Tanshinone IIA Attenuates Bleomycin-Induced Pulmonary Fibrosis via Modulating Angiotensin-Converting Enzyme 2/ Angiotensin-(1-7) Axis in Rats

Wu, Huajie; Li, Yan; Wang, Yanxia; Xu, Dunquan; Li, Congcong; Liu, Manling; Sun, Xin; Li, Zhichao

After being taken out, the lungs were dissected into 3-mm-thick slices at the same part (the lower lobe of the right lungs) and fixed in 10% formaldehyde. Then the lungs were embedded in paraffin and sectioned into 4-μm-thick sections. Hematoxylin and eosin staining (HE) and Trichrome Masson's staining were performed. Aiming to evaluate the collagen and fibrosis changes during PF, a modified picrosirius procedure based on the classic sirius red staining was performed [19, 20] . In brief, after regular deparaffinization and rehydration, the lung specimens were immersed in 1% phosphomolybdic acid for 5 min and then covered with 0.1% (w/v) sirius red F3B (Sigma-Aldrich, St. Louis, MO, USA) in saturated picric acid solution for 1 h at room temperature. The specimens were mounted after being dehydrated and cleared in xylene. The specimens were analyzed by using a light microscope (Olympus BX50, Tokyo, Japan) under normal and polarized light obtained with polarizing filters (Olympus U-ANT, Tokyo, Japan) in order to analyze the initial collagen organization and then the fibrosis. The lung alveolar inflammation and PF were scaled by the HE staining and the Trichrome Masson's staining as described by the previous study of Szapiel [21] . Three independent pathologists undertook the histological examination and applied the scoring system in a blinded fashion.