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

TMD 患者滑液中硝化基质金属蛋白酶 (MMP) 的生化分析

• 批准号: 12671955
• 负责人: KAWAKAMI Tetsuji
• 金额: $ 2.18万
• 依托单位: Nara Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671955/
• 关键词: TM disorders; Synovial fluid; Matrix metalloproteinase (MMP); Free radical; Nitric Oxide (NO); Peroxynitrite; 蛍光基質ペプチド; 活性型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.

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。近年来的研究表明，颞下颌关节（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，我们开发了一种简单的测定系统，使用一种雌激素肽来测量滑液（SF）中的基质金属蛋白酶（MMP）活性，并发现MMP-3活化是TM疾病（TMD）早期退行性变化的标志。然而，MMP在体内的活化途径仍然没有得到解释。近年来有研究报道，在体外激活MMPs。在这项研究中，我们研究了是否蛋白质SF从TMD患者硝基酪氨酸，这是已知的是稳定的和特定的足迹显示硝化过氧亚硝酸盐。此外，为了鉴定SF中的硝化蛋白，用免疫沉淀法分析SF蛋白。作为结果，我们证明了SF中的硝化蛋白被鉴定为MMP-2和MMP-3，并且SF中的过氧亚硝酸盐激活MMP-3。