Bleomycin-induced pulmonary fibrosis mouse model

Pulmonary fibrosis was induced using bleomycin treatment (5 mg/kg or 2 mg/kg) as described previously¹⁴. Briefly, male C57BL/6 mice were randomly divided into four groups: a Control group, a bleomycin (BLM) group, a BLM + PM014 group and a BLM + DEX (dexamethasone, 0.45 mg/kg). In the BLM + PM014 group, mice in three subgroups were treated with PM014 at a dose of 50, 100, or 200 mg/kg. At day 0, the mice were lightly anesthetized using isoflurane, and BLM was dissolved in 40 µl of PBS and administered intratracheally as previously described³⁹. To test preventive effect, on day 2 after BLM challenge PM014 was administered orally daily for 12 days (inflammatory phase) on mice given 5 mg/kg of BLM and 21 days (fibrotic phase) on mice received 2 mg/kg of BLM, respectively. Further, to test therapeutic effect of PM014, mice were examined under two different administration schedules. One group received with 5 mg/kg of BLM was administered once daily with PM014 (200 mg/kg) from day 7 to day 14 after BLM challenge. The second group given with 5 mg/kg of BLM was treated once daily with PM014 (200 mg/kg) from day 10 to day 21 after BLM exposure. All mice were killed 24 h after the last injection and the lungs were harvested for the assays described below.