Ethyl acetate fraction of aqueous extract of Lentinula edodes inhibits RANKL-mediated osteoclastogenesis. (A) Schematic representation showing the extraction and fractionation of anti-osteoclastic compounds from L. edodes. (B) BMMs were treated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of 10 μg/mL three extract (water extract, ethyl acetate extract, ethanol extract) of L. edodes for three days, and osteoclast formation was examined by tartrate-resistant acid phosphatase (TRAP) staining (left) and counting the number of TRAP-positive multinuclear osteoclasts (right). (C) BMMs were treated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of the extracts as in (B), and cell proliferation was measured by MTT assays. (D) BMMs were treated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of four fractions (10 μg/mL) of water extract of L. edodes for three days, and osteoclast differentiation was assessed by TRAP staining (left) and the number of TRAP-positive multinuclear osteoclasts (right) was counted. (E) BMMs were treated with the fractions as in (D), and cell proliferation was measured by MTT assays. Error bars represent the mean result ± SD of three independent experiments; * p < 0.05, *** p < 0.001.
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