Figure 3. NOX4 is induced during the fibrogenic phase of bleomycin-induced lung injury in mice and inhibition of NOX4 expression/activity attenuates pulmonary fibrosis.

(a) C57BL/6 mice were subjected to acute lung injury by airway (intra-tracheal) administration of bleomycin or saline/control on day 0. Following bleomycin injury, mice were euthanized at the indicated time intervals, whole lungs were harvested, and tissue homogenates analyzed by SDS-PAGE and Western immunoblotting for NOX4, NOX2, and β-actin. (b–e) NOX4 siRNA or a nontargeting control siRNA was instilled directly down the trachea of mice at the time of bleomycin injury (day 0), and lungs were analyzed on day 14 or 21. (b) NOX4 expression on day 21 was determined by Western immunoblotting of whole lung homogenates. (c) Fibrosis was assessed on day 14 by H & E staining and Masson’s trichrome blue staining for collagen (top panels); NOX4 and α-SMA expression were assessed by immunohistochemical (IHC) analysis (bottom panels); length bar = 100 μm. (d,e) Whole lung homogenates were analyzed on day 21 for hydroxyproline content (d), and on day 14 for acid-soluble collagen using the Sircol assay (e). Values represent mean± S.E.M.; n = 4–6; ^*P < 0.01 compared to all other groups. (f) C57BL/6 mice were administered intra-tracheal (IT) bleomycin on day 0. Diphenyleneiodonium (DPI; 1.6 mg/kg) or vehicle control was administered by daily intraperitoneal (IP) injections starting on day 7 for 14 days. Whole lung homogenates were analyzed for acid-soluble collagen using the Sircol assay on day 21. Values represent mean ± S.E.M.; n = 6; *P < 0.05 compared to saline control.