Busschers Evita, Holt Jeff P, Richardson Dean W
Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19348, USA.
Am J Vet Res. 2010 Feb;71(2):176-85. doi: 10.2460/ajvr.71.2.176.
To determine effects of interleukin (IL)-1 beta and glucocorticoids on total glycosaminoglycan (GAG) loss and aggrecanase-mediated matrix degradation in equine cartilage.
Cartilage from 24 equine cadavers free of sepsis and musculoskeletal disease.
Effects of IL-1 beta, IL-1 beta with glucocorticoids (dexamethasone and triamcinolone, 10(-6) and 10(-7)M), and glucocorticoids alone on degradation of equine articular and nasal cartilage explants were assessed by measuring GAG release in media and GAG content in cartilage. Aggrecanase-mediated cleavage within the interglobular domain at Glu373-Ala374 was evaluated via western blot analysis and ELISAs. Steady-state mRNA concentrations of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, and ADAMTS5 were assessed by use of real-time reverse transcriptase PCR assay (cartilage explants) and northern blot analysis (cell culture).
IL-1 beta increased GAG release and aggrecanase activity (11-fold). The MMP-3, MMP-13, and ADAMTS4 mRNA were upregulated with IL-1 beta, whereas ADAMTS5 mRNA was increased (13-fold), but significantly less than ADAMTS4 mRNA (27-fold), suggesting a role for both ADAMTS4 and ADAMTS5 in degradation of cytokine-stimulated cartilage. Despite downregulation of MMP-3 and MMP-13 mRNA, glucocorticoids did not alter GAG degradation. A further increase in aggrecanase activity was detected with ELISAs and western blot analysis, whereas ADAMTS4 mRNA was downregulated and ADAMTS5 mRNA was maintained or upregulated.
MMP-3, MMP-13, and ADAMTS4 were regulated differently than ADAMTS5. Glucocorticoids increased aggrecanase activity despite down-regulation of ADAMTS4 mRNA, suggesting a major role of ADAMTS5. Effects of glucocorticoids on aggrecanase activity have important implications in terms of treatment.
确定白细胞介素（IL）-1β和糖皮质激素对马软骨中总糖胺聚糖（GAG）损失和聚集蛋白聚糖酶介导的基质降解的影响。
取自24匹无败血症和肌肉骨骼疾病的马尸体的软骨。
通过测量培养基中的GAG释放量和软骨中的GAG含量，评估IL-1β、IL-1β与糖皮质激素（地塞米松和曲安奈德，10⁻⁶和10⁻⁷M）以及单独的糖皮质激素对马关节软骨和鼻软骨外植体降解的影响。通过蛋白质印迹分析和酶联免疫吸附测定（ELISA）评估聚集蛋白聚糖酶在球间结构域内Glu373-Ala374处的切割情况。使用实时逆转录聚合酶链反应测定（软骨外植体）和Northern印迹分析（细胞培养）评估基质金属蛋白酶（MMP）-3、MMP-13、含血小板反应蛋白基序的解聚素和金属蛋白酶（ADAMTS）4以及ADAMTS5的稳态mRNA浓度。
IL-1β增加了GAG释放和聚集蛋白聚糖酶活性（11倍）。IL-1β使MMP-3、MMP-13和ADAMTS4 mRNA上调，而ADAMTS5 mRNA增加（13倍），但显著低于ADAMTS4 mRNA（27倍），表明ADAMTS4和ADAMTS5在细胞因子刺激的软骨降解中均起作用。尽管MMP-3和MMP-13 mRNA下调，但糖皮质激素并未改变GAG降解。通过ELISA和蛋白质印迹分析检测到聚集蛋白聚糖酶活性进一步增加，而ADAMTS4 mRNA下调，ADAMTS5 mRNA维持不变或上调。
MMP-3、MMP-13和ADAMTS4的调节方式与ADAMTS5不同。尽管ADAMTS4 mRNA下调，但糖皮质激素增加了聚集蛋白聚糖酶活性，表明ADAMTS5起主要作用。糖皮质激素对聚集蛋白聚糖酶活性的影响在治疗方面具有重要意义。
