Fluvastatin attenuates hepatic steatosis-induced fibrogenesis in rats through inhibiting paracrine effect of hepatocyte on hepatic stellate cells

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, a-smooth muscle actin (a-SMA), and NF kappa B 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-alpha). 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-beta 1, alpha-SMA) and protein expression of a-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 (a-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, a-SMA, TIMP-1) genes. Results: In vitro, Flu (1-20 mu M) inhibited PA-induced free-radical production, gp91phox expression, and NF kappa B p65 translocation in HepG2 and PRHs, while CM-induced a-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, a-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, a-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.