Temporal Dynamics of ADSC Therapy in Skin Fibrosis: Unraveling the Roles of ROS/NF-κB/TSG-6 Signaling Axis

Background: Cutaneous fibrosis, particularly in localized scleroderma (LoS), poses a considerable therapeutic challenge owing to its progressive characteristics and the subsequent effects on quality of life. Although ADSCs exhibit therapeutic potential for fibrosis, their spatiotemporal mechanisms of action, particularly within fibrotic microenvironments, remain poorly characterized. This study sought to clarify the spatiotemporal dynamics and molecular mechanisms of ADSC-mediated fibrosis resolution in bleomycin (BLM)-induced murine LoS model. Methods: Skin fibrosis was induced in C57BL/6J mice through daily subcutaneous injections of bleomycin (BLM) administered over a period of four weeks. GFP-labeled mouse or human ADSCs were injected into the fibrotic dorsum. ADSC distribution was tracked using fluorescence imaging and flow cytometry. Skin fibrosis was assessed histologically (H&E, Masson's trichrome, α-SMA, COL1) and molecularly (qRT-PCR for cytokines). Transcriptomic profiling (RNA-seq) of sorted GFP+ ADSCs was performed on days 1, 7, and 14 post-injection. Key pathways (ROS, NF-κB, TSG-6) were validated in vitro using ADSCs and human LoS-derived fibroblasts (LoSFs) via pharmacological inhibition, gene knockdown (shTSG-6), co-culture, Western blotting, and dual-luciferase assays. Results: ADSCs mitigated dermal thickening, collagen deposition, α-SMA expression, and inflammation (TNF-α, IL-6, IL-1β) over 21 days. Transcriptomics revealed a temporal hierarchy: early oxidative stress response (Day 1), followed by immunomodulation (Day 7, NF-κB, cytokine pathways), and later ECM remodeling (Day 14). Mechanistically, TGF-β induced ROS via NOX4, activating NF-κB, which directly bound the TSG-6 promoter to drive its expression. TSG-6 knockdown in ADSCs (ADSCshTSG-6) abolished their ability to suppress TGF-β/Smad signaling, collagen production, α-SMA expression, and inflammation in vitro and in vivo. Conclusion: ADSCs resolve skin fibrosis through a biphasic mechanism involving initial adaptation and subsequent immunomodulation/ECM remodeling, centrally governed by a ROS-NF-κB-TSG-6 axis. TSG-6 is the critical downstream effector, disrupting the TGF-β/Smad pathway and inflammation. This study identifies TSG-6 as a key therapeutic mediator and a potential biomarker for optimizing ADSC-based therapies for fibrotic skin disorders.