Murphy-Marshman Hannah, Quensel Katherine, Shi-Wen Xu, Barnfield Rebecca, Kelly Jacalyn, Peidl Alex, Stratton Richard J, Leask Andrew
Department of Dentistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
Centre for Rheumatology, Royal Free and University College Medical School, London, United Kingdom.
PLoS One. 2017 Oct 19;12(10):e0186740. doi: 10.1371/journal.pone.0186740. eCollection 2017.
TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents.
转化生长因子β（TGFβ）可诱导成纤维细胞发生纤维化反应。活性氧（ROS）/烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶（NOX）可能参与纤维化反应。在此，我们研究抗氧化剂N-乙酰半胱氨酸（NAC）、NOX抑制剂二苯碘鎓（DPI）以及选择性NOX1/NOX4抑制剂GKT-137831是否会损害TGFβ诱导人牙龈成纤维细胞（HGF）和真皮成纤维细胞（HDF）中促纤维化基因表达的能力。我们还评估GKT-137831是否能阻断硬皮病（SSc）病变成纤维细胞的持续性纤维化表型。我们使用实时聚合酶链反应和蛋白质印迹分析来评估NAC和DPI是否会损害TGFβ1诱导成纤维细胞中纤维化基因表达的能力。使用蛋白质印迹和胶原凝胶收缩分析测试了GKT-137831对TGFβ诱导的蛋白质表达以及硬皮病（SSc）病变成纤维细胞持续性纤维化表型的影响。在HDF和HGF中，TGFβ1以对NAC敏感的方式诱导CCN2、CCN1、内皮素-1和α-平滑肌肌动蛋白（SMA）。COL1A1 mRNA的诱导不受影响。DPI也得到了类似结果。NAC和DPI损害了TGFβ1诱导HDF和HGF中CCN2和α-SMA蛋白质表达的能力。GKT-137831损害了TGFβ诱导的HGF和HDF中CCN2和α-SMA蛋白质表达。在硬皮病（SSc）病变的真皮成纤维细胞中，GKT-137831降低了α-SMA和CCN2蛋白的过表达以及胶原凝胶收缩。这些结果与以下假设一致，即抗氧化剂或NOX1/4抑制可能有助于阻断TGFβ对真皮和牙龈成纤维细胞的促纤维化作用，值得作为潜在的抗纤维化药物进一步开发考虑。
