LEMON-DERIVED EXTRACELLULAR VESICLES EXERT ANTI-INFLAMMATORY EFFECTS BY INHIBITING THE ERK/NF-KB PATHWAY

Abstract

nflammation can be the leading cause of several diseases, including cancer, diabetes, and ulcerative colitis; however, the existing anti-inflammatory drugs cause side effects. For these reasons, it is necessary to find new supportive therapeutic agents. In recent years, plant-derived extracellular vesicles (PDEVs) are gaining increasing interest in the scientific community as they have been found to possess a variety of biological properties; among those, the anti-inflammatory effects have been described by different groups that evaluated the effects of PDEVs in in vitro and in vivo models. We successfully isolated and characterized extracellular vesicles from Citrus limon juice (LEVs) and we studied their anti-inflammatory properties on in vitro and ex-vivo models: murine macrophages (RAW264.7) and immune cells isolated from healthy donors. LEVs were isolated by differential centrifugation followed by a final ultracentrifugation step. Further, biophysical analyses confirmed the size and morphology of LEVs. Also, through metabolomics analysis by means of HPLCESI-Q-ToF-MS, we characterized flavonoids, limonoids and lipids contained in the LEVs. Comparing the vesicle content with the total juice and LEVs-deprived juice, we observed an enrichment in the lipid component for LEVs. We then tested the toxicity of LEVs on target cells at different doses and time points and we observed that LEVs did not affect RAW and primary immune cell growth. To assess the role of EVs on inflammatory stimuli, we pre-treated both cell types for 24h with LEVs and subsequently stimulated them with LPS. Through RTPCR, ELISA and FACS analysis, we saw that the pre-treatment with LEVs decreased the gene and protein expression levels of the pro-inflammatory cytokines IL-6, IL-1β, and TNFα. Besides, we observed a reduction in the gene expression of prostaglandin COX-2, compared to LPS alone. Similarly, pretreatment with LEVs followed by stimulation with LPS in human monocytes, down-regulated TNFα levels compared to LPS alone. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is a key pro-inflammatory pathway that can be activated by the ERK 1/2 signalling cascade. By confocal analysis and western blot, we observed that the pre-treatment with LEVs reduced the nuclear translocation of NF-kB in RAW stimulated with LPS as well as its phosphorylation. NF-kB modulation is associated with the decrease of ERK phosphorylation. In conclusion, LEVs showed promising anti-inflammatory properties, both in vitro and ex vivo, by reducing the production of pro-inflammatory cytokines through the ERK/NF-kB pathway