Melatonin inhibits nitric oxide production and microsomal prostaglandin E synthase-1 expression in LPS-activated murine peritoneal macrophages via NFÒšB signalling pathway

M Aparicio-Soto, C Alarcón de la Lastra, A Cárdeno, M Sánchez-Hidalgo. University of Seville, Department of Pharmacology, 41012, Spain

Background: Melatonin (MLT), an indolamine derived from the aminoacid tryptophan, has in recent years been the subject of intense research within the fields of cancer and inflammation [1]. Aim: In this sense, we investigated the effects of MLT (12.5, 25, 50 and 100 µM) on lipopolysaccharide (LPS)-stimulated inflammatory response in murine peritoneal macrophages according to the procedure described by De la Puerta et al., 2009 [2] and deep insight into the action mechanisms involved in its anti-inflammatory effect. Material and methods: Cell viability was determined using sulphorhodamine B (SRB) assay and nitric oxide (NO) production was measured using the Griess reaction. Moreover, changes in the protein expression of the proinflammatory enzymes cyclooxygenase (COX)-2, the inducible synthase nitric oxide (iNOS) and microsomal prostaglandin (PG)E synthase-1 (mPGEs-1) were determined by western blot. Finally, the role of the nuclear transcription factor kappa B (NFÒšB) signalling pathway was also analyzed by western blot. Results: LPS-induced inflammatory response was characterized by an increase in NO production and an upregulation of COX-2, iNOS and mPGEs-1 protein expressions followed by a remarkable increase in nuclear p65 protein the expression, as a parameter for NF-κB activation. On the contrary, MLT treatment was found to reduce significantly the levels of nitrites in LPS-induced murine peritoneal macrophages (12.5 μM: 35.3± 0.1% p<0.001; 25 μM: 22.6±0.1% p<0.001; 50 μM: 14.1± 0.2% p<0.001; 100μM: 2.8±0.2% p<0.001) without affecting cell viability. Similarly, MLT induced a significant decrease in COX-2 (p<0.05 and p<0.001) iNOS (p<0.05 and p<0.01) and mPGEs-1 (p<0.05) protein expressions, prevented the IƘβα protein degradation (p<0.01) and reduced the expression of the nuclear p65 protein (p<0.01 and p<0.001). Conclusion: We conclude that MLT could exert a protective/preventive role in inflammatory based diseases inhibiting the LPS-induced NO production and mPGEs-1 expression via NFÒšB signalling pathway and thus, providing a molecular basis for its anti-inflammatory activity. References: [1] De la Puerta R, Marquez-Martin A, Fernandez-Arche A, Ruiz-Gutierrez V. Influence of dietary fat on oxidative stress and inflammation in murine macrophages. Nutrition. 2009. (5): 548-54. [2] Radogna F, Diederich M, Ghibelli L. Melatonin: a pleiotropic molecule regulating inflammation. Biochem Pharmacol. 2010. 80(12): 1844-52.