title: Biphasic modulation of NOS expression, protein and nitrite products by hydroxocobalamin underlies its protective effect in endotoxemic shock: downstream regulation of COX-2, IL-1β, TNF-α, IL-6, and HMGB1 expression.
abstract: BACKGROUND
  NOS/•NO inhibitors are potential therapeutics for sepsis, yet they increase clinical mortality. However, there has been no in vivo investigation of the (in vitro) •NO scavenger, cobalamin's (Cbl) endogenous effects on NOS/•NO/inflammatory mediators during the immune response to sepsis.
  
  
  METHODS
  We used quantitative polymerase chain reaction (qPCR), ELISA, Western blot, and NOS Griess assays, in a C57BL/6 mouse, acute endotoxaemia model.
  
  
  RESULTS
  During the immune response, pro-inflammatory phase, parenteral hydroxocobalamin (HOCbl) treatment partially inhibits hepatic, but not lung, iNOS mRNA and promotes lung eNOS mRNA, but attenuates the LPS hepatic rise in eNOS mRNA, whilst paradoxically promoting high iNOS/eNOS protein translation, but relatively moderate •NO production. HOCbl/NOS/•NO regulation is reciprocally associated with lower 4 h expression of TNF-α, IL-1β, COX-2, and lower circulating TNF-α, but not IL-6. In resolution, 24 h after LPS, HOCbl completely abrogates a major late mediator of sepsis mortality, high mobility group box 1 (HMGB1) mRNA, inhibits iNOS mRNA, and attenuates LPS-induced hepatic inhibition of eNOS mRNA, whilst showing increased, but still moderate, NOS activity, relative to LPS only. experiments (LPS+D-Galactosamine) HOCbl afforded significant, dose-dependent protection in mice.
  
  
  CONCLUSIONS
  HOCbl produces a complex, time- and organ-dependent, selective regulation of NOS/•NO during endotoxaemia, corollary regulation of downstream inflammatory mediators, and increased survival. This merits clinical evaluation.
authors.affiliation: The William Harvey Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, UK.
authors.forename: André L F
authors.initials: AL
authors.lastname: Sampaio
authors.forename: Jesmond
authors.initials: J
authors.lastname: Dalli
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Brancaleone
authors.forename: Fulvio
authors.initials: F
authors.lastname: D'Acquisto
authors.forename: Mauro
authors.initials: M
authors.lastname: Perretti
authors.forename: Carmen
authors.initials: C
authors.lastname: Wheatley
mesh_terms: D000818:Animals; D015153:Blotting, Western; D051546:Cyclooxygenase 2 / Q000378:metabolism; D019446:Endotoxemia / Q000378:metabolism; D005688:Galactosamine / Q000378:metabolism; D024243:HMGB1 Protein / Q000378:metabolism*; D006879:Hydroxocobalamin / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008168:Lung / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D024243:HMGB1 Protein; D053583:Interleukin-1beta; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D005688:Galactosamine; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D051546:Cyclooxygenase 2; D006879:Hydroxocobalamin