title: In silico analysis and experimental validation of molecular mechanisms of salvianolic acid A-inhibited LPS-stimulated inflammation, in RAW264.7 macrophages.
abstract: OBJECTIVE
  The aim of this study was to explore mechanisms by which salvianolic acid A (SAA) revealed its anti-inflammatory activity, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.
  
  
  METHODS
  Nitric oxide (NO) concentration was determined by the Griess reaction and cell viability was assessed by MTT assay. Interleukin-6, TNFα and interleukin-1β were determined by ELISA. The RAW264.7 cells were transfected with siRNA against p38 or HO-1. Expressions of COX-2, inducible NO synthase (iNOS), NF-κB, HO-1, p-p38 and phosphorylation of IκB kinase α/β were detected by western blotting. Potential targets of SAA were analysed by homology modelling, target prediction, protein-protein interaction prediction and docking studies.
  
  
  RESULTS
  Salvianolic acid A suppressed LPS-triggered production of NO, TNFα and Interleukin-6. It also reduced protein expression of inducible NO synthase and COX-2, and reduced translocation of NF-κB to nuclei. Moreover, SAA promoted expression of phosphorylated p38, and downstream HO-1. Zn (II) protoporphyrin IX, a specific inhibitor of HO-1, or siRNA against HO-1 could effectively increase transfer of NF-κB. SAA was predicted to target amyloid-beta protein-like protein and arachidonate 5-lipoxygenase, that could regulate p38 and HO-1.
  
  
  CONCLUSIONS
  In silico analysis and experimental validation together demonstrated that SAA exhibited its anti-inflammatory effect via the p38-HO-1 pathway in LPS-stimulated RAW264.7 cells, reduced transfer of NF-κB to the nuclei and thus reduced production of inflammatory mediators.
authors.affiliation: School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, China.
authors.forename: J
authors.initials: J
authors.lastname: Huang
authors.forename: Y
authors.initials: Y
authors.lastname: Qin
authors.forename: B
authors.initials: B
authors.lastname: Liu
authors.forename: G Y
authors.initials: GY
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Ouyang
authors.forename: J H
authors.initials: JH
authors.lastname: Wang
mesh_terms: D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002109:Caffeic Acids / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004798:Enzymes / Q000235:genetics / Q000378:metabolism; D007249:Inflammation; D015850:Interleukin-6 / Q000378:metabolism; D007773:Lactates / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000633:toxicity*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010766:Phosphorylation; D060066:Protein Interaction Maps; D011524:Protoporphyrins / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D016229:Amyloid beta-Peptides; D002109:Caffeic Acids; D004798:Enzymes; D015850:Interleukin-6; D007773:Lactates; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011524:Protoporphyrins; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; C066201:salvianolic acid A; C028025:protoporphyrin IX
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