Whole genome expression profiling of hepatitis B virus-transfected cell line reveals the potential targets of anti-HBV drugs.

Hepatitis B virus (HBV) infection is a major health concern world wide, and few effective treatments have been developed. It has recently been reported that inhibiting host-cell proteins can prevent viral infection. The human genome may contain more genes required for HBV infection and replication than the viral genome. A systematic approach to find these potential antiviral targets is by host gene expression analysis using DNA microarrays. The aim of this study was to identify and validate novel cellular anti-HBV targets. The Human Whole Genome Bioarray was used to analyze differentially expressed genes in HepG2.2.15 cells and HepG2 cells. Altered gene expression in HepG2.2.15 cells was studied following treatment with the anti-HBV drug, lamivudine. Genes that were differentially expressed during HBV infection and reversed with anti-HBV drugs were validated by semiquantitative reverse transcription-PCR. Bioinformatics analysis revealed ABHD2, EREG, ACVR2B, CDC34, KHDRBS3 and RORA as potential cellular anti-HBV targets. Antisense oligodeoxynucleotides were used to test the antiviral activity of these potential targets. Results strongly suggested that inhibition of ABHD2 or EREG significantly blocked HBV propagation in HepG2.2.15 cells. This study demonstrates that ABHD2 and EREG are essential for HBV propagation and provides strong evidence that these proteins could be used as potential targets for anti-HBV drugs.

Antisense oligonucleotides targeted against asialoglycoprotein receptor 1 block human hepatitis B virus replication.

Chronic hepatitis B virus (HBV) infection is a major worldwide public health problem. Better therapeutics and treatment strategies are urgently needed because of ineffective clinical treatment. Our previous study showed that asialoglycoprotein receptor 1 (ASGPR1) was upregulated by HBV but downregulated by lamivudine in HepG2.2.15 cells. It has also been reported that ASGPR is a candidate receptor for HBV attachment to hepatocytes. Therefore, as a major subunit of ASGPR, ASGPR1, might be a potential target for anti-HBV drugs. To validate this hypothesis, antisense oligonucleiotides (ASODNs) were used to downregulate ASGPR1 level in HepG2.2.15 cells. By using the MFOLD web server and BLAST searches, five ASODNs theoretically targeting ASGPR1 were selected. After 72 h post-transfection, HBV-DNA level in cell medium were examined by real-time polymerase chain reaction (PCR). Hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg) were detected using enzyme-linked immunosorbent assay (ELISA). ASGPR1 mRNA and protein level were measured by semi-quantitative reverse transcriptase (RT)-PCR and Western blot analysis respectively. The results showed that ASODN2 significantly downregulated ASGPR1 level. It also reduced HBV-DNA, HBsAg and HBeAg level in cell medium as observed with lamivudine. In contrast, the sense sequence and scrambled sequence of ASODN2 had no effect on ASGPR1 and HBV markers in HepG2.2.15 cells. This indicated that ASODN2 could specifically reduce HBV replication in vitro. Additionally, cell proliferation and apoptosis assay suggested that downregulation of ASGPR1 did not affect cell viability. We, therefore, proposed that ASODNs targeted against ASGPR1 could block HBV replication without the influence of other changes, and ASGPR1 could be targeted for anti-HBV drug development.

Hemodynamic effect of norepinephrine and serotonin with and without indomethacin.

Injections of serotonin (5-HT) into the aortic arch of rats caused greater reductions of blood pressure (BP) and less increases in renal vascular resistance (RVR) than that into jugular veins, in contrast to norepinephrine (NE) which tended to cause larger increases in BP and RVR by the intra-aortic route. Indomethacin (INDO) enhanced renal vasoconstrictor responses to NE, but did not affect those of 5-HT. These data support a central nervous system (CNS) hypotensive effect of 5-HT not shared by NE, and a less interaction with vasodilator prostaglandins for 5-HT, when compared with NE.

Serotonin and 5-hydroxytryptophan on blood pressure and renal blood flow in anesthetized rats.

The effects of exogenous (injected) serotonin on mean blood pressure and renal blood flow in male Sprague-Dawley rats were compared with the effects of enhanced endogenous serotonin, accomplished by injections of 5-hydroxytryptophan (5-HTP). Responses were evaluated with and without carbidopa, which blocks peripheral conversion of 5-HTP to serotonin, and with and without indomethacin to assess the contribution of prostanoids. Both serotonin (0.25-1.0 micrograms) and 5-HTP (50-200 micrograms) decreased blood pressure and renal blood flow and increased renal vascular resistance in dose-dependent fashion. Hypotensive responses with both agents were greater after arterial injections into the aortic arch than after intravenous injections (into the jugular vein). Carbidopa had no significant effects on responses to serotonin but ablated the ability of 5-HTP to increase renal vascular resistance and decrease renal blood flow. Carbidopa did not alter the hypotensive action of 5-HTP. Indomethacin had no significant effect on responses to serotonin or 5-HTP. These results support a direct renal vasoconstrictor effect for exogenous and endogenously formed serotonin and a hypotensive effect probably arising in the central nervous system.