Supplementary Material for: Oxidative Stress-Mediated Modulation of Fibrosis and Inflammation in Keloid Fibroblasts by Cold Atmospheric Plasma

Introduction: Despite numerous therapeutic approaches, keloid treatment remains a challenge. Clinical studies have demonstrated the possible use of cold atmospheric plasma (CAP) to treat hypertrophic scars and keloids. This study investigated the effects and relative mechanisms of CAP treatment on primary keloid fibroblasts (PKF) in vitro.
Method: PKF cells from 10 patients with keloid and human dermal fibroblast (HDFa) cell line were cultured to compare CAP treatment effects. Cell proliferation, migration via scratch assay, and reactive oxygen species (ROS) levels were measured using standard assays, while cell apoptosis was quantified by flow cytometry. A quantitative reverse transcription polymerase chain reaction was performed to analyze the effect of CAP on gene regulation in fibrosis and inflammation. Finally, CAP’s mode of action was compared to H2O2 treatment.
Result: CAP treatment in medium mode (CAP-mid), specifically for 30 and 60 s, significantly inhibited PKF proliferation and migration. No significant effects were seen in HDFa cells. Genetic analysis of pro-fibrotic components and inflammatory cytokines revealed that CAP-mid significantly reduced α-sma, periostin, h-col1, tgf-β, IL-6, and IL-31 expression in PKF cells, while it enhanced IL-10 expression. However, it had opposite effects on HDFa. Time-dependent analysis showed that CAP-mid at 60 and 30 s exerted the maximum effects on those molecules. Simultaneous analysis of CAP and H2O2 treatment on PKF cells demonstrated that CAP-mediated alterations in gene expression are primarily linked to enhanced ROS production in PKF cells.
Conclusion: These findings suggest that CAP may mitigate keloid formation by modifying fibrotic and inflammatory profiles through ROS production and inhibition of cell proliferation.