Src/Syk/IRAK1-targeted anti-inflammatory action of Torreya nucifera butanol fraction in lipopolysaccharide-activated RAW264.7 cells

• Published in: Journal of ethnopharmacology 2016-07, Vol.188, p.167-176
• Main Authors: Kim, Shi Hyoung, Park, Jae Gwang, Hong, Yong Deog, Kim, Eunji, Baik, Kwang-Soo, Yoon, Deok Hyo, Kim, Sunggyu, Lee, Mi-nam, Rho, Ho Sik, Shin, Song Seok, Cho, Jae Youl
• Format: Article
• Language: English
• Subjects: Animals; Anti-inflammatory activities; Anti-Inflammatory Agents - isolation & purification; Anti-Inflammatory Agents - pharmacology; AP-1; Butanols - chemistry; Cyclooxygenase 2 - genetics; Cyclooxygenase 2 - metabolism; Dinoprostone - metabolism; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Inflammation Mediators - metabolism; Interleukin-1 Receptor-Associated Kinases - metabolism; IRAK1; Lipopolysaccharides - pharmacology; Macrophage Activation - drug effects; Macrophages - drug effects; Macrophages - enzymology; Mice; NF-κB; Nitric Oxide; Nitric Oxide Synthase Type II - genetics; Nitric Oxide Synthase Type II - metabolism; Phosphorylation; Phytotherapy; Plant Extracts - isolation & purification; Plant Extracts - pharmacology; Plants, Medicinal; RAW 264.7 Cells; Signal Transduction - drug effects; Solvents - chemistry; Src; src-Family Kinases - metabolism; Syk; Syk Kinase - metabolism; Taxaceae; Taxaceae - chemistry; Torreya; Torreya nucifera; Transcription Factor AP-1 - metabolism; Transcription Factor RelA - metabolism; Transfection; Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - metabolism
• ISSN: 0378-8741; 1872-7573
• DOI: 10.1016/j.jep.2016.05.008

Seed of Torreya nucifera (L.) Siebold & Zucc is used to treat several diseases in Asia. Reports document that T. nucifera has anti-cancer, anti-inflammatory, anti-oxidative activities. In spite of numerous findings on its pharmacological effects, the understanding of the molecular inhibitory mechanisms of the plant remains to be studied. Therefore, we aimed to explore in vitro anti-inflammatory mechanisms of ethyl acetate fraction (Tn-EE-BF) prepared from the seed of T. nucifera in LPS-stimulated macrophage inflammatory responses. For this purpose, we measured nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages. Additionally, using RT-PCR, luciferase reporter gene assay, immunoblotting analysis, and kinase assay, the levels of inflammatory genes, transcription factors, and inflammatory signal-regulatory proteins were investigated. Finally, the constituent of Tn-EE-BF was identified using HPLC. Tn-EE-BF inhibits NO and PGE2 production and also blocks mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in a dose dependent manner. Tn-EE-BF reduces nuclear levels of the transcriptional factors NF-κB (p65) and AP-1 (c-Jun and FRA-1). Surprisingly, we found that Tn-EE-BF inhibits phosphorylation levels of Src and Syk in the NF-κB pathway, as well as, IRAK1 at the protein level, part of the AP-1 pathway. By kinase assay, we confirmed that Src, Syk, and IRAK1 are suppressed directly. HPLC analysis indicates that arctigenin, amentoflavone, and quercetin may be active components with anti-inflammatory activities. Tn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1. [Display omitted]