Inhibition of T-Cell Inflammatory Cytokines, Hepatocyte NF-κB Signaling, and HCV Infection by Standardized Silymarin
2007; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.02.038
ISSN1528-0012
AutoresStephen J. Polyak, Chihiro Morishima, Margaret C. Shuhart, Chia C. Wang, Yanze Liu, David Y.W. Lee,
Tópico(s)Gout, Hyperuricemia, Uric Acid
ResumoBackground & Aims: Chronic hepatitis C is a serious global medical problem necessitating effective treatment. Because standard of care with pegylated interferon plus ribavirin therapy is costly, has significant side effects, and fails to cure about half of all infections, many patients seek complementary and alternative medicine to improve their health, such as Silymarin, derived from milk thistle (Silybum marianum). Milk thistle’s clinical benefits for chronic hepatitis C are unsettled due to variability in standardization of the herbal product. Methods: In the current study, we focused on the anti-inflammatory and antiviral properties of a standardized Silymarin extract (MK-001). Results: MK-001 inhibited expression of tumor necrosis factor-alpha in anti-CD3 stimulated human peripheral blood mononuclear cells and nuclear factor kappa B-dependent transcription in human hepatoma Huh7 cells. Moreover, MK-001 dose dependently inhibited infection of Huh7 and Huh7.5.1 cells by JFH-1 virus. MK-001 displayed both prophylactic and therapeutic effects against HCV infection, and when combined with interferon-α, inhibited HCV replication more than interferon-α alone. Commercial preparations of Silymarin also displayed antiviral activity, although the effects were not as potent as MK-001. Antiviral effects of the extract were attributable in part to induction of Stat1 phosphorylation, while interferon-independent mechanisms were suggested when the extract was biochemically fractionated by high-performance liquid chromatography. Silybin A, silybin B, and isosilybin A, isosilybin B elicited the strongest anti-NF-κB and anti-HCV actions. These effects were independent of MK-001-induced cytotoxicity. Conclusions: The data indicate that Silymarin exerts anti-inflammatory and antiviral effects, and suggest that complementary and alternative medicine-based approaches may assist in the management of patients with chronic hepatitis C. Background & Aims: Chronic hepatitis C is a serious global medical problem necessitating effective treatment. Because standard of care with pegylated interferon plus ribavirin therapy is costly, has significant side effects, and fails to cure about half of all infections, many patients seek complementary and alternative medicine to improve their health, such as Silymarin, derived from milk thistle (Silybum marianum). Milk thistle’s clinical benefits for chronic hepatitis C are unsettled due to variability in standardization of the herbal product. Methods: In the current study, we focused on the anti-inflammatory and antiviral properties of a standardized Silymarin extract (MK-001). Results: MK-001 inhibited expression of tumor necrosis factor-alpha in anti-CD3 stimulated human peripheral blood mononuclear cells and nuclear factor kappa B-dependent transcription in human hepatoma Huh7 cells. Moreover, MK-001 dose dependently inhibited infection of Huh7 and Huh7.5.1 cells by JFH-1 virus. MK-001 displayed both prophylactic and therapeutic effects against HCV infection, and when combined with interferon-α, inhibited HCV replication more than interferon-α alone. Commercial preparations of Silymarin also displayed antiviral activity, although the effects were not as potent as MK-001. Antiviral effects of the extract were attributable in part to induction of Stat1 phosphorylation, while interferon-independent mechanisms were suggested when the extract was biochemically fractionated by high-performance liquid chromatography. Silybin A, silybin B, and isosilybin A, isosilybin B elicited the strongest anti-NF-κB and anti-HCV actions. These effects were independent of MK-001-induced cytotoxicity. Conclusions: The data indicate that Silymarin exerts anti-inflammatory and antiviral effects, and suggest that complementary and alternative medicine-based approaches may assist in the management of patients with chronic hepatitis C. Globally, HCV infects an estimated 170 million people, and causes an estimated 500,000 deaths per year due to complications of late-stage liver disease. In the United States, about 1.8% of the general population (∼4 million persons) is infected. Of those acutely infected with HCV, around 85% develop chronic infection. Approximately 70% of patients with chronic viremia develop histologic evidence of chronic liver disease. Hepatitis C is the most frequent indication for liver transplantation in the United States.1Di Bisceglie A.M. Liver transplantation for hepatitis C: the promise and the challenge.Hepatology. 1995; 22: 660-662Crossref PubMed Google Scholar, 2Ferrell L.D. Wright T.L. Roberts J. 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Liver biopsy data were available on 3 HCV-infected subjects. The necroinflammatory activity (grade) and degree of fibrosis (stage) of the liver disease were scored semiquantitatively, each on a scale of 0–4, using the Batts and Ludwig method.42Batts K. Ludwig J. Chronic hepatitis: an update on terminology and reporting.Am J Surg Pathol. 1995; 19: 1409-1417Crossref PubMed Scopus (922) Google Scholar All had at least grade 1 inflammation. Fibrosis stage was scored as 1, 1–2, and 2 for each of the 3 subjects, respectively. This study was approved by the institutional review boards at the University of Washington. Informed consent was obtained from all study participants. Human hepatoma Huh7 cells were grown in Huh7 medium that contained DMEM, 10% fetal bovine serum, 1× penicillin, streptomycin, fungizone, 10 mmol/L L-glutamine, and 1× nonessential amino acids (all reagents were from Invitrogen, Carlsbad, CA). Huh7.5.1 cells were obtained from Francis Chisari30Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. Wieland S.F. Uprichard S.L. Wakita T. Chisari F.V. Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci U S A. 2005; 102: 9294-9299Crossref PubMed Scopus (1511) Google Scholar and were cultured in Huh7 medium. All cell lines were checked for mycoplasma using MycoAlert assay (Cambrex Bio Science, Rockland, ME) and found to be mycoplasma-free. JFH-1 viral stock preparation, cell infection, and titration was performed exactly as described.27Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2405) Google Scholar, 30Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. Wieland S.F. Uprichard S.L. Wakita T. Chisari F.V. 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Extraction of nutraceuticals from milk thistle: I Hot water extraction.Appl Biochem Biotechnol. 2003; 105–108: 881-889Crossref PubMed Scopus (33) Google Scholar and the effects of defatted and nondefatted seeds have been investigated,46Wallace S.N. Carrier D.J. Clausen E. Extraction of nutraceuticals from milk thistle: part II Extraction with organic solvents.Appl Biochem Biotechnol. 2003; 105–108: 891-903Crossref PubMed Scopus (46) Google Scholar extraction with organic solvent is superior.47Wallace S.N. Carrier D.J. Clausen E.C. Batch solvent extraction of flavanolignans from milk thistle (Silybum marianum L. Gaertner).Phytochem Anal. 2005; 16: 7-16Crossref PubMed Scopus (61) Google Scholar Therefore, defatted seed meal of Silybum marianum was extracted with aqueous acetone. The extract was concentrated to remove acetone, and then washed by hexane to wash away hydrophobic impurities. The remaining concentrate was treated with 1% NaCl solution to remove water-soluble impurities. The precipitate and solid obtained through spray drying were combined together to form crude Silymarin. The crude Silymarin was washed with aqueous ethanol and then dried completely to give the refined yellowish powder (MK-001). This MK-001was standardized with a significant reduction of polar nonflavonolignans impurities compared to commercial products. MK-001 and the purified fractions of MK-001 were solubilized in DMSO at 50 mg/mL. To compare anti-HCV action of MK-001 with other preparations of Silymarin, we tested Ultrathistle (Natural Wellness, Montgomery, NY) and Silybinin (Indena SpA, Milan, Italy), kindly provided by Dr Leanna Standish (Bastyr University). Capsule contents were solubilized in 100% EtOH at 150 mg/mL and vortexed for 1 minute. Samples were heated at 75°C for 5 minutes, insoluble excipients were removed by a 10-second spin in a microcentrifuge, and supernatants were transferred to new tubes. Generation of JFH-1 RNA and transfection of cells was performed as described.27Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Krausslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2405) Google Scholar To measure NF-κB-dependent transcription, we used 2 different luciferase reporter genes. NF-κB-luc (Stratagene, La Jolla, CA) contains consensus DNA sequences for NF-κB binding. We also measured CXCL-8 transcription, because CXCL-8 is a well-known NF-κB responsive gene.48Holtmann H. Winzen R. Holland P. Eickemeier S. Hoffmann E. Wallach D. Malinin N.L. Cooper J.A. Resch K. Kracht M. Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least 3 different cytokine- or stress-activated signal transduction pathways.Mol Cell Biol. 1999; 19: 6742-6753Crossref PubMed Scopus (268) Google Scholar In this case, the luciferase gene was placed under control of the full-length CXCL-8 promoter and referred to as −1481-luc. This plasmid was obtained from Naofumi Mukaida.49Mukaida N. Shiroo M. Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8.J Immunol. 1989; 143: 1366-1371PubMed Google Scholar Reporter gene assays were performed as described.50Wagoner J. Austin M. Green J. Imaizumi T. Casola A. Brasier A. Khabar K.S. Wakita T. Gale Jr, M. Polyak S.J. Regulation of CXCL-8 (interleukin-8) induction by double-stranded RNA signaling pathways during hepatitis C virus infection.J Virol. 2007; 81: 309-318Crossref PubMed Scopus (59) Google Scholar Briefly, the day before transfection, 3 × 104 cells were plated in black, clear-bottomed, 96-well tissue culture plates. Endotoxin-free plasmid DNA was purified (Endofree kit, Qiagen, Valencia, CA), and was introduced into cells with lipofectamine 2000 according to manufacturer’s recommendations (Invitrogen). For reporter gene studies, unless otherwise indicated, 100 ng of the luciferase gene under control of promoter construct of interest was transfected into cells in quadruplicate. Eighteen hours later, stimuli such as rhTNF-α (15 ng/mL; Pierce Biotechnology, Rockford, IL) was added. Four hours later, luciferase activity was measured on cell lysates using the Britelite assay system (Perkin-Elmer, Boston, MA). We used the luminescence ATP detection assay system (ATPlite, Perkin-Elmer) as described by the manufacturer. Huh7 or Huh7.5.1 were grown overnight in black 96-well view plates (1 × 104 cells per well). After a 24-hour incubation with the compound the wells were washed twice with 0.2 mL of phosphate-buffered saline followed by addition of 0.1 mL of phosphate-bufered saline and 50 μL of lysis solution (provided with the kit) to each well. The microplate was shaken for 5 minutes at 600 rpm on an orbital shaker to allow cell lysis and ATP stabilization. Fifty microliters of the substrate solution were then added, and the microplate was shaken for 5 minutes at 600 rpm. Luminescence was measured on a TopCount NXT microplate scintillation and luminescence counter (Packard; Perkin-Elmer) after a 10-minute dark adaptation. Cytotoxicity for PBMC was tested after 24-hour incubation with MK-001 at various concentrations using Live/Dead Fixable Dead Cell Stain Kit (Molecular Probes, Eugene, OR), α-CD3-FITC (BD Biosciences, San Jose, CA). After fixing cells at a final concentration of 1% paraformaldehyde, samples were analyzed on a BD LSR II (BD Biosciences) at the University of Washington Department of Immunology Cell Analysis Facility (Seattle, WA) with data analysis performed using FlowJo software for Macintosh (Tree Star, Inc., Ashland, OR). Protein lysates were quantitated (BCA Protein Assay, Pierce) and equal amounts of total protein (10–20 μg) was separated on 4%–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Stat proteins were detected using polyclonal antiserum (Santa Cruz Biotechnology, Santa Cruz, CA). NS5A was detected using a polyclonal antibody to NS5A (Chiron, Emeryville, CA), while the HCV core protein was detected with a monoclonal antibody (Affinity Bioreagents, Golden, CO). GAPDH was detected with polyclonal antiserum (Santa Cruz Biotechnology). Blot images were scanned and protein pixel intensity measured using Image J as described.43Koo B.C. McPoland P. Wagoner J.P. Kane O.J. Lohmann V. Polyak S.J. Relationships between hepatitis C virus replication and CXCL-8 production in vitro.J Virol. 2006; 80: 7885-7893Crossref PubMed Scopus (30) Google Scholar HCV RNA was quantitated by real time reverse-transcriptase polymerase chain reaction, as previously described.51Plumlee C.R. Lazaro C.A. Fausto N. Polyak S.J. Effect of ethanol on innate antiviral pathways and HCV replication in human liver cells.Virol J. 2005; 2: 89Crossref PubMed Scopus (51) Google Scholar PBMC were freshly isolated from whole blood using standard Ficoll-Hypaque centrifugation and cultured for 24 hours in the presence of plate-bound anti-CD3 (10 mg/mL) and MK-001 (20 μg/mL) or DMSO (0.5%) control. Supernatants were tested for TNF-α levels using a
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