Tissue Engineering and Cellular Therapy Group, Department of Physiotherapy, Medicine and Biological Sciences, University of A Coruña, 15006 A Coruña, Spain.
Laboratory of Rheumatology, GIGA Research, University and CHU of Liège, 4000 Liège, Belgium.
Biochem Pharmacol. 2019 Jul;165:66-78. doi: 10.1016/j.bcp.2019.03.039. Epub 2019 Mar 29.
BACKGROUND/AIMS: Synovial fibrosis is a pathological process that is observed in several musculoskeletal disorders and characterized by the excessive deposition of extracellular matrix, as well as cell migration and proliferation. Despite the fact that glucocorticoids are widely employed in the treatment of rheumatic pathologies such as osteoarthritis (OA) and rheumatoid arthritis, the mechanisms by which glucocorticoids act in the joint and their impacts on pro-fibrotic pathways are still unclear.
Human OA synovial fibroblasts were obtained from knee and hip joints. Cells were treated with prednisolone (1 mM) or transforming growth factor-beta 1 (TGF-β1) (10 ng/ml) for 1 and 7 days for quantification of RNA and protein expression (by real-time quantitative reverse transcription-PCR and western blot, respectively), 72 h for immunocytochemistry analysis, and 48 h for proliferation (by BrdU assay) and migration (by wound assay) studies. In addition, cells were preincubated with prednisolone and/or the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) for 6 h before adding TGF-β1. pSmad1/5, pSmad2 and β-catenin levels were analyzed by Western blot. The activin receptor-like kinase-5 (ALK-5) inhibitor (SB-431542) was employed for the mechanistic assays.
Prednisolone showed a predominant anti-fibrotic impact on fibroblast-like synoviocytes as it attenuated the spontaneous and TGF-β-induced gene expression of pro-fibrotic markers. Prednisolone also reduced α-sma protein and type III collagen levels, as well as cell proliferation and migration after TGF-β stimulation. However, prednisolone did not downregulate the gene expression of all the pro-fibrotic markers tested and did not restore the reduced PPAR-γ levels after TGF-β stimulation. Interestingly, anti-fibrotic actions of the glucocorticoid were reinforced in the presence of the PPAR-γ agonist 15d-PGJ2. Combined pretreatment modulated Smad2/3 levels and, similar to the ALK-5 inhibitor, blocked β-catenin accumulation elicited by TGF-β.
Prednisolone, along with 15d-PGJ2, modulates pro-fibrotic pathways activated by TGF-β in synovial fibroblasts at least partially through the inhibition of ALK5/Smad2 signaling and subsequent β-catenin accumulation. These findings shed light on the potential therapeutic effects of glucocorticoids treatment combined with a PPAR-γ agonist against synovial fibrosis, although future studies are warranted to further evaluate this concern.
背景/目的：滑膜纤维化是一种在几种肌肉骨骼疾病中观察到的病理过程，其特征在于细胞外基质的过度沉积，以及细胞迁移和增殖。尽管糖皮质激素广泛用于治疗骨关节炎（OA）和类风湿关节炎等风湿性疾病，但糖皮质激素在关节中的作用机制及其对促纤维化途径的影响仍不清楚。
从膝关节和髋关节获得人 OA 滑膜成纤维细胞。用泼尼松龙（1mM）或转化生长因子-β1（TGF-β1）（10ng/ml）处理细胞 1 和 7 天以定量 RNA 和蛋白质表达（分别通过实时定量逆转录-PCR 和 Western blot），72 小时进行免疫细胞化学分析，48 小时进行增殖（通过 BrdU 测定）和迁移（通过划痕测定）研究。此外，细胞在用泼尼松龙和/或过氧化物酶体增殖物激活受体 γ（PPAR-γ）激动剂 15-脱氧-Δ-12,14-前列腺素 J2（15d-PGJ2）预处理 6 小时后再加入 TGF-β1。通过 Western blot 分析 pSmad1/5、pSmad2 和 β-连环蛋白水平。使用激活素受体样激酶-5（ALK-5）抑制剂（SB-431542）进行机制研究。
泼尼松龙对成纤维细胞样滑膜细胞表现出主要的抗纤维化作用，因为它减弱了自发和 TGF-β诱导的促纤维化标志物的基因表达。泼尼松龙还降低了α-SMA 蛋白和 III 型胶原水平，以及 TGF-β刺激后的细胞增殖和迁移。然而，泼尼松龙并没有下调所有测试的促纤维化标志物的基因表达，也没有恢复 TGF-β刺激后降低的 PPAR-γ 水平。有趣的是，在存在 PPAR-γ 激动剂 15d-PGJ2 的情况下，糖皮质激素的抗纤维化作用得到了加强。联合预处理调节 Smad2/3 水平，并且与 ALK-5 抑制剂一样，阻断了 TGF-β引起的β-连环蛋白积累。
泼尼松龙与 15d-PGJ2 一起，通过抑制 ALK5/Smad2 信号转导和随后的β-连环蛋白积累，至少部分调节 TGF-β激活的滑膜成纤维细胞中的促纤维化途径。这些发现揭示了糖皮质激素联合 PPAR-γ 激动剂治疗对滑膜纤维化的潜在治疗效果，尽管需要进一步研究来进一步评估这一问题。
