Punica granatum L. Leaf Extract Attenuates Lung Inflammation in Mice with Acute Lung Injury

Pinheiro, Aruanã Joaquim Matheus Costa Rodrigues; Gonçalves, Jaciara Sá; Dourado, Ádylla Wilenna Alves; de Sousa, Eduardo Martins; Brito, Natilene Mesquita; Silva, Lanna Karinny; Batista, Marisa Cristina Aranha; de Sá, Joicy Cortez; Monteiro, Cinara Regina Aragão Vieira; Fernandes, Elizabeth Soares; Monteiro-Neto, Valério; Campbell, Lee Ann; Zago, Patrícia Maria Wiziack; Lima-Neto, Lidio Gonçalves

Treatments. Animals were randomly distributed into six groups (n = 6/group) for lung analysis and five groups (n = 6/group) for bronchoalveolar lavage fluid (BALF) analysis. Mice received a single oral administration (p.o.) of vehicle (saline, 10 ml/kg), EAFPg (30-300 mg/kg), or dexamethasone (5 mg/kg) 30 min prior to LPS challenge. ALI was induced by a single intranasal (i.n.) instillation of LPS (30,000 EU in 50 μl/animal and 25 μl/narine). Vehicletreated mice were used as controls. Four hours after the challenge, mice were culled by anesthetic overdose with ketamine (120 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), and the bronchoalveolar lavage fluid (BALF) was collected. For this, the trachea of each mouse was cannulated and the alveoli were washed three times with 0.5 ml of ice-cold phosphate buffered saline (PBS) containing 0.5% sodium citrate. An aliquot of the BALF was separated and used for quantification of leukocyte counts and the rest centrifuged at 400 ×g for 10 min at 4°C. The supernatant was immediately frozen and stored at −80°C for further analysis. In a separate series of experiments, the lungs were collected from animals that did not undergo BALF collection and were processed for histological analysis.