Effects of Myrcia pubipetala Miq (Myrtaceae) extract on innate inflammatory response

Abstract Myrcia is a genus widespread in South America with many species presenting anti-inflammatory and biological properties. We investigated the anti-inflammatory activity of crude hydroalcoholic extract of Myrcia pubipetala leaves (CHE-MP) using macrophages (RAW 264.7), and the air pouch model in mice to evaluate leukocyte migration and mediator’s release. Adhesion molecule expression, CD49 and CD18, was evaluated in neutrophils. In vitro, the CHE-MP significantly reduced nitric oxide (NO), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) levels in the exudate and the supernatant culture. CHE-MP did not present cytotoxicity and modulated the percentage of positive neutrophils for CD18 and its expression per cell, without modifying the expression of CD49, which corroborated with significantly reduced neutrophil migration to inflammatory exudate and subcutaneous tissue. Taken together, the data demonstrate that CHE-MP presents a potential activity on innate inflammatory. Graphical Abstract


Introduction
inflammation is a complex process with the function of protecting the body in situations of homeostasis impairment.this process is mediated by a series of chemical substances that are simultaneously produced and released by innate immune cells, such as cytokines, arachidonic-acid cascade derivatives, and nitric oxide among others, with multiple immune functions, playing important roles in the inflammatory process (Ebbo et al. 2017).the long-term use of anti-inflammatory drugs can cause serious adverse reactions, such as changes in the gastrointestinal tract, and cardiovascular and liver systems. in this context, bioprospection of natural products plays a key role in the drug development process to find safe and effective medicines to regulate inflammation (Fullerton and Gilroy 2016).
Some species of Myrcia (myrtaceae) have been used in popular medicine for a long time as an infusion to treat gastrointestinal disorders, hemorrhagic states, and infectious diseases (Santos et al. 2009).Myrcia pubipetala is popularly known as 'guamirim ' , 'araçá' or 'goiabão' (Sobral et al. 2015).the only data available regarding this species describes the characterization of essential oil, which presents 25.2% of bicyclogermacrene and 32% of spathulenol (Limberger et al. 2004).
data from the literature demonstrate the high anti-inflammatory potential of the Myrcia genus (araújo et al. 2019). in this context, we present for the first time a biological study with Myrcia pubipetala, with the purpose to evaluate the anti-inflammatory effect of the hydroalcoholic extract obtained from Myrcia pubipetala leaves using in vivo and in vitro approaches.
it is important to mention that the CHE-mp did not present a cytotoxic effect, once the macrophage viability was not reduced in all evaluated concentrations (Figure S1).data presented in Figure S1B demonstrates that LpS induced the release of no by macrophages and the CHE-mp treatment was able to inhibit the production in all tested concentrations, especially in the high concentration, which was chosen to perform the following in vitro experiments.
it is well known that LpS modulates the early release of pro-inflammatory mediators and inflammatory-related enzymes on macrophages through toll-like receptor 4 (tLR4) activation, such as tnF, iL-1β, iL-6, no, CoX-2 (dorrington and Fraser 2019). in this context, our data clearly shows the direct inhibitory actions of CHE-mp on LpS-induced macrophage secretion of cytokines and no.
the modulation of expression of these proteins on neutrophil membranes is triggered by LpS during the inflammation, whether by shedding of Cd62L or increased expression of Cd18. the neutrophils stimulated by LpS and treated with CHE-mp presented inhibition of expression of Cd18, i.e. decrease in the percentage of neutrophils Cd18 + (Figure S2d) and its expression per cell (Figure S2E).CHE-mp did not alter the Cd62L expression (Figure S2B-C).the extract activity on Cd18 expression was not correlated to the cytotoxic effect once the neutrophil viability was not reduced by CHE-mp (Figure S2a).
at the first moment of the inflammatory process occurs hemodynamic changes or production of inflammatory mediators, which increases the Cd62L expression on the leukocyte's membrane.after the activation process, Cd62L is cleaved by adam-17, allowing the activation of Cd18, which is responsible for the steady adhesion of leukocytes to the endothelium (Liew and Kubes, 2019).Based on this knowledge, it is possible that CHE-mp reduces the cell adhesion to the endothelium, and consequently the leukocyte migration to the inflammatory focus.
to elucidate the mechanism throughout which CHE-mp impairs the leukocyte migration, the air pouch model was used to evaluate the in vivo effects of CHE-mp on carrageenan-induced inflammation.in the histological sections (Figure S3a) of air pouch tissue from animals of the vehicle group presented morphological changes of the inflammatory process, such as the thickness of the air pouch tissue, oedema, leukocyte infiltrate, and connective tissue injury.the leukocyte infiltrate was composed mainly of neutrophils, as can be observed by their morphology.
in the other hand, animals treated with CHE-mp the thickness of the air pouches was visibly reduced, as well as oedema and neutrophil migration, similarly to indomethacin.together, the data suggest that the acute inflammatory response in the pouch line tissue was significantly suppressed by CHE-mp.
additionally, the data presented in Figure S3B-d demonstrate that similarly to indomethacin, CHE-mp 3, 30, and 300 mg/kg was able to reduce cell counts, mainly neutrophil (Figure S3C), in the exudate in comparison with the control group.the CHE-mp also reduced the volume of exudate by 30 and 300 mg/kg when compared to the control group (Figure S3d).CHE-mp also reduced no 2 −, iL-1β, iL-6, and tnF levels in the exudate (Figure S4a-d).indomethacin-treated animals presented similar results; however, this compound did not affect no production (Figure S4a).
previous studies demonstrated that some Myrtaceae species can reduce the inflammatory process (araújo et al. 2019).the anti-inflammatory effect observed in CHE-mp may be due to phenolic compounds.Studies have shown that phenolic compounds reduced the translocation of the transcription factor nF-ĸB, necessary for the translation of inflammatory mediators such as cytokines.Flavonoids have demonstrated a decreasing proinflammatory cytokine, pGE 2 , and no levels (Baena and Salinas 2015).