School of Engineering and Materials Science, Queen Mary University of London, Mile End, London, E1 4NS, UK.
Arthritis Res Ther. 2010;12(6):R207. doi: 10.1186/ar3183. Epub 2010 Nov 8.
In inflammatory joint disease, such as osteoarthritis (OA), there is an increased level of proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines stimulate the production of matrix metalloproteinases (MMPs), which leads to the degradation of the cartilage extracellular matrix and the loss of key structural components such as sulphated glycosaminoglycan (sGAG) and collagen II. The aim of this study was to examine the therapeutic potential of n-3 polyunsaturated fatty acids (PUFAs) in an in vitro model of cartilage inflammation.
Two specific n-3 compounds were tested, namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), each at 0.1, 1 and 10 μM. Full thickness bovine cartilage explants, 5 mm in diameter, were cultured for 5 days with or without IL-1β and in the presence or absence of each n-3 compound. The media were replaced every 24 hours and assayed for sGAG content using the 1,9-dimethylmethylene blue (DMB) method. Chondrocyte viability was determined at the end of the culture period using fluorescence microscopy to visualise cells labelled with calcein AM and ethidium homodimer.
Treatment with IL-1β (10 ng.ml⁻¹) produced a large increase in sGAG release compared to untreated controls, but with no effect on cell viability, which was maintained above 80% for all treatments. In the absence of IL-1β, both n-3 compounds induced a mild catabolic response with increased loss of sGAG, particularly at 10 μM. By contrast, in the presence of IL-1β, both EPA and DHA at 0.1 and 1 μM significantly reduced IL-1β-mediated sGAG loss. The efficacy of the EPA treatment was maintained at approximately 75% throughout the 5-day period. However, at the same concentrations, the efficacy of DHA, although initially greater, reduced to approximately half that of EPA after 5 days. For both EPA and DHA, the highest dose of 10 μM was less effective.
The results support the hypothesis that n-3 compounds are anti-inflammatory through competitive inhibition of the arachidonic acid oxidation pathway. The efficacy of these compounds is likely to be even greater at more physiological levels of IL-1β. Thus we suggest that n-3 PUFAs, particularly EPA, have exciting therapeutic potential for preventing cartilage degradation associated with chronic inflammatory joint disease.
在炎症性关节疾病（如骨关节炎（OA））中，存在促炎细胞因子（如白细胞介素（IL）-1β）水平升高。这些细胞因子刺激基质金属蛋白酶（MMPs）的产生，导致软骨细胞外基质的降解和关键结构成分（如硫酸氨基葡萄糖（sGAG）和胶原 II）的丢失。本研究的目的是在软骨炎症的体外模型中研究 n-3 多不饱和脂肪酸（PUFAs）的治疗潜力。
测试了两种特定的 n-3 化合物，即二十碳五烯酸（EPA）和二十二碳六烯酸（DHA），每种浓度为 0.1、1 和 10 μM。直径为 5 毫米的全厚牛软骨标本在有或没有 IL-1β和存在或不存在每种 n-3 化合物的情况下培养 5 天。每隔 24 小时更换培养基，并使用 1,9-二甲基亚甲蓝（DMB）法测定 sGAG 含量。在培养期末，使用荧光显微镜观察用 calcein AM 和 ethidium homodimer 标记的细胞，以确定软骨细胞活力。
与未处理的对照组相比，用 10 ng.ml-1 的 IL-1β（10 ng.ml-1）处理会导致 sGAG 释放大量增加，但对细胞活力没有影响，所有处理的细胞活力均保持在 80%以上。在没有 IL-1β的情况下，两种 n-3 化合物均诱导轻度分解代谢反应，导致 sGAG 丢失增加，尤其是在 10 μM 时。相比之下，在存在 IL-1β的情况下，0.1 和 1 μM 的 EPA 和 DHA 均显著减少了 IL-1β 介导的 sGAG 丢失。在整个 5 天期间，EPA 治疗的疗效维持在约 75%。然而，在相同浓度下，DHA 的疗效虽然最初更高，但在 5 天后降低到 EPA 的约一半。对于 EPA 和 DHA，10 μM 的最高剂量效果较差。
这些结果支持了 n-3 化合物通过竞争性抑制花生四烯酸氧化途径发挥抗炎作用的假说。在更接近生理水平的 IL-1β 下，这些化合物的疗效可能更大。因此，我们认为 n-3 PUFAs，特别是 EPA，在预防与慢性炎症性关节疾病相关的软骨降解方面具有令人兴奋的治疗潜力。
