Effects of a combination of phenolic CATs and various inhibitors on HCV replication.

<p>Additive antiviral effect of the EC isomers combined with either (A) IFN-α or the viral enzyme inhibitors (B) telaprevir and (C) NM-107. Ava5 cells were treated with (a) (+)-EC or (b) (−)-EC in combination with each inhibitor at the indicated concentrations for 3 days. HCV RNA levels were quantified by qRT-PCR and normalized to <i>gapdh</i> mRNA levels. The efficacy of inhibition is expressed as the percentage relative to the RNA levels quantified without ECs. Error bars represent the SD from three experiments. Asterisks indicate a significant difference compared with single-compound treatment. *P<0.05; **P<0.01.</p

Green Tea Phenolic Epicatechins Inhibit Hepatitis C Virus Replication via Cycloxygenase-2 and Attenuate Virus-Induced Inflammation

<div><p>Chronic hepatitis C virus (HCV) infection is the leading risk factor for hepatocellular carcinoma (HCC) and chronic liver disease worldwide. Green tea, in addition to being consumed as a healthy beverage, contains phenolic catechins that have been used as medicinal substances. In the present study, we illustrated that the epicatechin isomers (+)-epicatechin and (−)-epicatechin concentration-dependently inhibited HCV replication at nontoxic concentrations by using <em>in vitro</em> cell-based HCV replicon and JFH-1 infectious systems. In addition to significantly suppressing virus-induced cyclooxygenase-2 (COX-2) expression, our results revealed that the anti-HCV activity of the epicatechin isomers occurred through the down-regulation of COX-2. Furthermore, both the epicatechin isomers additively inhibited HCV replication in combination with either interferon-α or viral enzyme inhibitors [2′-C-methylcytidine (NM-107) or telaprevir]. They also had prominent anti-inflammatory effects by inhibiting the gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and inducible nitrite oxide synthase as well as the COX-2 in viral protein-expressing hepatoma Huh-7 cells. Collectively, (+)-epicatechin and (−)-epicatechin may serve as therapeutic supplements for treating HCV-related diseases.</p> </div

Concentration-dependent restoration of EC-reduced HCV protein synthesis and RNA replication by extraneous COX-2 expression.

<p>Ava5 cells were transfected with the indicated amounts of the COX-2 expression plasmid pCMV-COX-2-Myc encoding <i>cox-2</i> for 6 h, followed by either (A) (+)-EC or (B) (−)-EC treatment at a concentration of 75 µM for 3 days. “Mock” indicated transfection of control vector pcDNA4/<i>myc</i>-His-A in the presence of 0.1% DMSO. (a) Cell lysates were subjected to Western blotting with anti-NS5B, anti-COX-2, and anti-GAPDH antibodies to evaluate protein expression levels. (b) Total RNAs were subjected to qRT-PCR to evaluate HCV RNA levels. Error bars represent the SD from three experiments. (C) Reduction of HCV protein synthesis by COX-2 gene knockdown. Ava5 cells were transfected with either different amounts (0.5–2 µg) of the COX-2 shRNA or 2 µg of LacZ shRNA vectors as a control group. After 3 days of incubation, cell lysates were prepared for Western blotting with anti-COX-2 and anti-NS5B antibodies.</p

Anti-HCV activity of phenolic CATs.

<p>Inhibitory effect of various phenolic CATs on HCV (A) protein synthesis and (B) RNA replication. Concentration-dependent reduction of HCV (C) protein synthesis and (D) RNA replication in (+)-EC- or (−)-EC-treated HCV replicon cells. Ava5 cells were treated with each CAT isomers [(+)-CAT, (−)-CAT, (+)-EC and (−)-EC] at the indicated concentrations (25, 50, and 75 µM). After 3 days, the cell lysates were collected and then subjected to Western blotting with antibodies against NS5B and GAPDH (loading control). IFN-α (100 U/mL) and DMSO (0.1%) treatment served as the positive and mock controls, respectively. HCV RNA levels was quantified by qRT-PCR and normalized to <i>gapdh</i> mRNA levels after CAT isomers treatment for 3 days. Cellular toxicity was evaluated by the MTS assay after 3 days in the presence of the indicated concentrations of each EC isomers. (E) Concentration-dependent reduction of infectious HCV JFH-1 replication in (+)-EC- or (−)-EC-treated Huh-7.5 cells. After 6 h of JFH-1 virus incubation, Huh-7.5-infected cells were treated with EC isomers for 3 days. The levels of intracellular HCV RNA were determined by qRT-PCR following normalization of cellular <i>gapdh</i> mRNA. The efficacy of inhibition is expressed as the percentage relative to the RNA levels quantified without ECs (mock control). (F) Time-dependent reduction of HCV RNA levels in Ava5 cells treated with EC isomers. Ava5 cells were treated with EC isomers at concentration of 25, 50 and 75 µM. HCV RNA levels were quantified by qRT-PCR after EC isomers treatment for 3, 6 and 9 days. DMSO (0.1%) treatment served as the mock controls. Error bars represent the SD from three experiments. *P<0.05; **P<0.01.</p

Inhibitory effect of the EC isomers on HCV-induced COX-2 expression.

<p>(A) The EC isomers reduce HCV-induced COX-2 gene promoter activity. Huh-7 cells or Ava5 were transfected with the pCOX-2-Luc reporter plasmid encoding firefly luciferase under control of the COX-2 promoter. After treatment with serial diluted concentrations of the EC isomers for 3 days, cell lysates were subjected to luciferase activity assays. The basal level of COX-2 promoter activity was identified in Huh-7 cells transfected with pCOX-2-Luc without EC isomers treatment, which is defined as 1 (B) The EC isomers reduce COX-2 protein expression. Ava5 cells were treated with the EC isomers at the indicated concentrations for 3 days. Cell lysates were subjected to Western blotting with anti-COX-2 and anti-GAPDH antibodies. (C) Concentration-dependent reduction of infectious HCV JFH-1-induced COX-2 gene promoter activity by EC isomers in Huh-7 cells. The pCOX-2-Luc transfected Huh-7 cells were incubated with JFH-1 virus for 6 h in the absence or presence of increasing concentrations of the EC isomers. After 3 days incubation, cell lysates were subjected to luciferase activity assays. (D) The EC isomers reduced COX-2 protein expression in JFH-1-infected Huh-7 cells. The JFH-1-infected Huh-7 cells were treated with the EC isomers at the indicated concentrations for 3 days. Cell lysates were subjected to Western blotting with anti-COX-2 and anti-GAPDH antibodies. Error bars represent the SD from three experiments. Asterisks indicate a significant difference compared with EC-untreated Ava5 or JFH-infected Huh-7 cells. *P<0.05; **P<0.01.</p

Anti-HCV activity and cytotoxicity of SFN and its analogs.

Anti-HCV activity and cytotoxicity of SFN and its analogs.</p

HCV NS5A Up-Regulates COX-2 Expression via IL-8-Mediated Activation of the ERK/JNK MAPK Pathway

<div><p>Chronic hepatitis C virus (HCV) infection leads to intrahepatic inflammation and liver cell injury, which are considered a risk factor for virus-associated hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. Inflammatory cytokines are critical components of the immune system and influence cellular signaling, and genetic imbalances. In this study, we found that cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) were significantly induced by HCV infection and HCV NS5A expression, and induction of COX-2 correlated with HCV-induced IL-8 production. We also found that the ERK and JNK signaling pathways were involved in the regulation of IL-8-mediated COX-2 induction in response to HCV infection. Using a promoter-linked reporter assay, we identified that the C/EBP regulatory element within the COX-2 promoter was the dominant factor responsible for the induction of COX-2 by HCV. Silencing C/EBP attenuated HCV-induced COX-2 expression. Our results revealed that HCV-induced inflammation promotes viral replication, providing new insights into the involvement of IL-8-mediated COX-2 induction in HCV replication.</p></div

Sulforaphane Suppresses Hepatitis C Virus Replication by Up-Regulating Heme Oxygenase-1 Expression through PI3K/Nrf2 Pathway

<div><p>Hepatitis C virus (HCV) infection-induced oxidative stress is a major risk factor for the development of HCV-associated liver disease. Sulforaphane (SFN) is an antioxidant phytocompound that acts against cellular oxidative stress and tumorigenesis. However, there is little known about its anti-viral activity. In this study, we demonstrated that SFN significantly suppressed HCV protein and RNA levels in HCV replicon cells and infectious system, with an IC₅₀ value of 5.7 ± 0.2 μM. Moreover, combination of SFN with anti-viral drugs displayed synergistic effects in the suppression of HCV replication. In addition, we found nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 induction in response to SFN and determined the signaling pathways involved in this process, including inhibition of NS3 protease activity and induction of IFN response. In contrast, the anti-viral activities were attenuated by knockdown of HO-1 with specific inhibitor (SnPP) and shRNA, suggesting that anti-HCV activity of SFN is dependent on HO-1 expression. Otherwise, SFN stimulated the phosphorylation of phosphoinositide 3-kinase (PI3K) leading Nrf2-mediated HO-1 expression against HCV replication. Overall, our results indicated that HO-1 is essential in SFN-mediated anti-HCV activity and provide new insights in the molecular mechanism of SFN in HCV replication.</p></div

Survival rate, body weight, and clinical score of ICR suckling mice infected with ZIKV by i.p. and i.c. injection.

Six-day-old ICR suckling mice received 102 to 105 PFU ZIKV by i.c. (n = 5) or i.p. (n = 5) injection. (A–D) Survival rate, (E–H) clinical score, and (I–L) body weight recorded every day. Mice were sacrificed at 8 dpi. Disease severity was scored as follows: 0: healthy, 1: body weight loss and ruffled fur, 2: lethargy and unsteady gait, 3: kinetic tremors and severe ataxia, 4: paralysis, and 5: death.</p

Monocyte infiltration in the brain of ICR suckling mice that received i.c. injection of various doses of ZIKV.

Six-day-old ICR suckling mice received 102 to 105 PFU ZIKV by i.c. or i.p. injection. Mice were sacrificed, and the brain tissues of mice that received ZIKV by (A) i.c. or (B) i.p. were collected and subjected to hematoxylin and eosin staining. The biopsy specimens were examined by microscope. (C, D) The quantitation of monocyte infiltration by IHC assay.</p