HUCMSCs-Derived Exosomes Attenuate Keloid Fibroblast Pathological Phenotypes with Associated Metabolic-Redox-Matrix Network Remodeling: A Proteomic and In Vivo Study.

Abstract

Background Keloids are fibroproliferative disorders characterized by excessive extracellular matrix deposition, and current treatments remain unsatisfactory because of high recurrence rates. Human umbilical cord mesenchymal stem cell-derived exosomes (HUCMSCs-Exo) have emerged as investigational cell-free candidates for the treatment of fibrotic diseases. Aims To examine the phenotypic effects of HUCMSCs-Exo on keloid fibroblasts (KFs) in vitro, characterize associated proteomic, hub-protein, and redox-mitochondrial changes, and evaluate their in vivo effects using a nude mouse xenograft model. Study Design In vitro and in vivo study using a nude mouse xenograft model. Methods HUCMSCs-Exo were isolated by differential ultracentrifugation and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting for CD9, CD63, and CD81. Label-free quantitative proteomics, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and protein-protein interaction (PPI) network analysis were performed. Western blot validation, DCFH-DA, JC-1, and MitoSOX assays, and peritumoral administration of HUCMSCs-Exo in vivo, together with biosafety evaluations based on hematology, serum biochemistry, and organ histopathology, were performed. Results PKH67-labeled exosomes were internalized by KFs in a time-dependent manner. In vitro, HUCMSCs-Exo inhibited KF proliferation in a dose- and time-dependent manner, induced apoptosis, caused cell-cycle arrest at the G0/G1 phase, and suppressed migration. The expression of fibrosis-associated markers, including fibronectin (FN1), alpha-smooth muscle actin (α-SMA), collagen type I (COL-I), and collagen type III (COL-III), was downregulated at both the mRNA and protein levels. Label-free quantitative proteomics identified 951 differentially expressed proteins. KEGG enrichment analysis demonstrated enrichment in metabolic pathways, carbon metabolism, and glycolysis/gluconeogenesis. PPI network analysis identified seven candidates hub proteins, of which APEX1, MMP2, and HMOX1 remained significant after Benjamini-Hochberg correction; the other four candidates, including TGFB1, did not survive correction. Therefore, the matrix- and TGF-beta-related findings are supported by orthogonal validation rather than by the corrected proteomic screen alone. Western blot validation demonstrated reduced abundance of TGFB1, MMP2, TIMP1, and TXN2 and increased abundance of APEX1 after HUCMSCs-Exo treatment. The DCFH-DA, JC-1, and MitoSOX assays indicated reduced intracellular reactive oxygen species levels, preservation of mitochondrial membrane potential, and reduced mitochondrial superoxide signals. In vivo, peritumoral administration of HUCMSCs-Exo suppressed keloid xenograft growth and was accompanied by reduced collagen deposition and lower expression of FN1, α-SMA, COL-I, and COL-III. Biosafety evaluation revealed no detectable abnormalities in hematology, serum biochemistry, or organ histopathology under the conditions tested. Conclusion The combined data indicate that HUCMSCs-Exo attenuate KF proliferation and fibrogenic phenotypes and are accompanied by proteomic, hub-protein, and redox-mitochondrial changes consistent with metabolic-redox-matrix network remodeling. These molecular associations are correlative and were not tested using perturbation experiments.