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Hesperidin methyl chalcone interacts with NFκB Ser276 and inhibits zymosan-induced joint pain and inflammation, and RAW 264.7 macrophage activation


Arthritis can be defined as a painful musculoskeletal disorder that affects the joints. Hesperidin methyl chalcone (HMC) is a flavonoid with analgesic, anti-inflammatory, and antioxidant effects. However, its effects on a specific cell type and in the zymosan-induced inflammation are unknown. We aimed at evaluating the effects of HMC in a zymosan-induced arthritis model. A dose–response curve of HMC (10, 30, or 100 mg/kg) was performed to determine the most effective analgesic dose after intra-articular zymosan stimuli. Knee joint oedema was determined using a calliper. Leukocyte recruitment was performed by cell counting on knee joint wash as well as histopathological analysis. Oxidative stress was measured by colorimetric assays (GSH, FRAP, ABTS and NBT) and RT-qPCR (gp91phox and HO-1 mRNA expression) performed. In vitro, oxidative stress was assessed by DCFDA assay using RAW 264.7 macrophages. Cytokine production was evaluated in vivo and in vitro by ELISA. In vitro NF-κB activation was analysed by immunofluorescence. We observed HMC reduced mechanical hypersensitivity and knee joint oedema, leukocyte recruitment, and pro-inflammatory cytokine levels. We also observed a reduction in zymosan-induced oxidative stress as per increase in total antioxidant capacity and reduction in gp91phox and increase in HO-1 mRNA expression. Accordingly, total ROS production and macrophage NFκB activation were diminished. HMC interaction with NFκB p65 at Ser276 was revealed using molecular docking analysis. Thus, data presented in this work suggest the usefulness of HMC as an analgesic and anti-inflammatory in a zymosan-induced arthritis model, possibly by targeting NFκB activation in macrophages.

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This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), National Council for Scientific and Technological Development (CNPq, Brazil), Funding Authority for Studies and Projects and State Secretariat of Science, Technology and Higher Education (MCTI/FINEP/CT-INFRA-PROINFRA, Brazil; grant agreements 01.12.0294.00 and 01.13.0049.00); the Department of Science and Technology from the Science, Technology and Strategic Inputs Secretariat of the Ministry of Health (Decit/SCTIE/MS, Brazil) intermediated by CNPq with support of Araucária Foundation and State Health Secretariat, Paraná (SESA-PR, Brazil) (PPSUS grant agreement 041/2017, protocol 48.095); Programa de Apoio a Grupos de Excelência (PRONEX) grant supported by SETI/Araucária Foundation and MCTI/CNPq, and Paraná State Government (agreement 014/2017, protocol 46.843).

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TSS and LS-F handled the mice and performed treatments and stimuli administration. FSR-O and MFM were responsible for behavioural testing. FSR-O, THZ, and VF were responsible for the analysis using confocal microscope. FSR-O, MFM, LS-F, TSS, NAA, and SB-G performed experiments. CRF performed molecular docking analysis. FSR-O, MFM, and WAVJ analysed and interpreted data set. WAVJ and RC delineated the study. RC, AdF, and WAVJ were responsible for grants and supplied utilized reagents. FSR-O wrote the first draft. FSR-O, VF, and WAVJ revised and modified the manuscript to its final version. The final version of the manuscript was read and approved by all Authors.

Correspondence to Waldiceu A. Verri Jr..

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted: Londrina State University Ethics Committee on Animal Research and Welfare (approval number 5943.2017.85). Animal care and handling procedures also followed the guideline of the International Association for Study of Pain (IASP) and Brazilian Council on Animal Experimentation (CONCEA).

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Rasquel-Oliveira, F.S., Manchope, M.F., Staurengo-Ferrari, L. et al. Hesperidin methyl chalcone interacts with NFκB Ser276 and inhibits zymosan-induced joint pain and inflammation, and RAW 264.7 macrophage activation. Inflammopharmacol (2020). https://doi.org/10.1007/s10787-020-00686-7

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  • HMC
  • Knee pain
  • Joint pain
  • Flavonoid
  • Molecular docking
  • Experimental arthritis