Luminex液相蛋白芯片检测一氧化氮合酶抑制剂对骨关节炎患者软骨基质金属蛋白酶表达的影响

Effects of nitric-oxide synthase inhibitor on matrix metalloproteinase＇s expression in osteoarthritis cartilage by Luminex analysis

目的应用Luminex液相蛋白芯片检测一氧化氮合酶（NOS）抑制剂对骨关节炎（OA）患者软骨基质金属蛋白酶（MMPs）表达的影响，以及NOS抑制剂改善OA代谢的途径。方法研究经本院医学伦理委员会批准并获取患者的知情同意。无菌条件下，取15例重度OA需行关节置换术患者的关节软骨，置人体外培养系统。应用随机数目表法分为：①对照组：不加药物干预；②L—N^6-亚氨乙基-赖氨酸（L-NIL）组：加入NOS抑制剂L-NIL干预。培养72h后，通过检测硝酸盐和亚硝酸盐的含量来观察软骨一氧化氮（NO）的释放量和NOS的活性；应用Luminex液相蛋白芯片检测OA患者软骨中MMPs（MMP-1，MMP-2，MMP-3，MMP-9，MMP-13）表达量的变化。数据采用配对t检验作均数的显著性检验。结果对照组软骨培养72h后，在其上清液中可检测到高浓度NO[（216±47）μmol／L]和高活性的NOS[（5．7±1．3）U／ml]，L—NIL组NO释放量[（55±20）μmol／L]较对照组明显减少，NOS活性[（1．7±0．7）U/ml]显著降低（P均〈0．01）。Luminex液相蛋白芯片显示对照组OA软骨中MMPs[（MMP-1（10．8±5．4）ng／ml，MMP-2（9．2±3．3）ng／ml，MMP-3（11．6±4．2）ng／ml，MMP-9（1．27±1．07）ng／ml，MMP-13（3．6±1．3）ng／ml]的表达异常，而L—NIL组OA软骨中MMPs表达量明显被抑制[分别为（3．6±1．8）ng／ml，（2．3±1．2）ng/ml，（3．6±1．4）ng／ml，（0．65±0．21）ng／ml，（1．8±0．5）．g／ml，P均〈0．05]。结论Luminex液相蛋白芯片检测系统表明NOS抑制剂通过减少NO的过度释放和降低NOS活性，进而抑制MMPs的过度表达来改善OA软骨的代谢。

Objective To investigate the effects of nitric-oxide synthase inhibitor on matrix metalloproteinases （MMPs） expression in osteoarthritis （OA） cartilage by Luminex analysis, and to explore the mechanism of nitric-oxide synthase inhibitor on modification of the metabolism of OA cartilage. Methods Fifteen specimens of articular cartilage taken from the patients with OA were cultured and divided in two groups. The control group was those with no intervention. L-NIL group was co-cultured with NOS inhibitor L-NIL. After 72 h cultivation, the release of NO and the activity of NOS on OA cartilage were measured by Griess reaction and spectrophotometrie methods. MMPs （MMP-1, MMP-2, MMP-3, MMP-9, MMP-13） expression was measured by Luminex analysis. Comparisons between groups were performed with paired samples t test. Results After cultured for 72 h, spectrophotometrie analysis showed high concentration of NO release [（216±47） μmol/L± and high level of active NOS [（5.7±1.3） U/ml] in supernatants of the control, 1 mmol/L concentration L-NIL could evidently reduce NO release [（55±20） μmol/L, P〈0.01] and NOS activity [（1.7±0.7）U/ml, P〈0.01 ]. Luminex analysis demonstrated high MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 expression in cartilages of the control group [respectively for （10.8±5.4） ng/ml, （9.2±3.3） ng/ml, （11.6±4.2） ng/ml, （1.27±1.07） ng/ml, （3.6±1.3） ng/ml] and 1 ng/ml/L concentration L-NIL could evidently inhibit MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 expression [respectively for （3.6±1.8）ng/ml, （2.3± 1.2） ng/ml, （3.6±1.4） ng/ml, （0.65±0.21） ng/ml, （1.8±0.5） ng/ml, P〈0.05]. Conclusion Luminex analysis has shown that NOS inhibitor can reduce NO release and NOS activity and modify metabolism of articular cartilage by inhibiting the over-expression of MMPs.