Biochemical Analysis of Nitrated Matrix Metalloproteinase (MMP) in Synovial Fluid from Patients with TMD

一氧化氮(NO)、超氧化物及其细胞毒性反应产物过氧亚硝酸盐(ONOO)是各种形式炎症中细胞损伤的终末介质。最近的研究表明，TMJ中的这些自由基可能会放大关节组织的损伤。在过去的研究中，我们开发了一种简易的检测系统，用来检测滑液中基质金属蛋白酶(MMPs)的活性，并发现MMP3的活性是TM疾病(TMD)早期退行性改变的标志。然而，基质金属蛋白酶在体内的激活途径尚不清楚。最近，一些研究报道了它在体外激活MMPs。在这项研究中，我们检测了TMD患者SF中的蛋白质是否存在于TMD患者的硝基酪氨酸中，这种已知的稳定和特异的足迹显示了过氧亚硝酸盐的硝化作用。此外，为了鉴定SF中的硝化蛋白，采用免疫沉淀法对SF蛋白进行了分析。结果表明，SF中的硝化蛋白被鉴定为MMP-2和-3，而SF中的过氧亚硝酸盐激活了MMP-3。

Nitric oxide (NO), super oxide, and their cytotoxic reaction product peroxynitrite (ONOO) are terminal mediators of cellular injury in various forms of inflammation. Recent studies suggested that these free radicals in the temporomandibular Joint (TMJ) might amplify damage of articular tissues. In the past study, we developed an easy assay system using a erogenic peptide for measuring matrix metalloproteinase (MMP) activity in synovial fluid (SF) and found that MMP-3 activatio was a hallmark indicating an early degenerative change in TM disorders (TMD). However, the activating pathway of MMP in vivo remains unexplained. Recently some studies have reported that activates MMPs in vitro. In this study, we examined whether proteins in SF from TMD patient nitro tyrosine, which was known to be stable and specific footprint showing nitration by peroxynitrite. Furthermore, in order to identify nitrated protein in SF, SF proteins were analyzed by immunoprecipitation method. As the results, we demonstrated that nitrated proteins in SF were identified as MMP-2 and -3, and that peroxynitrite in SF activated MMP-3.