title: Syk and Src are major pharmacological targets of a Cerbera manghas methanol extract with kaempferol-based anti-inflammatory activity.
abstract: BACKGROUND
  Cerbera manghas L. (Apocynaceae), a semi-mangrove medicinal plant distributed throughout tropical and subtropical countries, is traditionally known to possess analgesic, anti-inflammatory, anti-convulsant, cardiotonic, and hypotensive activity. In vitro and in vivo anti-inflammatory activities of a methanol extract of the leaves of Cerbera manghas and the underlying molecular mechanisms were investigated to validate the ethnopharmacological use of this plant.
  
  
  METHODS
  The effect of Cerbera manghas methanol extract (Cm-ME) on the production of inflammatory mediators and the induction of HCl/EtOH-treated gastritis was explored using macrophages, HEK293 cells, and ICR mice. The molecular targets of this extract and potential active components in Cm-ME were also investigated.
  
  
  RESULTS
  Cm-ME inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS)-treated RAW264.7 cells and peritoneal macrophages in a dose-dependent manner. This extract also suppressed the expression of NO synthase (iNOS) and cyclooxygenase (COX)-2. NF-κB-mediated enhancement of luciferase activity, nuclear translocation of p50 and p65, and phosphorylation of IκBα were markedly reduced by Cm-ME treatment. Direct enzyme assays, reporter gene assays, and immunoprecipitation analysis of kinases revealed Syk and Src as immunopharmacological targets of Cm-ME. Moreover, this extract strongly ameliorated the gastric symptoms induced by HCl/EtOH treatment of mice. Finally, HPLC analysis and pharmacological tests identified kaempferol as an active component of the extract with Src/Syk inhibitory activities.
  
  
  CONCLUSIONS
  Inhibition of Syk/Src and the NF-κB pathway by kaempferol could play a key role in the anti-inflammatory pharmacological action of Cerbera manghas.
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Hye Yoon
authors.initials: HY
authors.lastname: Jeong
authors.affiliation: Department of Herbal Crop Research, National Institutes of Horticultural & Herbal Science, Rural Development Administration, Suwon 441-707, Republic of Korea.
authors.forename: Gi-Ho
authors.initials: GH
authors.lastname: Sung
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Ji Hye
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Ju Young
authors.initials: JY
authors.lastname: Yoon
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Jae Gwang
authors.initials: JG
authors.lastname: Park
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Shi Hyoung
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Young-Su
authors.initials: YS
authors.lastname: Yi
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Woo Seok
authors.initials: WS
authors.lastname: Yang
authors.affiliation: Department of Biochemistry, Kangwon National University, Chuncheon 200-701, Republic of Korea.
authors.forename: Deok Hyo
authors.initials: DH
authors.lastname: Yoon
authors.affiliation: Department of Biochemistry, Kangwon National University, Chuncheon 200-701, Republic of Korea.
authors.forename: Tae Woong
authors.initials: TW
authors.lastname: Kim
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Biosafety Research Institute, Chonbuk National University, Jeonju 561-756, Republic of Korea. Electronic address: jhkim1@chonbuk.ac.kr.
authors.forename: Jong-Hoon
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, 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 / Q000494:pharmacology* / Q000627:therapeutic use; D029062:Apocynaceae / Q000737:chemistry; D002460:Cell Line; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics; D000431:Ethanol; D005756:Gastritis / Q000139:chemically induced / Q000188:drug therapy; D057809:HEK293 Cells; D006801:Humans; D006851:Hydrochloric Acid; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D044949:Kaempferols / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008297:Male; D000432:Methanol / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D010936:Plant Extracts / Q000494:pharmacology* / Q000627:therapeutic use; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D000072377:Syk Kinase; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D000893:Anti-Inflammatory Agents; D047908:Intracellular Signaling Peptides and Proteins; D044949:Kaempferols; D016328:NF-kappa B; D010936:Plant Extracts; D012333:RNA, Messenger; D012997:Solvents; D009569:Nitric Oxide; D000431:Ethanol; C006552:kaempferol; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D019061:src-Family Kinases; D006851:Hydrochloric Acid; D000432:Methanol
keywords: 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole; AP-1; Anti-inflammatory effect; Apocynaceae; CMC; COX; Cerbera manghas L.; EIA; IKK; Inflammatory mediator; IκB kinase; IκBα; JNK; LPS; MTT; MyD88; NF-κB; NO; PEI; PG; PI3K; PMA; Phorbol 12-myristate 13-acetate; RT-PCR; Src; Syk; TLR; activator protein-1; c-Jun N-terminal kinase; cyclooxygenase; enzyme immunoassay; iNOS; inducible NO synthase; inhibitor of kappa B alpha; lipopolysaccharide; myeloid differentiation primary response gene (88); nitric oxide; nuclear factor-κB; phosphoinositide-3-kinase; polyethylenimine.; prostaglandin; reverse transcriptase-polymerase chain reaction; sodium carboxymethylcellulose; spleen tyrosine kinase; toll-like receptors (TLR)