title: Ethanol extract from a Chinese herbal formula, "Zuojin Pill", inhibit the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 mouse macrophages.
abstract: BACKGROUND
  Zuojin Pill (ZJP), a traditional Chinese medicinal decoction that has been used in treating gastritis, gastric ulcer since 15th century, contains two herbs: Rhizoma Coptidis and Fructus Evodiae in the ratio of 6:1 (w/w). Alkaloids are the main active principles contributing to ZJP's efficacy, but anti-inflammatory mechanism has not been fully clarified.
  
  
  OBJECTIVE
  The objective of the study is to reveal anti-inflammatory molecular mechanism of ethanol extract from ZJP, which would form an additional proof to the traditional experience of ZJP in clinical administration.
  
  
  METHODS
  Seven alkaloids were determined from the ethanol extract of ZJP using high performance liquid chromatography (HPLC) with the gradient mobile phase. The ethanol extract from ZJP were used to evaluate the anti-inflammatory action in murine macrophage cell line RAW 264.7 treated with lipopolysaccharide (LPS). Production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. Proteome profiler array was analyzed to evaluate 40 cytokines at protein level. In addition, interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) synthesis were analyzed using ELISA to confirm the result of the Proteome profiler array. The gene expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-6, and interleukin 1β (IL-1β) were detected by quantitative real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). Furthermore, the nuclear translocation of the NF-κB p50 and p65 subunits was detected with ELISA.
  
  
  RESULTS
  The secretions of NO, PGE(2) and the mRNA expression of iNOS, COX-2 were significantly inhibited, moreover, the protein and mRNA expressions of IL-6, IL-1β and TNF-α were inhibited by preventing the nuclear translocation of the NF-κB p50 and p65 subunits. The proteome profiler array showed that 15 cytokines and chemokines involved in the inflammatory process were down-regulated by ZJP.
  
  
  CONCLUSIONS
  These results suggest that the anti-inflammatory properties of ethanol extract from ZJP might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1β, and TNF-α expression through preventing the nuclear translocation of the NF-κB p50 and p65 subunits in RAW 264.7 cells. In addition, these results provided evidence to understand the therapeutic effects of ZJP on gastritis, gastric ulcer, and other inflammatory diseases in clinic.
authors.affiliation: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China.
authors.forename: Qiang-Song
authors.initials: QS
authors.lastname: Wang
authors.forename: Yuan-Lu
authors.initials: YL
authors.lastname: Cui
authors.forename: Tian-Jiao
authors.initials: TJ
authors.lastname: Dong
authors.forename: Xin-Fang
authors.initials: XF
authors.lastname: Zhang
authors.forename: Ke-Ming
authors.initials: KM
authors.lastname: Lin
mesh_terms: D000470:Alkaloids / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D000431:Ethanol / Q000737:chemistry*; D018836:Inflammation Mediators / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology; D051379:Mice; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D008517:Phytotherapy; D010946:Plants, Medicinal; D040081:Protein Array Analysis; D021381:Protein Transport; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012997:Solvents / Q000737:chemistry*; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism
chemical_list: D000470:Alkaloids; D000893:Anti-Inflammatory Agents; D016207:Cytokines; D004365:Drugs, Chinese Herbal; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D052002:NF-kappa B p50 Subunit; D012333:RNA, Messenger; C498144:Rela protein, mouse; D012997:Solvents; D051996:Transcription Factor RelA; C545320:zuojin; D009569:Nitric Oxide; D000431:Ethanol; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D015232:Dinoprostone