| Antrodia camphorata(syn Taiwanofungus camphoratus and Antrodia cinnamomea), a rare and precious edible fungus which only grows on its host [Cinnamomum kanehirai(Hay)(Lauraceae), has many biological activities, such as anti-tumor, anti-inflammatory and hepatoprotective effect. Hepatic fibrosis is a pathological response of the liver to a variety of chronic stimuli and leads to massive accumulation of extracellular matrix(ECM), ultimately leading to liver cirrhosis and hepatocellular carcinoma. Blocking or reversing hepatic fibrosis is an important treatment for chronic liver disease. The previous study in our laboratory established a rapid colorimetric assay suitable for screening of anti-hepatofibrotic reagents, and found hexane extract from A. camphorata has anti-hepatic fibrosis activity and isolated and identified an active compound Antrodin B. In this study, we continued to isolate anti-hepatofibrotic compounds(Antrodin B and Antrodin C) in hexane extract from A. camphorata and established carbon tetrachloride(CCl4) induced mouse model of liver fibrosis to clarify the anti-hepatofibrotic mechanism of Antrodin B and Antrodin C. The main results are as follows:(1) Study of separation of Antrodin C from A. camphorata and its anti-hepatofibrotic activity. In this study, we isolated and identified a compound Antrodin C in n-hexane extract from A. camphorata by silica gel chromatography and HPLC purification, which structure was confirmed using UV, mass spectrometry and nuclear magnetic resonance analysis as well as comparison with literature reports. We established a cell prolferation model of liver fibrosis by using TGF-β1 stimulated CFSC-8B. The IC50 value of Antrodin C was 147.91 μM in CFSC-8B for 24 h; Antrodin C(12, 25, 50 μM) significantly inhibited cell proliferation induced by TGF-β1 in a dosage-dependent manner.(2) Study of anti-hepatofibrotic mechanism of Antrodin C in vitro. In order to clarify the anti-hepatofibrotic mechanism of Antrodin C, cell proliferation, cell migration, extracellular matrix(ECM) production and protein expression in the TGF-β1 or PDGF-BB signaling pathways were detected by WST-1 assay, cell migration assay, qRT-PCR and Western blotting analyses. The results showed that Antrodin C(12, 25, 50 μM) significantly inhibited TGF-β1 or PDGF-BB stimulated cell migration and ECM production. Furthermore, Antrodin C blocked the phosphorylation of Smad2(p-Smad2), serine/threonine kinase(p-AKT), extracellular signal-regulated kinase(ERK) and mitogen-activated protein kinases(P38) expression stimulated by TGF-β1 and also blocked p-AKT and p-ERK protein expression levels induced by PDGF-BB.(3) Study of anti-liver fibrosis activity of Antrodin B and Antrodin C from A. camphorata in vivo. We eatablished in vivo liver fibrosis model by administrating carbon tetrachloride(CCl4) in mice. The mouse liver fibrosis model was induced by CCl4. Meanwhile, Antrodin B or Antrodin C was administrated to evaluate their pharmaceutical values. The results showed that administration of Antrodin B and Antrodin C both significantly increased body weight compared with CCl4 induced model group. The H&E and Sirius-red staining revealed a marked increase in the extent of liver fibrosis. Antrodin B and Antrodin C treatment reduced the degree of liver fibrosis and ameliorated the increase of ALT, AST and Hyp induced by CCl4 injection. In addition, Antrodin B and Antrodin C also downregulated α-SMA, Col1, Col3, Fn, Tgf-β1 and Pdgf mRNA expression compared with that of CCl4 induced model group. Mechanistically, Antrodin B and Antrodin C suppressed protein expression of α-SMA, Col1, p-Smad2, p-ERK, p-P38 and p-AKT induced by CCl4 in liver. Thus, Antrodin B and Antrodin C could negatively regulate phosphorylation of protein in Smad, PI3 K, MAPK signaling pathways to ameliorate liver fibrosis. |
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