title: Celastrol blocks binding of lipopolysaccharides to a Toll-like receptor4/myeloid differentiation factor2 complex in a thiol-dependent manner.
abstract: BACKGROUND
  Tripterygium wilfordii (lei gong teng; Thunder of God Vine), which belongs to the Celastraceae family, has long been used in traditional Chinese medicine to treat inflammation and rheumatoid arthritis. Celastrol is a bioactive compound isolated from T. wilfordii.
  
  
  OBJECTIVE
  We investigated whether celastrol suppressed binding of lipopolysaccharides (LPS) to myeloid differentiation factor 2 (MD2), thereby downregulating Toll-like receptor4 (TLR4) activation in mouse primary macrophages.
  
  
  METHODS
  Cytokine expression was determined by polymerase chain reaction analysis and enzyme-linked immunosorbent assay in bone marrow-derived primary macrophages (BMDMs). The kinase activity of tank-binding kinase 1 (TBK1) was examined by a luciferase reporter assay and an in vitro kinase assay. LPS binding to MD2 was examined by an in vitro binding assay and confocal microscopy analysis.
  
  
  RESULTS
  Celastrol reduced LPS-induced expression of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, and IL-1β, at both the mRNA and protein levels in BMDMs. Celastrol suppressed LPS binding to MD2, as shown by the in vitro binding assay, whereas it did not inhibit TBK1. In addition, co-localization of LPS with MD2 in BMDMs was blocked by celastrol. The inhibitory effects of celastrol on LPS binding to MD2 were reversed by thiol donors (N-acetyl-L-cysteine and dithiothreitol), suggesting that the thiol reactivity of celastrol contributes to its inhibitory effects on TLR4 activation in macrophages.
  
  
  CONCLUSIONS
  Our results demonstrate that celastrol suppresses TLR4 activation through the inhibition of LPS binding to the TLR4/MD2 complex. These results provide a novel mechanism of action by which celastrol contributes to the anti-inflammatory activity of T. wilfordii.
authors.affiliation: Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon, 420-743 Republic of Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Pharmacology Research Center, Korea Research Institute of Chemical Technology, Daejeon, 305-343 Republic of Korea.
authors.forename: Byung Ho
authors.initials: BH
authors.lastname: Lee
authors.affiliation: Daegu-Gyeongbuk Medical Innovation Foundation, New Drug Development Center, Daegu, 706-010 Republic of Korea.
authors.forename: Nam Doo
authors.initials: ND
authors.lastname: Kim
authors.affiliation: Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon, 420-743 Republic of Korea. Electronic address: joolee@catholic.ac.kr.
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Lee
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000302:isolation & purification / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D008070:Lipopolysaccharides / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008516:Medicine, Chinese Traditional; D051379:Mice; D008810:Mice, Inbred C57BL; D053978:Pentacyclic Triterpenes; D016133:Polymerase Chain Reaction; D013438:Sulfhydryl Compounds / Q000737:chemistry; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D029757:Tripterygium / Q000737:chemistry; D014315:Triterpenes / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D000893:Anti-Inflammatory Agents; D016207:Cytokines; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D053978:Pentacyclic Triterpenes; D013438:Sulfhydryl Compounds; D051197:Toll-Like Receptor 4; D014315:Triterpenes; C050414:celastrol; D004229:Dithiothreitol; D000111:Acetylcysteine
keywords: Celastrol (PubChem CID: 122724); Dithiothreitol (PubChem CID: 446094); Inflammation; Innate immune signaling; Ligand binding to receptor; Lipopolysaccharide (PubChem CID: 11970143); Macrophages; N-Acetyl-(L)-cysteine (PubChem CID: 12035); Pattern-recognition receptor