Maiara Piva,1 Marília F Manchope,1,2 Fernanda Barbosa-Costa,1 Beatriz H S Bianchini,1 Ketlem C Andrade,1 Letícia Coelho Silva,1 Cássia Calixto-Campos,1 Fernanda S Rasquel-Oliveira,1,3 Victor Fattori,1,3 Ana Carla Zarpelon-Schutz,1 Doumit Camilios-Neto,4 Sergio M Borghi,1 Rubia Casagrande,5 Waldiceu A Verri1 1Department of Immunology, Parasitology and General Pathology, State University of Londrina, Londrina, Paraná, Brazil; 2Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Bavaria, Germany; 3Department of Surgery, Boston Children’s Hospital-Harvard Medical School, Boston, MA, USA; 4Department of Biochemistry and Biotechnology, Londrina State University, Londrina, Paraná, Brazil; 5Department of Pharmaceutical Sciences, Londrina State University, Londrina, Paraná, BrazilCorrespondence: Sergio M Borghi; Waldiceu A Verri, Email sergio.borghi@cogna.com.br; sergio_borghi@yahoo.com.br; waldiceujr@yahoo.com.br; waverri@uel.brBackground: Potassium superoxide (KO2), a superoxide anion donor, can be applied to induce reactive oxygen species (ROS) triggered pain and inflammation. trans-Chalcone (TC) is an atypical flavonoid because its molecular structure does not possess intrinsic antioxidant properties. This characteristic allows investigating the mechanisms of action of flavonoids excluding inherent chemical antioxidant effect. In the present study, we investigated the activity and mechanisms of TC in a model of inflammation and pain triggered by a superoxide anion donor, which to our knowledge have not been assessed yet.Methods: Overt pain-like behavior, mechanical hyperalgesia, edema, leukocyte recruitment, oxidative stress markers, cytokine dosage by enzyme-linked immunosorbent assay (ELISA), nuclear factor kappa B (NF-κB) phosphorylation by Western blotting, mRNA expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and neuronal activity by calcium levels were assessed. TC was administered orally 30 min before stimulation with KO2, and a dose of 30 mg/kg was selected based on previous study.Results: TC inhibited abdominal contortion, mechanical hyperalgesia, paw edema, and myeloperoxidase activity (an indirect marker of macrophage/neutrophil recruitment). TC induced antioxidant activity (assessed by ferric reducing ability and free radical scavenging), while reducing superoxide anion production and lipid peroxidation, at least in part, by upregulating Nrf2 and downregulating Gp91phox and Cox-2 mRNA expression. TC inhibited KO2-induced NF-κβ phosphorylation as well as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-33 production. Finally, TC reduced the activation of transient receptor potential vanilloid 1 (TRPV1+) and transient receptor potential ankyrin 1 (TRPA1+) nociceptive neurons in the dorsal root ganglia.Conclusion: These results demonstrate that the in vivo anti-inflammatory and analgesic activities of TC involve neuronal and non-neuronal mechanisms, and that non-antioxidant flavonoids are still biologically active.Keywords: analgesic, potassium superoxide, TRPV1, TRPA1, nociception, flavonoids