Autophagy inhibition enhances antifibrotic potential of placental mesenchymal stem cells of fetal origin via regulating TGF-β1 mediated protein degradation of HGF.

Mesenchymal stem cell (MSC) therapy represents a promising strategy for pulmonary fibrosis (PF) treatment, with hepatocyte growth factor (HGF) serving as a key mediator of MSC-mediated protection. However, the therapeutic efficacy of MSCs is limited by the complex PF microenvironment, and the mechanisms underlying this limitation remain unclear. This study investigates how the PF pathological microenvironment modulates the antifibrotic potential of placental mesenchymal stem cells of fetal origin (fPMSCs) through HGF regulation and elucidates the molecular mechanisms involved. Morphological analysis, flow cytometry, and multilineage differentiation assays were employed to characterize fPMSCs. Transforming growth factor-β1 (TGF-β1) was employed to simulate the PF microenvironment and activate fPMSCs in vitro. ELISA and Western blotting were used to analyze HGF expression, autophagy markers, and Smad signaling. Autophagosome formation was visualized via confocal microscopy and transmission electron microscopy. Co-immunoprecipitation (Co-IP) assays were performed to assess the interaction between p62 and HGF. The antifibrotic function of fPMSCs was further evaluated using a transwell co-culture system with MRC-5 fibroblasts in vitro and a bleomycin-induced PF mouse model in vivo. Phenotypic characterization confirmed that fPMSCs exhibited canonical MSC morphology, expressed CD73/CD90/CD105, lacked CD14/CD34/CD45/HLA-DR, and differentiated into adipogenic, osteogenic, and chondrogenic lineages. TGF-β1 treatment robustly downregulated the antifibrotic capacity, HGF protein expression, and paracrine secretion in fPMSCs. Recombinant HGF enhanced antifibrotic effects, while an HGF-neutralizing antibody abolished them. TGF-β1 induced autophagy in fPMSCs, promoting HGF degradation via p62 interaction and impairing antifibrotic function in vitro and in vivo. Mechanistically, Smad3 phosphorylation mediated the regulation of autophagy and HGF expression in TGF-β1-treated fPMSCs. Our findings demonstrate that TGF-β1 impairs the antifibrotic function of fPMSCs via autophagy-dependent HGF degradation and Smad3 signaling. Conversely, autophagy inhibition restores HGF levels and enhances fPMSCs' therapeutic efficacy in a preclinical PF model. Targeting autophagy inhibition emerges as a promising therapeutic strategy to counteract pulmonary fibrosis.