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.

引言：尽管已有多种治疗手段，瘢痕疙瘩（keloid）的治疗仍是临床难题。临床研究已证实，冷大气等离子体（cold atmospheric plasma, CAP）可用于治疗增生性瘢痕与瘢痕疙瘩。本研究旨在探究冷大气等离子体治疗对体外培养的原代瘢痕疙瘩成纤维细胞（primary keloid fibroblasts, PKF）的作用及其潜在机制。
方法：本实验培养了10例瘢痕疙瘩患者来源的原代瘢痕疙瘩成纤维细胞，以及人真皮成纤维细胞（human dermal fibroblast, HDFa）细胞系，以对比冷大气等离子体治疗的作用效果。采用标准实验方法检测细胞增殖能力，通过划痕实验检测细胞迁移能力，并测定活性氧（reactive oxygen species, ROS）水平；通过流式细胞术定量分析细胞凋亡情况。采用定量反转录聚合酶链反应，分析冷大气等离子体对纤维化与炎症相关基因的调控作用。最后，将冷大气等离子体的作用机制与过氧化氢（hydrogen peroxide, H₂O₂）处理进行对比分析。
结果：采用中等参数模式的冷大气等离子体（CAP-mid）处理30秒与60秒时，可显著抑制原代瘢痕疙瘩成纤维细胞的增殖与迁移能力，但该处理对人真皮成纤维细胞无显著影响。对促纤维化因子与炎症细胞因子的基因表达分析显示，中等参数模式冷大气等离子体可显著下调原代瘢痕疙瘩成纤维细胞中α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）、骨膜蛋白（periostin）、I型胶原（human collagen type I, h-Col1）、转化生长因子-β（transforming growth factor-β, TGF-β）、白细胞介素-6（interleukin-6, IL-6）以及白细胞介素-31（interleukin-31, IL-31）的表达，同时上调白细胞介素-10（interleukin-10, IL-10）的表达；但该处理对人真皮成纤维细胞的基因表达则产生相反的调控作用。时间依赖性分析结果显示，中等参数模式冷大气等离子体处理60秒与30秒时，对上述分子的调控效果最为显著。同时对原代瘢痕疙瘩成纤维细胞开展冷大气等离子体与过氧化氢处理的对比分析，结果显示冷大气等离子体诱导的基因表达改变，主要与原代瘢痕疙瘩成纤维细胞内活性氧水平升高密切相关。
结论：本研究结果表明，冷大气等离子体可通过提升细胞内活性氧水平、抑制细胞增殖，调控纤维化与炎症相关因子的表达谱，从而缓解瘢痕疙瘩的形成。