title: Esculentoside A suppresses lipopolysaccharide-induced pro-inflammatory molecule production partially by casein kinase 2.
abstract: BACKGROUND
  Esculentoside A (EsA) is a saponin isolated from the root of Phytolacca esculenta, an herb which has long been used in Traditional Chinese Medicine for various inflammatory diseases. EsA has been reported to have potent anti-inflammatory properties both in vitro and in vivo.
  
  
  OBJECTIVE
  The present study focused on the molecular mechanism of EsA for its anti-inflammatory effects in RAW264.7 cells stimulated with lipopolysaccharide (LPS).
  
  
  RESULTS
  Enzyme Linked Immunosorbent Assay (ELISA) showed EsA dose dependently inhibited the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide in RAW264.7 cells. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay further confirmed the suppression of LPS-induced TNF-α, IL-6 and iNOS gene expression by EsA on a transcriptional level. Moreover, EsA treatment markedly suppressed LPS-stimulated IκB phosphorylation and degradation as well as LPS-stimulated luciferase reporter construct driven by κB response elements in RAW264.7 cells. In addition, EsA significantly reduced LPS-induced stimulation of p38 and JNK, but not ERK1/2, phosphorylation. Furthermore, we used a computational method called "reverse docking" to search the possible binding proteins of EsA from the potential drug target database (PDTD), and focused on CK2 as the primary binding protein of EsA. Afterward, we further tested EsA directly interacts with recombinant CK2 using SPR assay. In CK2 kinase activity assay, EsA inhibited recombinant CK2 holoenzyme activity obviously in a dose-dependent manner. In addition, TBB (4, 5, 6, 7-tetrabromo-2-benzotriazole, a pharmacological inhibitor of CK2) blocked IL-6 release in a dose-dependent manner, whereas co-treatment of cells with EsA and TBB did not have an additive effect.
  
  
  CONCLUSIONS
  Taken together, these results indicate that EsA blocks the LPS-induced pro-inflammatory molecules expression, at least in part, by impediment of LPS-triggered activation of NF-κB and p38/JNK MAPK pathways in macrophages. Furthermore, we discovered for the first time EsA as a ligand for CK2, which was involved in the inhibition of EsA to the expression of inflammatory cytokines. These findings extended our understanding on the cellular and molecular mechanisms responsible for the anti-inflammatory activity of EsA.
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Li
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Cao
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Qiu
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Weiheng
authors.initials: W
authors.lastname: Xu
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Song
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Lu
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China. Electronic address: zhenlinhu@hotmail.com.
authors.forename: Zhenlin
authors.initials: Z
authors.lastname: Hu
authors.affiliation: School of Pharmacy, Second Military Medical University, Shanghai 200433, China. Electronic address: jpzhang08@163.com.
authors.forename: Junping
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D047390:Casein Kinase II / Q000502:physiology*; D002478:Cells, Cultured; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D015850:Interleukin-6 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D012503:Saponins / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D004365:Drugs, Chinese Herbal; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012503:Saponins; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; C076297:esculentoside A; D009828:Oleanolic Acid; D047390:Casein Kinase II; D020928:Mitogen-Activated Protein Kinases
keywords: Casein kinase 2; Esculentoside A; Immunological suppression; Molecular target