Identification of antrodin B from Antrodia camphorata as a new anti-hepatofibrotic compound using a rapid cell screening method and biological evaluation.

Abstract

Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) resulting from chronic liver diseases. Efficient and well-tolerated drugs for its treatment are urgently needed. This study aims to identify the active ingredients of Antrodia camphorata by a bioassay-guided fractionation approach and explore the acting mechanism by using a hepatic stellate cell (HSC) line CFSC-8B stimulated by transforming growth factor-β1 (TGF-β1). The accumulation of collagens was evaluated using chromogenic precipitation reaction with picro-sirius red (PSR) dye solution and quantified by spectrophotometric analysis of the dissolved stain. MTT assay, cell migration assay, quantitative polymerase chain reaction and western blotting analysis were used to determine the cell viability, cell migration and gene expression. We established a rapid colorimetric assay suitable for screening of anti-hepatofibrotic reagents. Stimulation with 10 ng/mL TGF-β1 for 48 h and 200 μL PSR dye solution were optimal for the colorimetric assay in CFSC-8B cells. We used SB431542, silybin and another 11 antifibrotic reagents to verify the cellular model. Within the safe doses, they attenuated ECM production induced by TGF-β1. Bioactivity-guided fractionation led to the identification of antrodin B from A. camphorata. Antrodin B significantly ameliorated cell proliferation, cell migration, suppressed HSC activation marker α-smooth muscle actin expression and ECM components Col1, Col3 and Fn expression, and blocked the phosphorylation of Smad2/3 induced by TGF-β1 in CFSC-8B cells in a dose-dependent manner. We developed a simple assay based on TGF-β1-induced total collagen accumulation in CFSC-8B cells and identified antrodin B which may serve as a potential candidate for treatment of liver fibrosis.