2.8. Hydroxyproline Assay

The hydroxyproline content of the lung tissues was determined with a Hydroxyproline Colorimetric assay kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions. In brief, lung tissues homogenized in dH₂O were hydrolyzed with concentrated HCl at 120 °C for 3 h and the supernatants were collected after centrifuging the hydrolyzed homogenates at 10,000× g for 3 min. Then, 10 μL of the supernatants was loaded onto a 96-well plate followed by a drying step, after which the samples were assessed for hydroxyproline at 560 nm. The data were expressed as micrograms of hydroxiproline per milligram wet lung weight (μg/mg wet tissue).