Bleomycin hydrochloride

Mice were divided randomly into two groups: (A) saline-only (PBS), or (B) bleomycin (Bleo). Lung injury and pulmonary fibrosis were induced by single-dose administration of bleomycin hydrochloride (Sigma Aldrich, Germany), which was dissolved in sterile PBS and given at 2 U/kg (oropharyngeal instillation) and 3U/kg (intratracheal instillation) bodyweight. The control group was treated with sterile PBS only. Mice were sacrificed at designated time points (days 1–14, 21, 28, 35, 56) after instillation. Treated animals were continuously under strict observation with respect to phenotypic changes, abnormal behavior and signs of body weight loss.

Six adult female Japanese white rabbits (3.0 kg; Japan SLC Inc., Tokyo, Japan) were randomly divided into two equal groups (n=3). One group served as the control and was treated with bleomycin only. At 30 min prior to the bleomycin injection, the second group (AEP group) was pretreated with 80 ng/g AEP (Chugai Pharmaceutical Co., Ltd., Tokyo, Japan) delivered by an intravenous bolus injection via the auricular vein.
All rabbits were anesthetized with ketamine hydrochloride and medetomidine hydrochloride. Bleomycin hydrochloride (30 mg; Sigma-Aldrich, Tokyo, Japan) was dissolved in 2 ml physiological saline and 8 U/kg bleomycin was subsequently injected into the trachea using a 22-gauge indwelling needle. To obtain equal drug distribution throughout the lungs, four injections of 2 U/kg each were delivered to each rabbit in the prone and dorsal positions, on the right and left lateral sites.

10–12 weeks old C57BL/6 N wild-type female mice were administered with bleomycin hydrochloride (Merck Sigma Aldrich Taufkirchen, Germany) diluted in 50 µL sterile PBS at 2 U/kg body weight of mice using a micro sprayer (Penn Century device distributed by BioJane Limited, China). Afterwards, mice were kept in O₂-enriched (30% V/V) cages for 14 days according to the recommendation received from the animal regulatory authorities.