Exosomal miR-194 from adipose-derived stem cells impedes hypertrophic scar formation through targeting TGF-ß1 [miRNA]
Hypertrophic scars, which result from aberrant fibrosis and disorganized collagen synthesis by skin fibroblasts, emerge due to disrupted wound healing processes. These scars present significant psychosocial and functional challenges to affected individuals. The current treatment limitations largely arise from an incomplete understanding of the underlying mechanisms of hypertrophic scar development. Recent studies, however, have shed light on the potential of exosomal non-coding RNAs interventions to mitigate hypertrophic scar proliferation. This research assesses the impact of exosomes derived from adipose-derived stem cells (ADSCs-Exos) on hypertrophic scar formation using a rabbit ear model. We employed Hematoxylin and Eosin staining, Masson's Trichrome staining, and Immunohistochemical staining techniques to track scar progression. Our comprehensive analysis encompassed the differential expression of non-coding RNAs, enrichment analyses of functional pathways, protein-protein interaction studies, and miRNA-mRNA interaction investigations. The results reveal a marked alteration in the expression levels of long non-coding RNAs and microRNAs following ADSCs-Exos treatment, with little changes observed in circular RNAs. Notably, miR-194 emerges as a critical regulator within the signaling pathways that govern hypertrophic scar formation. Dual-luciferase assays indicated a significant reduction in the promoter activity of TGF-ß1 after miR-194 overexpression. Quantitative reverse transcription PCR and Western blotting assays further validated the decrease in TGF-ß1 expression in the treated samples. Moreover, the treatment resulted in diminished levels of inflammatory markers IL-1ß, TNF-a, and IL-10. In vivo evidence strongly supports the role of miR-194 in attenuating hypertrophic scar formation through the suppression of TGF-ß1. Our findings endorse the strategic use of ADSCs-Exos, particularly through miR-194 modulation, as an effective strategy for reducing scar formation and lowering pro-inflammatory and fibrotic indicators like TGF-ß1. Therefore, this study advocates for the targeted application of ADSCs-Exos, with an emphasis on miR-194 modulation, as a promising approach to managing proliferative scarring. Overall design: Six female New Zealand White rabbits underwent a one-week acclimatization. After aseptic preparation, six full-thickness wounds were created on the ventral surface of each ear. Each rabbit was housed individually and given antibiotics and analgesics post-surgery. The rabbits were randomly assigned to either a control group or the ADSCs-Exos treatment group (three rabbits per group). Wound care included the application of saline or ADSCs-Exos (100 µg/2 ml) for the first three days post-injury. Scar progression was monitored, and tissue samples were collected for histological analysis on days 3, 7, 14, and 21. Scar tissues from both the control group and the ADSCs-Exos treated group were collected on post-treatment day 21 to extract total RNA, followed by RNA-seq library construction. Small RNA sequencing libraries were then constructed from the extracted scar tissues. Small RNAs were selected, ligated to adaptors, reverse-transcribed to cDNA, and amplified. Specific fragments were isolated using PAGE gel electrophoresis, and quality was assessed with Kapa qPCR and Agilent TapeStation. Libraries meeting quality standards were sequenced on an Illumina NovaSeq 6000. miRNA expression profiling was subsequently conducted using the sequencing data.