Disruption of the NF-κB/NLRP3 connection by melatonin requires retinoid-related orphan receptor-α and blocks the septic response in mice García, José A Volt, Huayqui Venegas, Carmen Doerrier, Carolina Escames, Germaine López, Luis C Acuña-Castroviejo, Darío This research was partially supported by grants PI08-1664 and RD12/0043/0005 from the Instituto de Salud Carlos III, Spain, and P07-CTS-03135 from the Conserjería de Innovación, Ciencia y Empresa, Junta de Andalucía, Spain. The results of this study constitute part of J.A.G.’s doctoral thesis under the Biotechnology Doctorate Program of the University of Granada. We determined the NF-kB- and NOD-like receptor (NLR)P3-dependent molecular mechanisms involved in sepsis and evaluated the role of retinoid-related orphan receptor (ROR)-a in melatonin’s anti-inflammatory actions. Western blot, RT-PCR, ELISA, and spectrophotometric analysis revealed that NF-kB and NLRP3 closely interact, leading to proinflammatory and pro-oxidant status in heart tissue of septic C57BL/6J mice. Moreover, mitochondrial oxygen consumption was reduced by 80% in septic mice. In vivo and in vitro analysis showed that melatonin administration blunts NF-kB transcriptional activity through a sirtuin1-dependent NF-kB deacetylation in septic mice. Melatonin also decreased NF-kB-dependent proinflammatory response and restored redox balance and mitochondrial homeostasis, thus inhibiting the NLRP3 inflammasome. In an important finding, the inhibition of NF-kB by melatonin, but not that of NLRP3, was blunted in RORasg/sg mice, indicating that functional RORa transcription factor is necessary for the initiation of the innate immune response against inflammation. Our results are evidence of the NF-kB/NLRP3 connection during sepsis and identify NLRP3 as a novel molecular target for melatonin. The multiple molecular targets of melatonin in this study explain its potent anti-inflammatory efficacy against systemic innate immune activation and herald a promising therapeutic application for melatonin in the treatment of sepsis. 2025-02-03T12:15:31Z 2025-02-03T12:15:31Z 2015-06-04 journal article García et al. The FASEB Journal article fj.15-273656. 2015. doi:10.1096/fj.15-273656 https://hdl.handle.net/10481/101909 10.1096/fj.15-273656 eng open access Wiley