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Aqueous extract of the edible Gracilaria tenuistipitata inhibits hepatitis C viral replication via cyclooxygenase-2 suppression and reduces virus-induced inflammation.

Chen KJ, Tseng CK, Chang FR, Yang JI, Yeh CC, Chen WC, Wu SF, Chang HW, Lee JC - PLoS ONE (2013)

Bottom Line: AEGT synergistically enhanced interferon-α (IFN-α) anti-HCV activity in a combination treatment.We found that AEGT also significantly suppressed virus-induced cyclooxygenase-2 (COX-2) expression at promoter transactivation and protein levels.Notably, addition of exogenous COX-2 expression in AEGT-treated HCV replicon cells gradually abolished AEGT anti-HCV activity, suggesting that COX-2 down-regulation was responsible for AEGT antiviral effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
Hepatitis C virus (HCV) is an important human pathogen leading to hepatocellular carcinoma. Using an in vitro cell-based HCV replicon and JFH-1 infection system, we demonstrated that an aqueous extract of the seaweed Gracilaria tenuistipitata (AEGT) concentration-dependently inhibited HCV replication at nontoxic concentrations. AEGT synergistically enhanced interferon-α (IFN-α) anti-HCV activity in a combination treatment. We found that AEGT also significantly suppressed virus-induced cyclooxygenase-2 (COX-2) expression at promoter transactivation and protein levels. Notably, addition of exogenous COX-2 expression in AEGT-treated HCV replicon cells gradually abolished AEGT anti-HCV activity, suggesting that COX-2 down-regulation was responsible for AEGT antiviral effects. Furthermore, we highlighted the inhibitory effect of AEGT in HCV-induced pro-inflammatory gene expression such as the expression of tumour necrosis factor-α, interleukin-1β, inducible nitrite oxide synthase and COX-2 in a concentration-dependent manner to evaluate the potential therapeutic supplement in the management of patients with chronic HCV infections.

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Inhibitory effect of AEGT on HCV protein synthesis, RNA replication and HCV infection.(A) Concentration-dependent reduction of HCV NS5B protein levels in AEGT-treated HCV replicon cells. Ava5 cells were treated with AEGT for 3 days at the indicated concentrations. Treatment with 100 U/ml IFN-α served as a positive control. Equal amounts of protein extracts were subjected to Western blotting with anti-NS5B and anti-GAPDH (a loading control) antibodies. (B) Concentration-dependent reduction of HCV RNA replication in AEGT-treated HCV replicon cells. HCV RNA levels were quantified by qRT-PCR and normalized to cellular gapdh mRNA levels following AEGT treatment for 3 days. Cellular toxicity was evaluated by the MTS assay after 3 days of incubation with AEGT. (C) Concentration-dependent reduction of infectious HCV JFH-1 replication in AEGT-treated Huh-7 cells. After 6 h of JFH-1 infection, Huh7-infected cells were treated with AEGT for 3 days. The levels of intracellular HCV RNA were determined by qRT-PCR following normalization of cellular gapdh mRNA. Results are represented as the mean ± SD (error bar) of three independent experiments. *P<0.05; ** P<0.01.
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pone-0057704-g001: Inhibitory effect of AEGT on HCV protein synthesis, RNA replication and HCV infection.(A) Concentration-dependent reduction of HCV NS5B protein levels in AEGT-treated HCV replicon cells. Ava5 cells were treated with AEGT for 3 days at the indicated concentrations. Treatment with 100 U/ml IFN-α served as a positive control. Equal amounts of protein extracts were subjected to Western blotting with anti-NS5B and anti-GAPDH (a loading control) antibodies. (B) Concentration-dependent reduction of HCV RNA replication in AEGT-treated HCV replicon cells. HCV RNA levels were quantified by qRT-PCR and normalized to cellular gapdh mRNA levels following AEGT treatment for 3 days. Cellular toxicity was evaluated by the MTS assay after 3 days of incubation with AEGT. (C) Concentration-dependent reduction of infectious HCV JFH-1 replication in AEGT-treated Huh-7 cells. After 6 h of JFH-1 infection, Huh7-infected cells were treated with AEGT for 3 days. The levels of intracellular HCV RNA were determined by qRT-PCR following normalization of cellular gapdh mRNA. Results are represented as the mean ± SD (error bar) of three independent experiments. *P<0.05; ** P<0.01.

Mentions: Initially, we assessed the effect of AEGT on HCV protein synthesis in Ava5 cells harboring an HCV subgenomic replicon [31]. HCV protein levels were determined by Western blotting using anti-HCV NS5B antibody. We found that different concentrations (100, 200, 400, 600, 800 and 1000 µg/ml) of AEGT markedly inhibited HCV protein synthesis in a concentration-dependent manner 3 days after treatment (Fig. 1A). To verify this finding, we next analyzed the effects of AEGT on HCV RNA replication using qRT-PCR analysis. Consistent with the results of the inhibitory effects of AEGT on viral protein synthesis, AEGT was also found to inhibit HCV RNA replication in a concentration-dependent manner, with an EC50 value of 300±0.3 µg/ml, as normalized by cellular gapdh mRNA (Fig. 1B). To rule out the possibility that AEGT anti-viral activity was caused by cytotoxic effects, cell proliferation was analyzed using the MTS assay. As shown in Fig. 1B, no significant cytotoxicity was detected at high AEGT concentrations (up to 1000 µg/ml). Using an HCV JFH-1 infection system [32], we confirmed the anti-HCV activity of AEGT, with an EC50 value of 325±0.7 µg/ml (Fig. 1C).


Aqueous extract of the edible Gracilaria tenuistipitata inhibits hepatitis C viral replication via cyclooxygenase-2 suppression and reduces virus-induced inflammation.

Chen KJ, Tseng CK, Chang FR, Yang JI, Yeh CC, Chen WC, Wu SF, Chang HW, Lee JC - PLoS ONE (2013)

Inhibitory effect of AEGT on HCV protein synthesis, RNA replication and HCV infection.(A) Concentration-dependent reduction of HCV NS5B protein levels in AEGT-treated HCV replicon cells. Ava5 cells were treated with AEGT for 3 days at the indicated concentrations. Treatment with 100 U/ml IFN-α served as a positive control. Equal amounts of protein extracts were subjected to Western blotting with anti-NS5B and anti-GAPDH (a loading control) antibodies. (B) Concentration-dependent reduction of HCV RNA replication in AEGT-treated HCV replicon cells. HCV RNA levels were quantified by qRT-PCR and normalized to cellular gapdh mRNA levels following AEGT treatment for 3 days. Cellular toxicity was evaluated by the MTS assay after 3 days of incubation with AEGT. (C) Concentration-dependent reduction of infectious HCV JFH-1 replication in AEGT-treated Huh-7 cells. After 6 h of JFH-1 infection, Huh7-infected cells were treated with AEGT for 3 days. The levels of intracellular HCV RNA were determined by qRT-PCR following normalization of cellular gapdh mRNA. Results are represented as the mean ± SD (error bar) of three independent experiments. *P<0.05; ** P<0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585194&req=5

pone-0057704-g001: Inhibitory effect of AEGT on HCV protein synthesis, RNA replication and HCV infection.(A) Concentration-dependent reduction of HCV NS5B protein levels in AEGT-treated HCV replicon cells. Ava5 cells were treated with AEGT for 3 days at the indicated concentrations. Treatment with 100 U/ml IFN-α served as a positive control. Equal amounts of protein extracts were subjected to Western blotting with anti-NS5B and anti-GAPDH (a loading control) antibodies. (B) Concentration-dependent reduction of HCV RNA replication in AEGT-treated HCV replicon cells. HCV RNA levels were quantified by qRT-PCR and normalized to cellular gapdh mRNA levels following AEGT treatment for 3 days. Cellular toxicity was evaluated by the MTS assay after 3 days of incubation with AEGT. (C) Concentration-dependent reduction of infectious HCV JFH-1 replication in AEGT-treated Huh-7 cells. After 6 h of JFH-1 infection, Huh7-infected cells were treated with AEGT for 3 days. The levels of intracellular HCV RNA were determined by qRT-PCR following normalization of cellular gapdh mRNA. Results are represented as the mean ± SD (error bar) of three independent experiments. *P<0.05; ** P<0.01.
Mentions: Initially, we assessed the effect of AEGT on HCV protein synthesis in Ava5 cells harboring an HCV subgenomic replicon [31]. HCV protein levels were determined by Western blotting using anti-HCV NS5B antibody. We found that different concentrations (100, 200, 400, 600, 800 and 1000 µg/ml) of AEGT markedly inhibited HCV protein synthesis in a concentration-dependent manner 3 days after treatment (Fig. 1A). To verify this finding, we next analyzed the effects of AEGT on HCV RNA replication using qRT-PCR analysis. Consistent with the results of the inhibitory effects of AEGT on viral protein synthesis, AEGT was also found to inhibit HCV RNA replication in a concentration-dependent manner, with an EC50 value of 300±0.3 µg/ml, as normalized by cellular gapdh mRNA (Fig. 1B). To rule out the possibility that AEGT anti-viral activity was caused by cytotoxic effects, cell proliferation was analyzed using the MTS assay. As shown in Fig. 1B, no significant cytotoxicity was detected at high AEGT concentrations (up to 1000 µg/ml). Using an HCV JFH-1 infection system [32], we confirmed the anti-HCV activity of AEGT, with an EC50 value of 325±0.7 µg/ml (Fig. 1C).

Bottom Line: AEGT synergistically enhanced interferon-α (IFN-α) anti-HCV activity in a combination treatment.We found that AEGT also significantly suppressed virus-induced cyclooxygenase-2 (COX-2) expression at promoter transactivation and protein levels.Notably, addition of exogenous COX-2 expression in AEGT-treated HCV replicon cells gradually abolished AEGT anti-HCV activity, suggesting that COX-2 down-regulation was responsible for AEGT antiviral effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
Hepatitis C virus (HCV) is an important human pathogen leading to hepatocellular carcinoma. Using an in vitro cell-based HCV replicon and JFH-1 infection system, we demonstrated that an aqueous extract of the seaweed Gracilaria tenuistipitata (AEGT) concentration-dependently inhibited HCV replication at nontoxic concentrations. AEGT synergistically enhanced interferon-α (IFN-α) anti-HCV activity in a combination treatment. We found that AEGT also significantly suppressed virus-induced cyclooxygenase-2 (COX-2) expression at promoter transactivation and protein levels. Notably, addition of exogenous COX-2 expression in AEGT-treated HCV replicon cells gradually abolished AEGT anti-HCV activity, suggesting that COX-2 down-regulation was responsible for AEGT antiviral effects. Furthermore, we highlighted the inhibitory effect of AEGT in HCV-induced pro-inflammatory gene expression such as the expression of tumour necrosis factor-α, interleukin-1β, inducible nitrite oxide synthase and COX-2 in a concentration-dependent manner to evaluate the potential therapeutic supplement in the management of patients with chronic HCV infections.

Show MeSH
Related in: MedlinePlus