Paclitaxel Ameliorates Pulmonary Fibrosis By Suppressing TGF-β1/Smad3Pathway Via MiR-140Regulation

Objective:This study aims to explore the mechanisms of PTX on TGF-β1/Smad3pathway via regulating miR-140, and to investigate the antifibrotic effect of low-dose paclitaxel (PTX) on pulmonary fibrosis.Methods:A549cells and primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) were incubated with TGF-β1(5ng/mL) for72h, producing a pulmonary fibrosis model in vitro, and then treated with low-dose PTX (10nM,50nM) with/without enforced expression of miR-140. The ultrastructure changes were observed under transmission electron microscope. The expressions of vimentin, E-cadherin and surfactant protein C(SP-C) were evaluated under a laser confocal microscopy. The GFP-positive cells were confirmed via a fluorescence microscope, flow cytometry measurements. Rats with bleomycin (BLM) intratracheal instillation produced a pulmonary fibrosis model in vivo, which began low-dose PTX (0.6mg/kg) treatment at the15th day post-BLM instillation. HE staining for histopathological observation. Masson’s trichrome staining, hydroxyproline and type I collagen assay were performed to assess collagen deposition. a-SMA, Smad3and phosphorylated Smad3(p-Smad3) were detected via immunohistochemistry. Western bloting and real-time RT-PCR analysis were used to confirme the levels of vimentin, E-cadherin, α-SMA, and Smad3/p-Smad3. The miR-140expression was monitored by real-time RT-PCR analysis.Results:TGF-β1treatment resulted in epithelial-mesenchymal transition (EMT) in A549cells and AEC Ⅱ, and resulted in miR-140downregulation in A549cells. PTX treatment reversed EMT and upregulated miR-140expression, especially, in50nM PTX-treated cultures. PTX treatment or miR-140overexpression could suppress the activity of TGF-β1/Smad3pathway, and miR-140overexpression amplifies the antifibrotic effect of PTX. BLM-instilled rats exhibited a pulmonary fibrosis, notable collagen deposition, significant increase of α-SMA and p-Smad3, and miR-140downregulation with its nadir on7d. The α-SMA-and p-Smad3-positive cells, majority, were AEC II. PTX treatment attenuated lung fibrosis, restored miR-140expression nearly up to normal level, and resulted in an obviously lower level of collagen, α-SMA and p-Smad3than that of BLM28d group.Conclusion:Our results demonstrate that low-dose PTX reverses EMT and ameliorates pulmonary fibrosis by suppressing the TGF-β1/Smad3pathway via upregulating miR-140.