Lipoxin A4 Attenuates Bronchopulmonary Dysplasia via Upregulation of Let-7c and Downregulation of TGF-β1 Signaling Pathway

Transforming growth factor-beta (TGF-beta) superfamily members are key regulators for lung development and progress of bronchopulmonary dysplasia (BPD). The mechanisms by which lipoxin A(4) (LXA(4)) attenuates development of BPD have not been clarified. Neonatal murine BPD models were inducted by hyperoxia treatment. Neonatal mice were exposed to room air or 85% O-2 hyperoxia with or without treatment with 5S,6R-methyl-LXA(4) or anti-TGF-beta antibodies. Mouse lung epithelial cells (MLE-12 cells) and mouse embryonic fibroblasts (NIH/3T3 cells) were cultured in room air or 85% O-2 followed by treatment of LXA(4), anti-TGF-beta antibodies, and let-7c mimic/anti-microRNA transfections. Treatment with 5S,6R-methyl-LXA(4) and anti-TGF-beta antibodies both attenuated the mice alveolar simplification induced by hyperoxia. Hyperoxia treatment significantly altered pulmonary basal mRNA and protein expressions of several important extracellular matrix (ECM) and ECM remodeling proteins including fibronectin, alpha-smooth muscle actin (alpha-SMA), tissue inhibitor of metalloproteinase-1 (TIMP-1), elastin, tenascin C, collagen I, and matrix metalloproteinase-1 (MMP-1). 5S,6R-methyl-LXA(4) and anti-TGF-beta antibodies suppressed the mRNA and protein expressions of TGF-beta(1) and TGF-beta R1 but not TGF-beta R2 in the lungs exposed to hyperoxia. Treatment with LXA(4) and anti-TGF-beta antibodies alleviated hyperoxia-induced injury of the NIH/3T3 cells identified by morphologic observation and flow cytometry, and expressions of ECM, ECM remodeling proteins, and TGF-beta(1) signaling pathway, but reversed by transfection with let-7c anti-miRNA. LXA(4) upregulated the let-7c expression in MLE-12 cells, transfection with let-7c anti-miRNA, inhibited the LXA(4)-induced let-7c expression in MLE-12 cells exposed to hyperoxia and reduced the relative luciferase activity of let-7c binding with let-7c binding sites of the TGF-beta R1 3' UTR. Treatment with 5S,6R-methyl-LXA(4) and anti-TGF-beta antibodies significantly improved histology, ECM, and ECM remodeling proteins in the lungs isolated from the murine BPD model induced by hyperoxia. The LXA(4)-imparted protective effects on hyperoxia-induced lung injury are mediated by upregulation of let-7c and inhibition of TGF-beta(1) and subsequent downregulation of TGF-beta(1) signaling pathway.