2.8. Bleomycin-induced pulmonary fibrosis animal model and treatment

The mouse pulmonary fibrosis model was created with a single lung administration of bleomycin (bleomycin sulfate; Tokyo Chemical Industry Co.) dissolved in 1x PBS. Each mouse received a weight-adjusted 1.5-mg/kg dose of bleomycin via oropharyngeal aspiration. Before administration, mice were anesthetized with 30 mg/kg of tiletamine/zolazepam and 10 mg/kg of xylazine. Mice in the control group received an equal volume (50 μL) of saline. During administration, the mouse's nostril was occluded with a thumb, forcing the mouse to breathe through its mouth and thus enhancing deposition into the lung. The thumb was released after the completion of five breaths. The UCB solution for intravenous injection was prepared by fully dissolving bilirubin in NaOH solution, followed by adjusting the solution pH to ~7–8 using an HCl solution. Starting on day 1, 50 μL of saline or UCB (4.64 mg/kg) or BRNPs (5, 20, 40 mg/kg) were intravenously injected via the retro-orbital route every other day for 14 days.