporin-M1, both HNF4α expression and HBV replication were recovered. Further more, Mucroporin-M1significantly activated MAPK pathway in HBV infected mouse.Here, we found that a scorpion derived peptide, Mucroporin-M1, activated the mitogen-activated protein kinases exogenous signal-regulated kinase1/2, and c-jun N-terminal kinase, and then inhibited the expression of HNF4α, a transcription factors essential for HBV expression and replication. As the result of HNF4α down-regulation, the transcriptional activities of HBV promoters were significantly reduced. When HBV RNA transcript reduced by Mucroporin-M1peptide, HBV DNA and protein productions were also decreased. In HBV infection mouse model, we found that Mucroporin-M1peptide also activated MAPKs and inhibited HBV replication in mouse hepatocytes. This study found that a scorpion derived activated peptide, Mucroporin-M1, inhibited HBV replication, and demonstrated the anti-HBV mechanism of Mucroporin-Ml in vitro and in vivo is that activating MAPKs, down-regulating of HNF4a, and reducing the transcriptional activities of HBV promoters. |
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