Sadowski Thorsten, Steinmeyer Jürgen
Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany.
Biochem Pharmacol. 2002 Jul 15;64(2):217-27. doi: 10.1016/s0006-2952(02)01073-0.
In this study we determined the in vitro effects of polysulfated glycosaminoglycan (PSGAG) and the glucocorticoid triamcinolone acetonid (TA) on the IL-1 altered expression and activity of matrix metalloproteinases (MMP-1, MMP-3), tissue inhibitor of metalloproteinases-1, the plasminogen activators tPA and uPA and plasminogen activator inhibitor 1 by articular chondrocytes. Bovine chondrocytes were cultured in alginate gel beads. Cells were treated with interleukin-1alpha (IL-1alpha) in the presence of vehicle or drugs at various concentrations. After 48hr mRNA expression of MMP-1, MMP-3, TIMP-1, uPA, tPA and PAI-1 was analyzed by RT-PCR-ELISA. The protein synthesis of TIMP-1 and MMP-3 was determined by immunoprecipitation, PAI-1 protein was quantitated by ELISA. The activity of enzymes and inhibitors was measured by functional assays. Treating chondrocytes with IL-1 induced the expression of MMPs and downregulated TIMP-1 but stimulated both the expression of PAs and PAI-1. Both drugs significantly reduced collagenase and proteoglycanase activities which was accompanied by inhibition of the expression of MMP-1 and MMP-3. The IL-1 decreased expression of TIMP-1 was further reduced by TA, which resulted in a significant loss of TIMP activity. No effects on TIMP activity or TIMP-1 biosynthesis were observed after treatment of chondrocytes with PSGAG. Both drugs inhibited the IL-1-induced mRNA expression of tPA, whereas expression of uPA was only mildly reduced by PSGAG, which also induced PAI-1 above IL-1 stimulated levels. As inhibition of collagenase activities and tPA expression by PSGAG occurred at physiological concentrations it might be of clinical relevance, indicating that PSGAG could help reducing cartilage degradation and has a strong anti-fibrinolytic potential. Due to their co-regulation of MMPs and TIMP(s) glucocorticoids should be carefully studied for their overall effect on extracellular matrix proteolysis.
在本研究中，我们测定了多硫酸化糖胺聚糖（PSGAG）和糖皮质激素曲安奈德（TA）对白细胞介素-1（IL-1）改变的基质金属蛋白酶（MMP-1、MMP-3）、金属蛋白酶组织抑制剂-1、纤溶酶原激活物tPA和uPA以及纤溶酶原激活物抑制剂1的表达和活性的体外影响，这些影响由关节软骨细胞产生。牛软骨细胞在藻酸盐凝胶珠中培养。细胞在不同浓度的溶剂或药物存在下用白细胞介素-1α（IL-1α）处理。48小时后，通过逆转录聚合酶链反应-酶联免疫吸附测定（RT-PCR-ELISA）分析MMP-1、MMP-3、TIMP-1、uPA、tPA和PAI-1的mRNA表达。通过免疫沉淀法测定TIMP-1和MMP-3的蛋白质合成，通过酶联免疫吸附测定法定量PAI-1蛋白。通过功能测定法测量酶和抑制剂的活性。用IL-1处理软骨细胞可诱导基质金属蛋白酶的表达并下调TIMP-1，但刺激纤溶酶原激活物和PAI-1的表达。两种药物均显著降低胶原酶和蛋白聚糖酶活性，同时抑制MMP-1和MMP-3的表达。TA进一步降低了IL-1诱导的TIMP-1表达降低，导致TIMP活性显著丧失。用PSGAG处理软骨细胞后，未观察到对TIMP活性或TIMP-1生物合成的影响。两种药物均抑制IL-1诱导的tPA的mRNA表达，而uPA的表达仅被PSGAG轻度降低，PSGAG还将PAI-1诱导至高于IL-1刺激的水平。由于PSGAG在生理浓度下可抑制胶原酶活性和tPA表达，这可能具有临床相关性，表明PSGAG有助于减少软骨降解并具有强大的抗纤溶潜力。由于它们对基质金属蛋白酶和金属蛋白酶组织抑制剂的共同调节作用，应仔细研究糖皮质激素对细胞外基质蛋白水解的总体影响。
