title: Src/Syk/IRAK1-targeted anti-inflammatory action of Torreya nucifera butanol fraction in lipopolysaccharide-activated RAW264.7 cells.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Tn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1.
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea.
authors.forename: Shi Hyoung
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea.
authors.forename: Jae Gwang
authors.initials: JG
authors.lastname: Park
authors.affiliation: Longevity Science Research Team, AmorePacific R&D Unit, Yongin 17074, Republic of Korea.
authors.forename: Yong Deog
authors.initials: YD
authors.lastname: Hong
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea.
authors.forename: Eunji
authors.initials: E
authors.lastname: Kim
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea.
authors.forename: Kwang-Soo
authors.initials: KS
authors.lastname: Baik
authors.affiliation: Department of Biochemistry, Kangwon National University, Chuncheon 24341, Republic of Korea.
authors.forename: Deok Hyo
authors.initials: DH
authors.lastname: Yoon
authors.affiliation: Research and Business Foundation, Sungkyunkwan University, Suwon 16419, Republic of Korea.
authors.forename: Sunggyu
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Food and Nutrition, School of Foodservice Industry, Chungkang College of Cultural industries, Icheon 17390, Republic of Korea.
authors.forename: Mi-Nam
authors.initials: MN
authors.lastname: Lee
authors.affiliation: Longevity Science Research Team, AmorePacific R&D Unit, Yongin 17074, Republic of Korea.
authors.forename: Ho Sik
authors.initials: HS
authors.lastname: Rho
authors.affiliation: Longevity Science Research Team, AmorePacific R&D Unit, Yongin 17074, Republic of Korea. Electronic address: ssshin@amorepacific.com.
authors.forename: Song Seok
authors.initials: SS
authors.lastname: Shin
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 16419, Republic of Korea. Electronic address: jaecho@skku.edu.
authors.forename: Jae Youl
authors.initials: JY
authors.lastname: Cho
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000302:isolation & purification / Q000494:pharmacology*; D000440:Butanols / Q000737:chemistry*; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D051379:Mice; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D008517:Phytotherapy; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D010946:Plants, Medicinal; D000067996:RAW 264.7 Cells; D015398:Signal Transduction / Q000187:drug effects; D012997:Solvents / Q000737:chemistry*; D000072377:Syk Kinase / Q000378:metabolism*; D032490:Taxaceae / Q000737:chemistry*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D000893:Anti-Inflammatory Agents; D000440:Butanols; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D010936:Plant Extracts; C498144:Rela protein, mouse; D012997:Solvents; D018808:Transcription Factor AP-1; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D019061:src-Family Kinases; D053592:Interleukin-1 Receptor-Associated Kinases; C507769:Irak1 protein, mouse; D015232:Dinoprostone
keywords: AP-1; Anti-inflammatory activities; IRAK1; NF-κB; Src; Syk; Taxaceae; Torreya nucifera