Disruption of the NF-κB/NLRP3 connection by melatonin requires retinoid-related orphan receptor-α and blocks the septic response in mice

Abstract

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.