Isolation Of Antrodin B From Antrodia Cinnamomea And Its Mechanisms For Treatment Of Liver Fibrosis

Liver fibrosis is characterized by changes in tissue architecture and widespread scarformation resulting from chronic, nonresolving inflammation, ultimately leading to livercirrhosis and hepatocellular carcinoma. Efficient and well-tolerated drugs for its treatment areurgently needed. The aim of this study was to screen anti-hepatofibrotic compounds from sixmedicinal mushrooms in submerged culture by using a cell based model in vitro, investigatethe anti-fibrotic mechanism, and provide the theoretical basis for the study of anti-fibroticdrugs.In this study, we established a rapid colorimetric assay suitable for screening ofanti-hepatofibrotic reagents by using a cell based model. An in vitro cell model was designedby using a hepatic stellate cell (HSC) line CFSC-8B stimulated by transforming growthfactor-β1(TGF-β1). The accumulation of collagen was evaluated using chromogenicprecipitation reaction with Picro-sirius red (PSR) dye solution and quantified byspectrophotometric analysis of the dissolved stain. We found that stimulation with10ng/mLTGF-β1for48h and200μL PSR dye solution were optimal for the TGF-β1induced cellularmodel of liver fibrosis. Treatment with two positive control compounds SB431542andSilybin, and other eleven anti-fibrotic compounds and proteins verified the reliability of thismethod. Bioactivity guided fractionation led to the identification of two compounds fromAntrodia cinnamomea in submerged culture by chromatographic methods (silica gel andHPLC). By ultraviolet, mass spectrometry and nuclear magnetic resonance analysis as well ascomparison with literature reports, the structures of the two compounds were identified asAntrodin A and Antrodin B. PSR staining showed that Antrodin B significantly decreasedcollagen production in a dosage-dependent manner.In order to further clarify the anti-hepatofibrotic mechanism of Antrodin B, the MTT,WST-1, wound-healing, transwell migration assay, qRT-PCR and Western Blotting analysiswere used to determine the cell viability, cell proliferation, cell migration, fibrosis relatedgene expression, and protein expression in the related signaling pathways. We found that theIC50value of Antrodin B incubated with CFSC-8B for48h was59.99μM. Stimulation with10ng/mL PDGF-BB for48h was identified as optimal for the PDGF-BB induced cellularproliferation model. Antrodin B (at6,12,25μM) significantly inhibited PDGF-BB stimulatedcell proliferation, markedly ameliorated cell migration induced by PDGF-BB or TGF-β1,significantly suppressed HSC activation markers α-SMA expression and extracellular matrix(ECM) components Col1, Col3and Fn mRNA expression induced by PDGF-BB or TGF-β1in CFSC-8B cells in a dose-dependent manner. Furthermore, Antrodin B (at6,12μM)blocked the phosphorylation of extracellular signal-regulated kinase (ERK), c-JunNH2-terminal kinase (JNK) and mitogen-activated protein kinases (P38) expressionstimulated by PDGF-BB and also blocked Smad2and Smad3phosphorylation induced byTGF-β1.Collectively, with the help of this simple and rapid method for screening potentialanti-hepatofibrotic drugs, we identified the active compound Antrodin B which has the anti-hepatofibrotic activity from Antrodia cinnamomea using bioactivity guided isolation. Thestudy indicates that Antrodin B has in vitro anti-hepatofibrotic activity by inhibitingPDGF-BB induced cell proliferation, TGF-β1or PDGF-BB induced cell activation, migrationand ECM accumulation, which in part, through negatively regulates PDGF-BB/MAPK andTGF-β1/Smad2/3signaling pathways. Hence, Antrodin B may have potential for further studyas a therapeutic option for hepatic fibrosis.