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

The collected lungs were fixed in 10% neutral formalin for 24 h and then embedded in paraffin. After deparaffinization and dehydration as previously described [10] , sections (4 μm) were stained with hematoxylin and eosin or Masson's trichrome and analyzed by microscopy under 100x and 400x magnification. The density of leukocytes/unit area (130 μm 2 ) was determined by counting the number of cells on the integrating eyepiece that fell into areas of the bronchioli and distal lung parenchyma using the ImageJ Program. Using the same program, the collagen area was measured also in areas of the bronchioli and distal lung parenchyma. All measurements were performed in ten random sections of 130 μm 2 per animal at 200x (for collagen area) and 400x magnification (for leukocyte count), and the researcher who performed the analysis was unaware of the experimental group designation. Two independent researchers performed blinded analyses; if there was discordance, a third researcher performed the analysis. TNF-α, IL-6, IL1-β, and IL-10 mRNA expression was evaluated in RAW 264.7 cells by RT-qPCR as described in Section 2.3.4.