Histology and Ashcroft score

The mouse lungs were fixed with a buffered formalin solution overnight, dehydrated, transparentized, and embedded in paraffin before sectioning into slices of 5 μm thick. The slides were stained with H&E for structured observation or with Masson's trichrome stain to detect collagen deposits according to the instructions given by the manufacturer (KeyGen no. KGA224/KGMST-8004, Nanjing, China). The determination of hydroxyproline content was carried out using a hydroxyproline assay kit by following the manufacturer's instructions (Nanjing Jian Cheng Bioengineering Institute, no. A030-3, Nanjing, China). The degree of pulmonary fibrosis was evaluated by a histopathologist blinded to the experimental groups using the validated semiquantitative Ashcroft method ^{36, 37}. Briefly, using 100× magnification, each of 10 successive fields was visually graded from 0 (normal lung) to 8 (total fibrous obliteration of the area). The mean value of the grades obtained for all of the areas was taken as the visual fibrotic score ³⁸.