Jang Mi Han1, Eun Kyeong Lee1, So Youn Gong1, Jae Kyung Sohng1, Yue Jai Kang2, Hye Jin Jung3. 1. Department of Pharmaceutical Engineering & Biotechnology, Sun Moon University, 70, Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 336-708, South Korea. 2. Department of Aquatic Life and Medical Sciences, Sun Moon University, 70, Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 336-708, South Korea. Electronic address: kyj5088@hanmail.net. 3. Department of Pharmaceutical Engineering & Biotechnology, Sun Moon University, 70, Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 336-708, South Korea. Electronic address: poka96@sunmoon.ac.kr.
Abstract
ETHNOPHARMACOLOGY RELEVANCE: Sparassis crispa, also known as cauliflower mushroom, has been used historically in traditional Asian medicine. It possesses various biological activities, such as immunopotentiation, anti-diabetes, anti-cancer, and anti-inflammatory effects. Recently, we isolated the non-aqueous fraction from methanol extract of S. crispa (SCF4) by using water-organic solvent mixtures and high-performance liquid chromatography (HPLC). In the present study, we identified the anti-inflammatory activity and action mechanism of SCF4 in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage cells. MATERIALS AND METHODS: The chloroform layer isolated from S. crispa methanol extract was separated into seven fractions using preparative HPLC. The fractions were then applied to NO assay to identify the fraction with the best anti-inflammatory activity. The inflammation inhibitory effect and underlying mechanism of SCF4 in LPS-stimulated RAW264.7 cells were assessed using WST-1 assay, enzyme-linked immunosorbent assay (ELISA), ROS assay, and Western blot analysis. RESULTS: SCF4 significantly suppressed LPS-induced production of pro-inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)- 6, and IL-1β, without cytotoxicity. In addition, SCF4 downregulated not only the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), but also the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) stimulated by LPS. SCF4 also blocked the nuclear translocation of NF-κB via reduction of inhibitor of κB alpha (IκBα) degradation. Furthermore, SCF4 inhibited the phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1), an important upstream factor of NF-κB and MAPK signaling mediated through toll-like receptor (TLR). CONCLUSIONS: These findings demonstrate for the first time the correlation between the anti-inflammatory activity of SCF4 and TLR-mediated NF-κB and MAPK signaling pathways in LPS-stimulated RAW264.7 macrophage cells, suggesting that the non-aqueous extract of S. crispa could be applied as a promising natural product for the prevention and treatment of inflammatory diseases.
ETHNOPHARMACOLOGY RELEVANCE: Sparassis crispa, also known as cauliflower mushroom, has been used historically in traditional Asian medicine. It possesses various biological activities, such as immunopotentiation, anti-diabetes, anti-cancer, and anti-inflammatory effects. Recently, we isolated the non-aqueous fraction from methanol extract of S. crispa (SCF4) by using water-organic solvent mixtures and high-performance liquid chromatography (HPLC). In the present study, we identified the anti-inflammatory activity and action mechanism of SCF4 in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage cells. MATERIALS AND METHODS: The chloroform layer isolated from S. crispa methanol extract was separated into seven fractions using preparative HPLC. The fractions were then applied to NO assay to identify the fraction with the best anti-inflammatory activity. The inflammation inhibitory effect and underlying mechanism of SCF4 in LPS-stimulated RAW264.7 cells were assessed using WST-1 assay, enzyme-linked immunosorbent assay (ELISA), ROS assay, and Western blot analysis. RESULTS: SCF4 significantly suppressed LPS-induced production of pro-inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)- 6, and IL-1β, without cytotoxicity. In addition, SCF4 downregulated not only the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), but also the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) stimulated by LPS. SCF4 also blocked the nuclear translocation of NF-κB via reduction of inhibitor of κB alpha (IκBα) degradation. Furthermore, SCF4 inhibited the phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1), an important upstream factor of NF-κB and MAPK signaling mediated through toll-like receptor (TLR). CONCLUSIONS: These findings demonstrate for the first time the correlation between the anti-inflammatory activity of SCF4 and TLR-mediated NF-κB and MAPK signaling pathways in LPS-stimulated RAW264.7 macrophage cells, suggesting that the non-aqueous extract of S. crispa could be applied as a promising natural product for the prevention and treatment of inflammatory diseases.
Authors: Thanh Q C Nguyen; Tran Duy Binh; Tuan L A Pham; Yen D H Nguyen; Dai Thi Xuan Trang; Trong Tuan Nguyen; Kenji Kanaori; Kaeko Kamei Journal: Int J Mol Sci Date: 2020-05-13 Impact factor: 5.923