ABSTRACT
Increasing evidence demonstrates that melatonin has an anti-inflammatory effect. Nevertheless, the molecular mechanisms remain obscure. In this study, we investigated the effect of melatonin on toll-like receptor 4 (TLR4)-mediated molecule myeloid differentiation factor 88 (MyD88)-dependent and TRIF-dependent signaling pathways in lipopolysaccharide (LPS)-stimulated macrophages. RAW264.7 cells were incubated with LPS (2.0 µg/mL) in the absence or presence of melatonin (10, 100, 1000 µm). As expected, melatonin inhibited TLR4-mediated tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, and IL-10 in LPS-stimulated macrophages. In addition, melatonin significantly attenuated LPS-induced upregulation of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in macrophages. Further analysis showed that melatonin inhibited the expression of MyD88 in LPS-stimulated macrophages. Although it had no effect on TLR4-mediated phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK), melatonin significantly attenuated the activation of nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages. In addition, melatonin inhibited TLR4-mediated Akt phosphorylation in LPS-stimulated macrophages. Moreover, melatonin significantly attenuated the elevation of interferon (IFN)-regulated factor-3 (IRF3), which was involved in TLR4-mediated TRIF-dependent signaling pathway, in LPS-stimulated macrophages. Correspondingly, melatonin significantly alleviated LPS-induced IFN-ß in macrophages. In conclusion, melatonin modulates TLR4-mediated inflammatory genes through MyD88-dependent and TRIF-dependent signaling pathways.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Anti-Inflammatory Agents/pharmacology , Inflammation Mediators/metabolism , Inflammation/prevention & control , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Melatonin/pharmacology , Myeloid Differentiation Factor 88/metabolism , Signal Transduction/drug effects , Toll-Like Receptor 4/drug effects , Adaptor Proteins, Vesicular Transport/genetics , Animals , Cell Line , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cytokines/genetics , Cytokines/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Gene Expression Regulation , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Mitogen-Activated Protein Kinases/metabolism , Myeloid Differentiation Factor 88/genetics , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/metabolism , Time Factors , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolismABSTRACT
Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure and is primarily caused by cytochrome P450 (CYP) 2E1-driven conversion of APAP into hepatotoxic metabolites. Several reports showed that melatonin attenuated APAP-induced acute liver failure. Nevertheless, the exact mechanism remains obscure. In the present study, we investigated the effects of melatonin on apoptosis-inducing factor (AIF)-dependent cell death in APAP-induced acute liver failure. Mice were intraperitoneally (i.p.) injected with different doses of melatonin (1.25, 5, 20 mg/kg) 30 min before APAP (300 mg/kg, i.p.). As expected, melatonin significantly alleviated APAP-induced cell death, as determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Further analysis showed that melatonin significantly attenuated APAP-induced activation of the serine/threonine kinase receptor interacting protein 1 (RIP1). In addition, melatonin inhibited APAP-induced hepatic c-Jun N-terminal kinase (JNK) phosphorylation and mitochondrial Bax translocation. Correspondingly, melatonin inhibited APAP-induced translocation of AIF from mitochondria to nuclei. Interestingly, no changes were induced by melatonin on hepatic CYP2E1 expression. In addition, melatonin had little effect on APAP-induced hepatic glutathione (GSH) depletion. In conclusion, melatonin protects against AIF-dependent cell death during APAP-induced acute liver failure through its direct inhibition of hepatic RIP1 and subsequent JNK phosphorylation and mitochondrial Bax translocation.