title: Fluvastatin attenuates hepatic steatosis-induced fibrogenesis in rats through inhibiting paracrine effect of hepatocyte on hepatic stellate cells.
abstract: BACKGROUND
  Non-alcoholic steatohepatitis (NASH) is associated with hepatic fibrogenesis. Despite well-known cholesterol-lowering action of statins, their mechanisms against NASH-mediated fibrogenesis remain unclear. This study aimed at investigating the in vitro and in vivo anti-fibrotic properties of fluvastatin (Flu).
  
  
  METHODS
  Palmitate (PA)-induced changes in intracellular hydrogen peroxide levels in primary rat hepatocytes (PRHs) and human hepatoma cell line (HepG2) were quantified by dichlorofluorescein diacetate (DCF-DA) dye assay, whereas changes in expressions of NADPH oxidase gp91 (phox) subunit, α-smooth muscle actin (α-SMA), and NFκB p65 nuclear translocation were quantified with Western blotting. Quantitative real-time polymerase chain reaction (q-PCR) was used to investigate mRNA expressions of pro-inflammatory genes (ICAM-1, IL-6, TNF-α). Conditioned medium (CM) from PA-treated PRHs was applied to cultured rat hepatic stellate cell line, HSC-T6, with or without Flu-pretreatment for 2 h. Pro-fibrogenic gene expressions (COL1, TIMP-1, TGF-β1, α-SMA) and protein expression of α-SMA were analyzed. In vivo study using choline-deficient L-amino acid defined (CDAA) diet-induced rat NASH model was performed by randomly assigning Wistar rats (n = 28) to normal controls (n = 4), CDAA diet with vehicles, and CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each) through gavage for 4 or 8 weeks. Livers were harvested for histological, Western blot (α-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, α-SMA, TIMP-1) genes.
  
  
  RESULTS
  In vitro, Flu (1-20 μM) inhibited PA-induced free-radical production, gp91 (phox) expression, and NFκB p65 translocation in HepG2 and PRHs, while CM-induced α-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs. Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats.
  
  
  CONCLUSIONS
  We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu. In vivo, Flu alleviated steatosis-induced HSC activation and hepatic fibrogenesis through mitigating inflammation and oxidative stress, suggesting possible therapeutic role of Flu against NASH.
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei, Taiwan. leewonch@ms52.hinet.net.|Division of Hepatology and Gastroenterology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan. leewonch@ms52.hinet.net.
authors.forename: Lee-Won
authors.initials: LW
authors.lastname: Chong
authors.affiliation: Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan. hsuyc@mmc.edu.tw.
authors.forename: Yi-Chao
authors.initials: YC
authors.lastname: Hsu
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei, Taiwan. leetingfang@hotmail.com.
authors.forename: Ting-Fang
authors.initials: TF
authors.lastname: Lee
authors.affiliation: National Research Institute of Chinese Medicine, No. 155-1, Li-Nong Street, Sec. 2, Taipei, 112, Taiwan. u9223015@gmail.com.
authors.forename: Yun
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Department of Medical Research and Education, Taichung Veterans General Hospital, Taichung, Taiwan. ytchiu@vghtc.gov.tw.
authors.forename: Yung-Tsung
authors.initials: YT
authors.lastname: Chiu
authors.affiliation: Division of Hepatology and Gastroenterology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan. M001003@ms.skh.org.tw.
authors.forename: Kuo-Ching
authors.initials: KC
authors.lastname: Yang
authors.affiliation: Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei, Taiwan. jcwu@vghtpe.gov.tw.|Translational Research Division, Medical Research Department, Taipei Veterans General Hospital, Taipei, Taiwan. jcwu@vghtpe.gov.tw.
authors.forename: Jaw-Ching
authors.initials: JC
authors.lastname: Wu
authors.affiliation: National Research Institute of Chinese Medicine, No. 155-1, Li-Nong Street, Sec. 2, Taipei, 112, Taiwan. huangyt@nricm.edu.tw.|Institute of Traditional Medicine, School of Medicine, National Yang Ming University, Taipei, Taiwan. huangyt@nricm.edu.tw.
authors.forename: Yi-Tsau
authors.initials: YT
authors.lastname: Huang
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002794:Choline / Q000008:administration & dosage; D024042:Collagen Type I / Q000235:genetics; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004032:Diet; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology* / Q000627:therapeutic use; D000077340:Fluvastatin; D015870:Gene Expression / Q000187:drug effects; D056945:Hep G2 Cells; D055166:Hepatic Stellate Cells / Q000502:physiology*; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D007211:Indoles / Q000494:pharmacology* / Q000627:therapeutic use; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D008103:Liver Cirrhosis / Q000209:etiology / Q000473:pathology / Q000517:prevention & control*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D065626:Non-alcoholic Fatty Liver Disease / Q000150:complications / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D019308:Palmitic Acid / Q000494:pharmacology; D019899:Paracrine Communication / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D051996:Transcription Factor RelA / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
chemical_list: D000199:Actins; D002794:Choline; D024042:Collagen Type I; D017077:Culture Media, Conditioned; D005229:Fatty Acids, Monounsaturated; D000077340:Fluvastatin; D006861:Hydrogen Peroxide; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007211:Indoles; D018799:Intercellular Adhesion Molecule-1; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D052247:Nitric Oxide Synthase Type II; D019308:Palmitic Acid; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D051996:Transcription Factor RelA; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; C541116:ACTA2 protein, human; C498862:Acta2 protein, rat; C056558:CYBB protein, human; C500593:Cybb protein, rat