Urine-derived stem cells reverse bleomycin‑induced experimental pulmonary fibrosis by inhibition of the TGF-β1-Smad2/3 pathway

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung interstitial lesions with the disease pathophysiology incompletely understood, which is a serious and fatal disorder with limited treatment options. Mesenchymal stem cells (MSCs) have exhibited promising therapeutic capability for IPF. While most types of MSCs are obtained invasively, urine-derived stem cells (USCs) can be gained in a safe, noninvasive and inexpensive procedure, which are readily available and reported to exhibit no risk of teratoma formation or oncogenic potential in vivo, sounding alternative to other MSCs. This study aims to investigate the therapeutic effect and mechanism of USCs on IPF, using a bleomycin (BLM)-induced IPF model in mice. Methods: Cell surface marker examination by flow cytometry analysis and cell differentiation culture were used to characterize USCs obtained from healthy individuals. BLM was instilled endotracheally in adult C57BL/6 mice, followed by USCs or human bone marrow-derived mesenchymal stem cells (BMSCs) treatment by tail vein injection on day 14. Mice were euthanized on day 14 before administration or day 21 for the evaluation of pulmonary histopathology and hydroxyproline (HYP) content. Inflammatory factors of lung, including transforming growth factor (TGF)-β1, TNF-α, IL-6, MMP2 were analyzed by quantitative real-time PCR (qRT-PCR). Additionally, immunohistochemistry (IHC) and western blotting (WB) were applied to evaluate the expression of α-SMA and activation of TGF-β1-Smad2/3 in lung. Results: USCs highly expressed CD29 and CD90, showing negative expression of haematopoietic stem cell markers (CD45, CD34) and could differentiate into, at least, bone and fat in vitro. In mice challenged with BLM, septal thickening and prominent fibrosis were observed on day 14, with higher HYP content and mRNA levels of TGF-β1, TNF-α and IL-6 exhibited, compared to untreated mice. USCs could migrate to lung and accumulate there in mouse model after intravenous injection. Transplantation of USCs into BLM-induced mice improved their pulmonary histopathology, decreasing Ashcroft score, Szapiel score, HYP content and mRNA levels of TGF-β1 and MMP2 of lung, similar to the effects of BMSCs. IHC and WB further revealed that USCs could inhibit activation of the TGF‑β1-Smad2/3 pathway of lung in vivo. Conclusions: Transplantation of USCs effectively reverses pulmonary fibrotic phenotype in an experimental IPF model, inhibiting the TGF-β1-Smad2/3 pathway, a key driver of fibrosis. These results suggest the therapeutic application of USCs for IPF, instead of other types of MSCs obtained invasively.