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

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.


Introduction
Hepatitis C virus (HCV) infection is the primary etiological agent of chronic hepatitis, liver cirrhosis and eventually hepatocellular carcinoma (HCC) [1,2]. Till date, no prophylactic vaccine is available for HCV infection, and current therapy is primarily restricted to a combination of pegylated interferon-a (IFN-a) and ribavirin [3,4]. However, IFN-based therapy is fraught with severe side effects and has various sustained virological response (SVR) rates, which are depended on the HCV genotype [5]. Despite the recent advancement of therapeutics with the approval of NS3/4A protease inhibitors, telaprevir and boceprevir, in combination with pegylated-interferon alpha (IFN-a) and ribavirin, the emergence of drug-resistant variants and the unfavourable side effects, such as anaemia and rash, may reduce the susceptibility and applicability of current HCV triple therapies [6]. Therefore, it is essential to develop alternate drugs or supplements for treating HCV infections.
HCV belongs to the Flaviviridae family and has a 9.6-kb singlestranded positive-sense RNA genome encoding a single precursor polyprotein of approximately 3000 amino acids, which generate 10 individual proteins, including structural proteins (core, E1, and E2) and non-structural (NS2, NS3, NS4A, NS5A and NS5B) proteins, processed by both host and viral proteases [7]. Among the latter, NS5A is a serine phosphoprotein, which exists as an important risk factor for hepatocarcinogenesis as a transcriptional activator of chronic inflammation by stimulating crucial mediators such as nuclear transcription factor-kappaB (NF-kB) and cyclooxygenase-2 (COX-2) [8,9,10]. COX-2, in particular, is an inducible Cox isozyme, and COX-2 overexpression is closely associated with human tumour formation by the production of large amounts of various prostaglandins (PGs), such as prostaglandin E2, thromboxane B2 and prostacyclin, which in turn cause the promotion of cellular proliferation, cancer invasiveness, angiogenesis and antiapoptosis [11,12]. Therefore, the inhibition of improper COX-2 expression is now considered to be a potential approach for cancer prevention and treatment [13]. In addition to developing a promising anti-tumour strategy by targeting the COX-2 signalling pathway, increasing evidence, including our previous studies, links the inhibition of COX-2 expression to anti-HCV activity at present [14,15,16,17]. Therefore, an attractive means of eliminating HCV-related HCC is by combining anti-HCV and anti-inflammatory agents.
Many species of marine algae comprise a rich source of active substances, such as diterpenes and sulfated polysaccharides, which are potentially used for treating infectious diseases [18,19]. Gracilaria, a genus of red algae, is abundantly produced in Taiwan. Numerous Gracilaria extracts promote various bioactivities including anti-oxidation [20,21,22], anti-hypercholesterolemia, antiinflammation [23] and anti-microbial activities [22,24]. More recently, our results indicated that the aqueous extracts of the Gracilaria tenuistipitata (AEGT) exhibited anti-oxidant activity and protective effect on H 2 O 2 -induced DNA damage [25]. In contrast, ethanolic and methanolic extracts of G. tenuistipitata exhibited antiproliferation and induction of apoptosis on oral cancer cells [26,27]. The effect of Gracilaria algae on human viral infection has not been investigated till date. The objectives of the present study were to evaluate the inhibitory effect of AEGT on HCV replication and to investigate the molecular mechanism underlying the anti-viral activity of AEGT by evaluating its inhibitory effect on COX-2 expression and upstream transcription factor, NF-kB. We subsequently examined whether the combination of AEGT and IFN-a produced synergistic effects that could inhibit HCV replication. Furthermore, we highlighted the inhibitory effect of AEGT on HCV-induced inflammatory mediators including tumour necrosis factor-a (TNF-a), interleukin-1b (IL-1b), inducible nitric oxide synthase (iNOS) and COX-2 to assess its potential supplement use against HCV-related HCC.

Ethics Statement
G. tenuistipitata is not an endangered or protected seaweed. G. tenuistipitata was collected in the No.129, Kouhu village, Kouhu Township, Yunlin County 653, Taiwan (R.O.C.), which is not a protected area but is privately owned by SHUI-TUI LI Taiwan. Mr. LI permits research in the reserves, and no specific permits are required for field studies such as the one described here.
Preparation of Aqueous Extract of G. tenuistipitata (AEGT) Raw materials. G. tenuistipitata was collected in the spring of 2009 from a culture farm at Kouhu beach, Yunlin County, Taiwan and delivered to our laboratory at 0uC. In the laboratory, the seaweeds were washed with tap water to remove epiphytes and encrusting material, soaked in distilled water twice and then lyophilised. The dried samples were pulverized, passed through a 60-mesh sieve, ground to a fine powder and stored at 240uC.

Analysis of AEGT Extract: Polyphenol, Flavonoid, and Ascorbic Acid
Determination of bioactive components of the AEGT extract was performed as described previously [25], which included polyphenol, flavonoid, and ascorbic acid. The amount of polyphenol, flavonoid, and ascorbic acid were respectively recorded as 98.9462.43 mg, 22.5961.08 mg, and 1.5960.18 mg, respectively, in 1 mg of dry extract.

Quantitative Real-Time Polymerase Chain Reaction (PCR)
HCV or cellular RNA was measured by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis as described previously [17]. The levels of HCV NS5B, TNF-a, IL-1b, iNOS and COX-2 RNA were detected by qRT-PCR with the following forward and reverse primer sets:

HCV JFH-1 Infection Assay
Production of infectious HCV genotype 2a JFH-1 particles and titration of generated HCV were performed as described [28]. By transfection of in vitro-transcribed full-length JFH-1 RNA into Huh-7.5, the infectious viral particles were generated. The inhibitory effect of AGET on HCV infection was assayed as described previously [29]. In brief, the Huh-7 cells were seeded at density of 4610 4 cells per well in a 24-wells culture plate and infected with 100 ml of HCV JFH-1 particles at a multiplicity of infection (MOI) of 0.1 for 6 h followed by incubation with various concentrations of AEGT for an additional 72 h. Subsequently, total RNA was collected and subjected to RT-qPCR for measuring mRNAs of HCV and GAPDH as described above.

Transient Transfection and Luciferase Activity Assay
Ava5 cells were seeded in 24-well plates at a density of 5610 4 per well and were transfected with 1 mg of plasmid pCOX-2-Luc or pNF-kB-Luc (BD Biosciences Clontech, Palo Alto, CA) using T-Pro TM reagent (Ji-Feng Biotechnology Co. Ltd., Taiwan) in accordance with the manufacturer's instructions. Six hours posttransfection, the cells were treated with various concentrations of AEGT for 3 days. To further investigate COX-2 regulation by AEGT, Ava5 cells were transfected with serially increased concentrations of COX-2 expression vector (pCMV-COX-2-Myc) from 0.5 to 2 mg, and then treated with 600 mg/ml of AEGT for 3 days. The luciferase activity of cell extracts from each sample was measured using the Bright-Glo TM Luciferase Assay System (Promega) according to the manufacturer's protocol. To determine the relationship between AEGT and NS5A-induced inflammatory genes (TNF-a, IL-1b, iNOS and COX-2), Huh-7 cells were transfected with 1 mg of plasmid pCMV-NS5A-Myc in the presence of AEGT (200 and 800 mg/ml) for 3 days. Subsequently, total RNA was subjected to qRT-PCR with specific primers.

Intracellular Prostaglandin E2 (PGE 2 ) Measurements
Ava5 cells were treated with AEGT at various concentrations for 3 days. Following drug treatment, cultured cells were thoroughly washed with cold phosphate-buffered saline (pH 7.4), and cell membranes were ruptured using lysis reagent containing C 15 H 34 BrN to release intracellular PGE 2 . The levels of PGE 2 levels were then analyzed by PGE 2 enzyme-linked immunosorbent assay system (Biotrak, Amersham Bioscience) according to the manufacturer's protocol.

Preparation of Nuclear Fraction
Nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc.,  USA) according to the manufacturer's instructions. In brief, approximately 1610 6 Ava5 cells were seeded onto 10-cm dish for 24 h and then treated with or without AEGT. After 4 days of incubation, the cells were homogenated in hypotonic buffer (10 mM HEPES, 1 mM MgCl 2 , 1 mM EDTA, 10 mM KCl, 0.5 mM DTT, 0.5% Nonidet P-40, 4 mg/ml leupeptin, 20 mg/ml aprotinin, and 0.2 mM PMSF), followed by centrifugation at 2000 g for 5 min. Nuclear extracts were prepared using the hypotonic buffer after centrifugation at 7000 g for 15 min, following which the pellets containing crude nuclei were resuspended in the hypertonic buffer at 4uC for 30 min. After centrifugation at 20000 g for 15 min, nuclear proteins were collected and stored at 280uC until use.

Statistical Analysis
The data were expressed as mean 6 SD of at least three independent experiments. Statistical calculations were analysed by the Student's t-test; p-values ,0.01 were considered statistically significant.

AEGT Inhibits HCV Protein Synthesis, RNA Replication and HCV Infection
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 mg/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 EC 50 value of 30060.3 mg/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 mg/ml). Using an HCV JFH-1 infection system [32], we confirmed the anti-HCV activity of AEGT, with an EC 50 value of 32560.7 mg/ml (Fig. 1C).

Combination of IFN-a and AEGT Synergistically Inhibits HCV Replication
Next, we examined the anti-HCV of AEGT in combination with IFN-a. Ava5 cells were incubated with a combination of fixed concentrations of AEGT and IFN-a. HCV RNA replication inhibition was detected by qRT-PCR analysis. Based on the results of CalcuSyn TM analysis, a combination of IFN-a and AEGT at concentrations of 1:5, 1:10 and 1:20 was found to have synergistic anti-HCV effects, as revealed by the combination index (CI) values of ,1 for ED 50 , ED 75 and ED 90 (range, 0.89-0.52) ( Table 1). These results indicated that AEGT may be a promising adjuvant for combination HCV therapy.

Anti-HCV Activity of AEGT Involves COX-2 and NF-kB Suppression
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 PGE 2 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 mg). Cells were incubated with AEGT (600 mg/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 AEGTreduced 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-kB 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-kB, we performed a luciferase assay specifically mediated via NF-kB activation. Ava5 and Huh-7 cells were transiently transfected with the cis-reporting plasmid pNF-kB-Luc and then incubated with or without AEGT for 3 days. As shown in Fig. 4A, increased NF-kB luciferase activity was significantly suppressed by AEGT in a concentration-dependent manner. Translocation of the NF-kB p65 subunit from the cytoplasm to the nucleus is required for NF-kB activation. We observed that compared with parental Huh-7 cells without AEGT treatment, the high level of virus-induced NF-kB p65 nuclear protein was gradually decreased to the basal level following AEGT  (Fig. 4B), suggesting that AEGT blocked HCV replication through the sustained suppression of NF-kB signalling pathways. We further performed the HCV JFH-1 infectious assay to confirm the anti-NF-kB activity of AEGT described above ( Fig. 4C and D).

AEGT Inhibits Gene Expression of Pro-inflammatory Mediators in HCV-infected and NS5A-expressing Cells
Chronic inflammation caused by HCV infection is considered as one of the major pathogenic mechanisms, A number of proinflammatory gene products as well cytokines, including COX-2, iNOS, TNF-a and IL-1b, are believed to play a critical role in inflammatory diseases [36,37]. To examine the potential AEGT hepatoprotective actions against the HCV-stimulated inflammatory mediators described above, HCV JFH-1-infected Huh-7 cells were treated with different concentrations of AEGT for 3 days. qRT-PCR analysis demonstrated that compared to the uninfected cells, the elevated mRNA levels of these induced pro-inflammatory mediators were reduced in a concentration-dependent manner by AEGT (Fig. 5A-D). Among HCV proteins, NS5A is one of risk factors involving hepatocarcinogenesis through chronic inflammation [38,39]. To further investigate the potential AEGT hepatoprotective actions against the NS5A-stimulated inflammatory mediators, NS5A-transfected Huh-7 cells were treated with different concentrations of AEGT for 3 days. Similar results for the reduction of NS5A-induced pro-inflammatory mediators by AEGT treatment were observed compared with the untreated cells ( Fig. 5E-H).

Discussion
In the present study, we showed that an active extract from G. tenuistipitata (AEGT) has potent inhibitory effects on HCV replication in both HCV subgenomic and infectious systems (Fig. 1). This is the first report of the potent anti-HCV activity Total cell lysates were prepared for luminescence detection using the Steady-Glo Luciferase Assay Kit (Promega). Nontreated Huh-7 cells served as the basal control, which is defined as 1. (B) Concentration-dependent reduction of nuclear p65 protein levels in AEGTtreated HCV replicon cells. Nuclear extracts were prepared from AEGT-treated Ava5 cells and subjected to Western blot analysis using anti-NF-kB p65 and laminB antibodies, in which lamin B was used as a nuclear fraction control. (C) Concentration-dependent reduction of NF-kB 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 6 SD of three independent experiments. *P,0.05; **P,0.01. doi:10.1371/journal.pone.0057704.g004 from algal extract. Notably, combining AEGT with IFNa synergistically inhibited HCV replication (Table 1), revealing that AEGT may be an effective therapeutic dietary alga in a combinational anti-HCV regimen. Studies on the mechanism of action of AEGT revealed that AEGT suppressed NF-kB-mediated COX-2 production at the transcriptional level (Fig. 2), and that this suppression revealed a critical mechanism underlying the attenuation of anti-HCV activity of AEGT by extraneous COX-2 overexpression in AEGT-treated HCV replicon cells (Fig. 3).
Recent advances in anti-HCV drug development have revealed many direct-acting antiviral agents (DAAs) that target HCV replication complexes, e.g. NS3/4A protease, NS5A and NS5B polymerase, at different stages of clinical development [40]. Due to the low fidelity of NS5B polymerase, increasing mutations throughout the viral genome usually reduces DAA susceptibility [41]. In this respect, cellular factors required for viral replication have emerged as promising drug targets because of lower mutation frequencies in the host genome than in the viral genome [42]. In addition, targeting the host factors provides an opportunity for the development of innovative drugs with broad-spectrum activity against all HCV genotypes. In addition to HCV infection, highly upregulated COX-2 levels during viral infection facilitated viral replication in other viruses such as cytomegalovirus [43], herpesvirus [44], respiratory syncytial virus (RSV) [45], enterovirus 71 [46] and West Nile virus [47]. In contrast, the elimination of virus-induced COX-2 expression by a selective inhibitor prevented the replication of other viruses such as human cytomegalovirus (HCMV) [48], H5N1 [49] and HCV [14,16]. Thus, COX-2 may be a promising target for viral treatment. In the present study, AEGT efficiently blocked HCV replication by suppressing COX-2 expression (Fig. 3). Therefore, detailed investigation on COX-2 and its downstream signalling pathways involved in HCV replication are essential to develop an efficient therapeutic target against viral diseases.
An important HCC risk factor is chronic inflammation caused by HCV infection, in which HCV core and NS5A proteins act as etiologic proteins that greatly stimulate inflammatory mediators for the initiation and maintenance of cancer cell survival and growth [1]. NF-kB plays a primary role in inflammatory gene regulation upon viral infection. Moreover, increase in COX-2/ PEG 2 expression is linked to the progression of inflammation, which leads to carcinogenesis. Till date, little is known about the HCV regulatory pathways leading to COX-2 expression. Our results demonstrated that HCV dramatically induced NF-kB and COX-2 activation, whereas a gradual suppression of NF-kB-and COX-2-mediated transcriptional activity was observed upon AEGT treatment in HCV replicon cells, as depicted in Fig. 2 and Fig. 4. Based on these results, we suggested that the inhibitory effect of AEGT on numerous HCV-induced pro-inflammatory gene products is partly mediated via an NF-kB-dependent signalling pathway (Fig. 4). However, we cannot exclude the involvement of other cellular targets in the regulation of anti-HCV and anti-inflammatory activities of AEGT because this crude extract may possibly contain various active constitutes. Therefore, further purification of active AEGT constitutes is essential. Indeed, it is possible that crude extracts contribute to the synergistic actions through multiple targets against viral pathophysiological effects, although it is arduous to elucidate the detailed mechanisms. Further studies will be conducted to separate and identify active components for testing therapeutic protection against HCVrelated diseases. In conclusion, the edible G. tenuistipitata extracts may be useful as a potential dietary supplement in the prevention and treatment of chronic HCV infection by simultaneous inhibition of viral replication, inflammation and carcinogenesis.