Background and purpose

Osteoarthritis (OA) is a chronic disease accompanied by severe inflammation. The inflammation activation in the chondrocytes and the degradation of the extracellular matrix were reported to be involved in the progress of OA. Roflumilast is a selective PDE4 inhibitor used for treating chronic obstructive pulmonary disease (COPD) and exerts significant anti-inflammation effects. The present study aims to investigate the effects of Roflumilast on tumor necrosis factor-β (TNF-β)-induced inflammation activation and degradation of type 2 collagen in chondrocytes.

Methods

TNF-β was used to establish the in-vitro inflammation model on ATDC5 chondrocytes. Quantitative real-time polymerase chain reaction (QRT-PCR) and western blot were used to determine the expression level of tumor necrosis factor receptor 2 (TNFR2), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinase 3 (MMP-3), matrix metalloproteinase 13 (MMP-13), type 2 collagen and nuclear factor kappa B (NF-κB) p65. The release of prostaglandin E2 (PGE₂), MMP-3, and MMP-13 were evaluated by ELISA. The production of NO was determined by DAF-FM DA staining and the function of the NF-κB promoter was evaluated by Luciferase activity assay.

Results

TNFR2 and COX-2 were upregulated and the release of PGE₂ was promoted by TNF-β stimulation, which were all inhibited by Roflumilast. Roflumilast suppressed the promoted iNOS expression and NO production induced by TNF-β. MMP-3 and MMP-13 were up-regulated, and type 2 collagen was down-regulated by TNF-β stimulation, which were all reversed by Roflumilast. Roflumilast inhibited the promoted releasing of Interleukin-8 (IL-8) and Interleukin-12 (IL-12), expression of up-regulated NF-κB, and activation of NF-κB transcriptional activity induced by TNF-β.

Conclusion

Roflumilast may prevent TNF-β-induced inflammation activation and degradation of type 2 collagen in chondrocytes.

中文翻译：

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罗氟司特预防淋巴毒素α（TNF-β）诱导的炎症激活和软骨细胞中2型胶原的降解

背景和目的

骨关节炎（OA）是一种伴随严重炎症的慢性疾病。据报道，软骨细胞的炎症激活和细胞外基质的降解与OA的进展有关。罗氟司特是一种选择性PDE4抑制剂，用于治疗慢性阻塞性肺疾病（COPD），并具有显着的抗炎作用。本研究旨在研究罗氟司特对肿瘤坏死因子-β（TNF-β）诱导的软骨细胞炎症激活和2型胶原降解的影响。

方法

TNF-β用于建立ATDC5软骨细胞的体外炎症模型。实时定量聚合酶链反应（QRT-PCR）和Western印迹法检测肿瘤坏死因子受体2（TNFR2），环氧合酶2（COX-2），诱导型一氧化氮合酶（iNOS），基质的表达水平金属蛋白酶3（MMP-3），基质金属蛋白酶13（MMP-13），2型胶原蛋白和核因子κB（NF-κB）p65。通过ELISA评估前列腺素E2（PGE ₂），MMP-3和MMP-13的释放。通过DAF-FM DA染色确定NO的产生，并通过荧光素酶活性测定评估NF-κB启动子的功能。

结果

TNF-β刺激上调了TNFR2和COX-2的表达，促进了PGE ₂的释放，而后者均被罗氟司特抑制。罗氟司特抑制了TNF-β诱导的iNOS表达和NO生成的促进。MMP-3和MMP-13被TNF-β刺激而上调，而2型胶原被TNF-β刺激而被下调，而Roflumilast均将其逆转。罗氟司特抑制白细胞介素8（IL-8）和白细胞介素12（IL-12）的释放释放，上调的NF-κB的表达以及由TNF-β诱导的NF-κB转录活性的激活。

结论

罗氟司特可预防软骨细胞中TNF-β诱导的炎症激活和2型胶原降解。