(3S-5S-6E)-7-[3-(4-fluorophenyl)-1-(propan-2-yl)-1H-indol-2-yl]-3-5-dihydroxyhept-6-enoic-acid and Non-alcoholic-Fatty-Liver-Disease

Free cholesterol (FC) accumulation in the liver is an important pathogenic mechanism of nonalcoholic steatohepatitis (NASH). Plasmalogens, key structural components of the cell membrane, act as endogenous antioxidants and are primarily synthesized in the liver. However, the role of hepatic plasmalogens in metabolic liver disease is unclear. In this study, we found that hepatic levels of docosahexaenoic acid (DHA)-containing plasmalogens, expression of glyceronephosphate O-acyltransferase (Gnpat; the rate-limiting enzyme in plasmalogen biosynthesis), and expression of Pparα were lower in mice with NASH caused by accumulation of FC in the liver. Cyclodextrin-induced depletion of FC transactivated Δ-6 desaturase by increasing sterol regulatory element-binding protein 2 expression in cultured hepatocytes. DHA, the major product of Δ-6 desaturase activation, activated GNPAT, thereby explaining the association between high hepatic FC and decreased Gnpat expression. Gnpat small interfering RNA treatment significantly decreased peroxisome proliferator-activated receptor α (Pparα) expression in cultured hepatocytes. In addition to GNPAT, DHA activated PPARα and increased expression of Pparα and its target genes, suggesting that DHA in the DHA-containing plasmalogens contributed to activation of PPARα. Accordingly, administration of the plasmalogen precursor, alkyl glycerol (AG), prevented hepatic steatosis and NASH through a PPARα-dependent increase in fatty acid oxidation. Gnpat. Increased hepatic FC in animals with NASH decreased plasmalogens, thereby sensitizing animals to hepatocyte injury and NASH. Our findings uncover a novel link between hepatic FC and plasmalogen homeostasis through GNPAT regulation. Further study of AG or other agents that increase hepatic plasmalogen levels may identify novel therapeutic strategies against NASH. (Hepatology 2017;66:416-431).

Topics: Analysis of Variance; Animals; Biomarkers; Biopsy, Needle; Disease Models, Animal; Fatty Acids, Monounsaturated; Fatty Liver; Fluvastatin; Glucosamine 6-Phosphate N-Acetyltransferase; Immunohistochemistry; Indoles; Male; Mediator Complex Subunit 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Non-alcoholic Fatty Liver Disease; Plasmalogens; Random Allocation; Sensitivity and Specificity; Signal Transduction

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).. 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.. 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.. 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.

Topics: Actins; Animals; Choline; Collagen Type I; Culture Media, Conditioned; Diet; Fatty Acids, Monounsaturated; Fluvastatin; Gene Expression; Hep G2 Cells; Hepatic Stellate Cells; Hepatocytes; Humans; Hydrogen Peroxide; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Intercellular Adhesion Molecule-1; Interleukin-6; Liver Cirrhosis; Male; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Nitric Oxide Synthase Type II; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Palmitic Acid; Paracrine Communication; Rats; Rats, Wistar; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Transcription Factor RelA; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha