Yan Geng1, Jing Wang1, Qing Sun1, Minfeng Xie1, Zhen-Ming Lu1, Hong-Yu Xu1, Jin-Song Shi1, Zheng-Hong Xu1,2. 1. School of Pharmaceutical Science, Jiangnan University, Wuxi, China. 2. Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
Abstract
AIM: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.
AIM: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.