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Objective To evaluate the effects of IR-61 on the development of pulmonary fibrosis and on the proliferation and migration of lung fibroblasts in rats. Methods Forty male SD rats (6 to 8 weeks old) were randomized equally into normal control group, IR-61 group, bleomycin group and bleomycin+IR-61 group. In the latter 2 groups, rat models of pulmonary fibrosis were established by intratracheal infusion of bleomycin (2.5 mg/kg). In the 2 groups with IR-61 treatment, IR-61 was intraperitoneally injected at the dose of 2.0 mg/kg 30 min following intratracheal bleomycin infusion and was then administered twice a week. At 10 d after bleomycin treatment, 3 rats from each group were selected for IR-61 injection (2 mg/kg) via the tail vein to observe the accumulation of IR-61 in each organ at 24 h using near infrared fluorescence imaging (NIR). After 28 d of treatment, the rats were euthanized and the lung tissue was collected for HE and Masson staining. RT-PCR and Western blotting were performed to detect the changes of the expression of the genes related with lung fibroblast transdifferentiation and proliferation (including Collagen-1, Collagen-3, α-SMA, fibronectin, FOXM1, CCNB1, CDC25b and AURKB) in human lung fibroblasts (HFL1) and primary rat lung fibroblasts in response to IR-61 and transforming growth factor-β1 (TGF-β1) treatments; the migration of the cells was assessed using cell scratch test. Results NIR imaging showed that IR-61 could target the injured lung tissue induced by bleomycin. Morphological observation showed that compared with bleomycin-treated rats, the rats with IR-61 treatment presented with better structural integrity of the lungs with less obvious lung collapse, fewer fibrotic nodules and a significantly milder of pulmonary fibrosis (0.103 3±0.014 53 vs 0.460 0±0.037 86, P < 0.05). In HFL1 cells and primary rat lung fibroblasts, IR-61 pretreatment prior to TGF-β1 stimulation resulted in significantly lowered mRNA and protein expression of collagen-1, collagen-3, α-SMA, and fibronectin as well as the proliferation-specific genes FOXM1, CCNB1, CDC25b and AURKB (P < 0.05). Cell scratch test showed that TGF-β1 treatment significantly increased the cell migration activity, which was effectively suppressed by IR-61 treatment. Conclusion IR-61 ameliorates bleomycin-induced pulmonary fibrosis in rats by targeting the injured lung tissue and inhibiting the transdifferentiation, proliferation and migration of lung fibroblasts |
