title: Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin.
abstract: Hyperbilirubinemia may lead to encephalopathy in neonatal life, particularly in premature infants. Although the mechanisms were never established, clinicians commonly consider sepsis as a risk factor for bilirubin-induced neurological dysfunction (BIND). Our previous studies showed that elevated levels of unconjugated bilirubin (UCB) have immunostimulant effects, which are potentiated by lipopolysaccharide (LPS), and that immature neural cells are more vulnerable to UCB. The present study was undertaken to explore the role of nitric oxide (NO)/NO synthase (NOS), c-Jun N-terminal kinases (JNK) 1/2 and caspase activation in BIND, as well as the additional effects of inflammation, in immature neurons, incubated from 1 h to 24 h, at 37°C. UCB, at conditions mimicking those of jaundiced newborns (UCB/serum albumin=0.5), induced NO production, neuronal NOS (nNOS) expression and JNK1/2 activation in 3 days in vitro neuron cultures. As a consequence of these events, mitochondrial and extrinsic pathways of apoptosis were initiated, ultimately leading to neuronal dysfunction. Co-incubation with TNF-α+IL-1β intensified the activation of NO/NOS, JNK1/2, caspase-8, caspase-9 and caspase-3 by UCB. Cleavage of Bid into truncated Bid (tBid), as well as increased cytotoxic potential, were also observed. Interestingly, both L-NAME (NOS inhibitor) and SP600125 (JNK1/2 inhibitor) reversed the effects produced by UCB either alone, or in association with pro-inflammatory cytokines. Taken together, our data reveal not only that activation of NO/NOS, JNK1/2 and caspase cascades are important determinants of BIND, but also that the association of TNF-α+IL-1β have cumulative effects. These events provide a reason for the risk of sepsis in BIND and point to potential targets for therapeutic intervention.
authors.affiliation: Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal.
authors.forename: Ana R
authors.initials: AR
authors.lastname: Vaz
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Silva
authors.forename: Andreia
authors.initials: A
authors.lastname: Barateiro
authors.forename: Adelaide
authors.initials: A
authors.lastname: Fernandes
authors.forename: Ana S
authors.initials: AS
authors.lastname: Falcão
authors.forename: Maria A
authors.initials: MA
authors.lastname: Brito
authors.forename: Dora
authors.initials: D
authors.lastname: Brites
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D001663:Bilirubin / Q000494:pharmacology*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007249:Inflammation / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism* / Q000494:pharmacology; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000873:Anthracenes; D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; C432165:pyrazolanthrone; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D048670:MAP Kinase Kinase 4; D020169:Caspases; D001663:Bilirubin; D019331:NG-Nitroarginine Methyl Ester