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Eriocitrin: implications on pro-resolutive markers of inflammation


We recently found that erythritrine (ERIOC) has anti-inflammatory, antioxidant properties and has been shown to stimulate pro-resolving factors. Therefore, information on the impact of ERIOC in stimulating markers and pro-resolving mechanisms requires careful clearance, which will contribute to the solid construction of more comprehensive and eventually applied scientific content. This proposal focuses on detecting the active exchange of pro-inflammatory mediators for the production of pro-resolution mediators using citrus flavonoids (ERIOC) in a strategy to accelerate the inflammation resolution phase, avoiding chronic inflammatory stimuli. The hypothesis is that dietary supplementation with ERIOC modulates the inflammatory immune process, stimulating anti-inflammatory and pro-resolving events.Subproject # 1 aims to assess, in vivo, the impact of dietary supplementation with ERIOC on the nature and action of the leukocyte infiltrate during the inflammatory reaction in periodontal tissues, as well as on the generation of chemical mediators, in an experimental protocol for inflammatory disease. periodontal induced by LPS injections. Mice will be treated with a standard diet or diet enriched with ERIOC for 60 days at concentrations of 25 and 50 mg/kg of body weight/day. On the 30th day, the animals will be submitted to LPS injections of Escherichia coli in the gingival tissues adjacent to the palatal surface of the first maxillary molars, 3x /week for 4 weeks. At the end of the experimental period, the mice will be euthanized and the samples collected for analysis of bone volume, through computerized microtomography; microscopic analysis of the jaws, liver tissue and gastric mucosa; quantification of lipoxin A4 and evaluation of the expression of cytokines IL-1², TNF±, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, TGF², GM-CSF by multiplex immunoassay; immunoenzymatic assay (ELISA) for the evaluation of leukotriene B4 and prostaglandin E2; immunohistochemistry for marking macrophages (M1 / M2) and the transcription factor PPAR-³ in the jaws. Subproject # 2: evaluate, in vitro, the action of ERIOC on the activity of fibroblasts and macrophages. For this, L929 fibroblasts will be cultured concomitantly with different treatments according to each group. Analyzes of cell proliferation and cytotoxicity (Alamar blue and MTT - 1, 3 and 7 days) will be performed; migratory activity (scratch test - 24 and 72hrs); collagen synthesis (Hydroxyproline and Picrosirius - 3 and 7 days); expression of FGF and TGF-²1 growth factors (ELISA - 3 and 7 days). In the macrophage assay (RAW264.7), cells will be incubated with Escherichia coli LPS + IFN-³ and simultaneously treated with eriocitrin for 48 hours. qPCR analyzes will be performed to evaluate the expression of M1 and M2 phenotype markers, quantification of M1 and M2 phenotypes by flow cytometry and evaluation of transcription factors STAT1, STAT6 and PPAR-³ by Western blotting. The expression of cytokines IL-1², IL-4, IL-13, TNF-±, GM-CSF, TGF-² and IL-10 will also be evaluated by multiplex immunoassay. At the end of the study, it is expected to propose strategies to promote the resolution process of inflammatory diseases. (AU)

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