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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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AEGT suppression of HCV-induced NF-κB activation.(A) Concentration-dependent reduction of NF-κB promoter-linked luciferase activity in AEGT-treated HCV replicon cells. Ava5 cells were transfected with the reporter plasmid pNF-κB-Luc in the presence of AEGT at indicated concentrations for 3 days. Total cell lysates were prepared for luminescence detection using the Steady-Glo Luciferase Assay Kit (Promega). Non-treated Huh-7 cells served as the basal control, which is defined as 1. (B) Concentration-dependent reduction of nuclear p65 protein levels in AEGT-treated HCV replicon cells. Nuclear extracts were prepared from AEGT-treated Ava5 cells and subjected to Western blot analysis using anti-NF-κB p65 and laminB antibodies, in which lamin B was used as a nuclear fraction control. (C) Concentration-dependent reduction of NF-κB promoter-linked luciferase activity by AEGT in JFH-1-infected Huh-7 cells. After 6 h of JFH-1 infection, Huh-7-infected cells were treated with AEGT at indicated concentrations for 3 days. Non-infected Huh-7 cells served as the basal control, which is defined as 1. Luciferase activity assay was performed as described above. (D) Concentration-dependent reduction of nuclear p65 protein levels by AEGT in JFH-1-infected Huh-7 cells. Western blot analysis was performed as described above. Each value was represented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01.
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pone-0057704-g004: AEGT suppression of HCV-induced NF-κB activation.(A) Concentration-dependent reduction of NF-κB promoter-linked luciferase activity in AEGT-treated HCV replicon cells. Ava5 cells were transfected with the reporter plasmid pNF-κB-Luc in the presence of AEGT at indicated concentrations for 3 days. Total cell lysates were prepared for luminescence detection using the Steady-Glo Luciferase Assay Kit (Promega). Non-treated Huh-7 cells served as the basal control, which is defined as 1. (B) Concentration-dependent reduction of nuclear p65 protein levels in AEGT-treated HCV replicon cells. Nuclear extracts were prepared from AEGT-treated Ava5 cells and subjected to Western blot analysis using anti-NF-κB p65 and laminB antibodies, in which lamin B was used as a nuclear fraction control. (C) Concentration-dependent reduction of NF-κB promoter-linked luciferase activity by AEGT in JFH-1-infected Huh-7 cells. After 6 h of JFH-1 infection, Huh-7-infected cells were treated with AEGT at indicated concentrations for 3 days. Non-infected Huh-7 cells served as the basal control, which is defined as 1. Luciferase activity assay was performed as described above. (D) Concentration-dependent reduction of nuclear p65 protein levels by AEGT in JFH-1-infected Huh-7 cells. Western blot analysis was performed as described above. Each value was represented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01.

Mentions: Several reports have demonstrated that some constituents of Gracilaria possess anti-inflammatory properties [23], [33], [34]. COX-2, a pro-inflammatory enzyme, is linked to HCV-associated liver carcinogenesis [10]. To investigate whether AEGT can inhibit HCV-stimulated COX-2 expression, we analyzed the promoter activity, protein synthesis and enzyme activity of COX-2 in AEGT-treated Ava5 cells. As shown in Fig. 2A, HCV-stimulated COX-2 promoter activity was suppressed by AEGT in a concentration-dependent manner compared with 0.1% DMSO-treated Ava5 and parental Huh-7 cells (the fold of control), which was observed by a COX-2 promoter-linked luciferase reporter assay. These results indicated that AEGT down-regulated COX-2 expression at the mRNA transcription level. AEGT-induced COX-2 reduction was further confirmed by Western blot analysis (Fig. 2B). In addition, AEGT caused a concentration-dependent decrease in COX-2-mediated PGE2 biogenesis (Fig. 2C). Recently, many reports, including our previous studies, have demonstrated that the suppression of virus-induced COX-2 expression inhibits HCV replication [8], [14], [15], [17]. To examine whether the elimination of COX-2 expression was responsible for AEGT inhibition of HCV replication, we transiently overexpressed COX-2 in AEGT-treated Ava5 cells. Ava5 cells were transfected with a control vector or a pCMV-COX-2-Myc vector encoding the cox-2 gene at increasing concentrations of transfected plasmid DNA (0.5, 1, 1.5 and 2 µg). Cells were incubated with AEGT (600 µg/ml), in which HCV-stimulated COX-2 expression and HCV replication were markedly blocked (Fig. 1 and Fig. 2). Western blot analysis revealed that AEGT-inhibited HCV NS5B protein synthesis (Fig. 3A, upper panel, lanes 3–6) was gradually attenuated by the increase in exogenous COX-2-Myc expression (middle panel) compared with the control transfected cells in the absence (lane 1) or presence of AEGT (lane 2). Consistent with previous results, qRT-PCR analysis revealed that exogenous COX-2-Myc augmentation significantly restored the AEGT-reduced HCV RNA levels in a concentration-dependent manner (Fig. 3B). Taken together, these findings suggest that COX-2 reduction was associated with AEGT anti-viral activity. NF-κB is a crucial transcription factor for COX-2 transactivation in response to viral infection and inflammation [17], [35]. To further elucidate whether the AEGT-mediated downregulation of COX-2 was modulated by NF-κB, we performed a luciferase assay specifically mediated via NF-κB activation. Ava5 and Huh-7 cells were transiently transfected with the cis-reporting plasmid pNF-κB-Luc and then incubated with or without AEGT for 3 days. As shown in Fig. 4A, increased NF-κB luciferase activity was significantly suppressed by AEGT in a concentration-dependent manner. Translocation of the NF-κB p65 subunit from the cytoplasm to the nucleus is required for NF-κB activation. We observed that compared with parental Huh-7 cells without AEGT treatment, the high level of virus-induced NF-κB p65 nuclear protein was gradually decreased to the basal level following AEGT treatment (Fig. 4B), suggesting that AEGT blocked HCV replication through the sustained suppression of NF-κB signalling pathways. We further performed the HCV JFH-1 infectious assay to confirm the anti-NF-κB activity of AEGT described above (Fig. 4C and D).


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)

AEGT suppression of HCV-induced NF-κB activation.(A) Concentration-dependent reduction of NF-κB promoter-linked luciferase activity in AEGT-treated HCV replicon cells. Ava5 cells were transfected with the reporter plasmid pNF-κB-Luc in the presence of AEGT at indicated concentrations for 3 days. Total cell lysates were prepared for luminescence detection using the Steady-Glo Luciferase Assay Kit (Promega). Non-treated Huh-7 cells served as the basal control, which is defined as 1. (B) Concentration-dependent reduction of nuclear p65 protein levels in AEGT-treated HCV replicon cells. Nuclear extracts were prepared from AEGT-treated Ava5 cells and subjected to Western blot analysis using anti-NF-κB p65 and laminB antibodies, in which lamin B was used as a nuclear fraction control. (C) Concentration-dependent reduction of NF-κB promoter-linked luciferase activity by AEGT in JFH-1-infected Huh-7 cells. After 6 h of JFH-1 infection, Huh-7-infected cells were treated with AEGT at indicated concentrations for 3 days. Non-infected Huh-7 cells served as the basal control, which is defined as 1. Luciferase activity assay was performed as described above. (D) Concentration-dependent reduction of nuclear p65 protein levels by AEGT in JFH-1-infected Huh-7 cells. Western blot analysis was performed as described above. Each value was represented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585194&req=5

pone-0057704-g004: AEGT suppression of HCV-induced NF-κB activation.(A) Concentration-dependent reduction of NF-κB promoter-linked luciferase activity in AEGT-treated HCV replicon cells. Ava5 cells were transfected with the reporter plasmid pNF-κB-Luc in the presence of AEGT at indicated concentrations for 3 days. Total cell lysates were prepared for luminescence detection using the Steady-Glo Luciferase Assay Kit (Promega). Non-treated Huh-7 cells served as the basal control, which is defined as 1. (B) Concentration-dependent reduction of nuclear p65 protein levels in AEGT-treated HCV replicon cells. Nuclear extracts were prepared from AEGT-treated Ava5 cells and subjected to Western blot analysis using anti-NF-κB p65 and laminB antibodies, in which lamin B was used as a nuclear fraction control. (C) Concentration-dependent reduction of NF-κB promoter-linked luciferase activity by AEGT in JFH-1-infected Huh-7 cells. After 6 h of JFH-1 infection, Huh-7-infected cells were treated with AEGT at indicated concentrations for 3 days. Non-infected Huh-7 cells served as the basal control, which is defined as 1. Luciferase activity assay was performed as described above. (D) Concentration-dependent reduction of nuclear p65 protein levels by AEGT in JFH-1-infected Huh-7 cells. Western blot analysis was performed as described above. Each value was represented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01.
Mentions: Several reports have demonstrated that some constituents of Gracilaria possess anti-inflammatory properties [23], [33], [34]. COX-2, a pro-inflammatory enzyme, is linked to HCV-associated liver carcinogenesis [10]. To investigate whether AEGT can inhibit HCV-stimulated COX-2 expression, we analyzed the promoter activity, protein synthesis and enzyme activity of COX-2 in AEGT-treated Ava5 cells. As shown in Fig. 2A, HCV-stimulated COX-2 promoter activity was suppressed by AEGT in a concentration-dependent manner compared with 0.1% DMSO-treated Ava5 and parental Huh-7 cells (the fold of control), which was observed by a COX-2 promoter-linked luciferase reporter assay. These results indicated that AEGT down-regulated COX-2 expression at the mRNA transcription level. AEGT-induced COX-2 reduction was further confirmed by Western blot analysis (Fig. 2B). In addition, AEGT caused a concentration-dependent decrease in COX-2-mediated PGE2 biogenesis (Fig. 2C). Recently, many reports, including our previous studies, have demonstrated that the suppression of virus-induced COX-2 expression inhibits HCV replication [8], [14], [15], [17]. To examine whether the elimination of COX-2 expression was responsible for AEGT inhibition of HCV replication, we transiently overexpressed COX-2 in AEGT-treated Ava5 cells. Ava5 cells were transfected with a control vector or a pCMV-COX-2-Myc vector encoding the cox-2 gene at increasing concentrations of transfected plasmid DNA (0.5, 1, 1.5 and 2 µg). Cells were incubated with AEGT (600 µg/ml), in which HCV-stimulated COX-2 expression and HCV replication were markedly blocked (Fig. 1 and Fig. 2). Western blot analysis revealed that AEGT-inhibited HCV NS5B protein synthesis (Fig. 3A, upper panel, lanes 3–6) was gradually attenuated by the increase in exogenous COX-2-Myc expression (middle panel) compared with the control transfected cells in the absence (lane 1) or presence of AEGT (lane 2). Consistent with previous results, qRT-PCR analysis revealed that exogenous COX-2-Myc augmentation significantly restored the AEGT-reduced HCV RNA levels in a concentration-dependent manner (Fig. 3B). Taken together, these findings suggest that COX-2 reduction was associated with AEGT anti-viral activity. NF-κB is a crucial transcription factor for COX-2 transactivation in response to viral infection and inflammation [17], [35]. To further elucidate whether the AEGT-mediated downregulation of COX-2 was modulated by NF-κB, we performed a luciferase assay specifically mediated via NF-κB activation. Ava5 and Huh-7 cells were transiently transfected with the cis-reporting plasmid pNF-κB-Luc and then incubated with or without AEGT for 3 days. As shown in Fig. 4A, increased NF-κB luciferase activity was significantly suppressed by AEGT in a concentration-dependent manner. Translocation of the NF-κB p65 subunit from the cytoplasm to the nucleus is required for NF-κB activation. We observed that compared with parental Huh-7 cells without AEGT treatment, the high level of virus-induced NF-κB p65 nuclear protein was gradually decreased to the basal level following AEGT treatment (Fig. 4B), suggesting that AEGT blocked HCV replication through the sustained suppression of NF-κB signalling pathways. We further performed the HCV JFH-1 infectious assay to confirm the anti-NF-κB activity of AEGT described above (Fig. 4C and D).

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