Participação dos mediadores pró-inflamatórios na inibição da migração celular induzida pelo extrato bruto da Cunila spicata em camundongos

Abstract

The respiratory system (RS) is affected by diseases and conditions of varying etiologies, each involving numerous mediators, biochemical, and immunological pathways. Several animal models have been developed to study and research acute RS alterations, such as the inhalation of a phlogistic agent in mice. Carrageenan is a polysaccharide capable of inducing an acute inflammatory response with nonselective release of inflammatory mediators. Cunila spicata is a phytotherapeutic plant belonging to the Spicatae section of the Lamiacae family, native to southern Latin America, also known as field pennyroyal. It has a strong presence in ethnopharmacology, being used to treat diseases of the digestive and respiratory systems, such as nausea, diarrhea, motion sickness, rhinitis, coughs, bronchitis, and flu, and has expectorant properties. Phytochemical analysis of C. spicata collected in the municipality of Santa Catarina's highlands revealed the highest concentrations of 2-Bornanone and Menthofuran. The dried plant was subjected to ultrasound-assisted extraction (30 min, amplitude 65% and cycle 1.0) and rotary evaporator (70°C at 100 RPM). The material was oven-dried, stored in an amber bottle and frozen until use, at which time it was resuspended in 5% DMSO and diluted with PBS to the desired concentration. Bronchoalveolar lavage (BAL) allows the evaluation of local inflammatory phenomena of the RS using different techniques, such as total leukocyte count (TLC) and differential leukocyte count (DLC). The present study analyzed the CTL and CDL of Balb/C mice pretreated with different doses of the hydroalcoholic extract of C. spicata (EHCS) (30mg/kg, 100mg/kg and 300mg/kg), meloxicam (positive control (CP) (2mg/kg)) and PBS (negative control (NC)) V.O by gavage one hour after nebulization with carrageenan (300μg/ml) for 3 minutes. After 2 hours, the animals were euthanized, the BAL was performed with 2ml of heparinized PBS and the samples were centrifuged for 5min at 2000 RPM. The supernatant was reserved for further analysis, and the pellets were resuspended in 1.0 ml of PBS, of which 100 μl was added to 100 μl of Turk's fluid and used in the CTL using a Neubauer chamber with a 40x objective. The remainder was used for the CDL, with cell sedimentation in the Suta chamber, using only enough to fill the hole in the filter paper. The pellet was dried in an oven at 30°C. The pellets were stained with a rapid simple panopticon, and up to 200 cells per slide were counted using an immersion objective lens (1000x magnification). A significant increase in total leukocyte recruitment was observed in the lungs of animals subjected to nebulized carrageenan. EHCS significantly and dose-dependently reduced the migration of total leukocytes in the BAL of mice subjected to nebulized carrageenan. The maximum inhibitions obtained were 49.84 ± 5.35%, 61.66 ± 4.55%, and 75.30 ± 2.86% for doses of 30, 100, and 300 mg/kg, respectively. The cells found in greatest numbers were mononuclear cells, macrophages, and lymphocytes. No signs of toxicity were observed during the period and at the doses used during the experiment. EHCS has potential as a phytotherapeutic product in airway inflammation.