第二型活化蛋白酶抑制胜肽調控環氧化酶-2 在退化性關節炎之分子機制

PAR-2 inhibition peptide modulated cyclooxygenase-2 molecular mechanisms of osteoarthritis

陳詩芸(Shi-Yun Chen)

指導教授 ： 陳建和

共同指導教授 ： 許明照

臺北醫學大學/醫學院/醫學科學研究所/碩士(2009年)

https://doi.org/10.6831/TMU.2009.00095

摘要

第二型蛋白酶受體可被絲胺酸類似的胰蛋白酶給活化，並且有研究指出會參予發炎反應的路徑。在之前的研究指出,在受損的退化性關節炎病人的軟骨細胞中，,第二型蛋白酶受體的表現會大於正常軟骨細胞。之前研究也已證實在病人滑膜液細胞當中第二型蛋白酶受體可在短時間受到第一型介白素的調控。這些結果顯示第二型蛋白酶受體可當作治療退化性關節炎的目標。本篇的研究目的要找出有效的第二型蛋白酶活化受體的抑制胜肽，,當作一種抑制劑來達到降低發炎反應的產生，,包括環氧化酶-2(COX-2)和基質金屬蛋白酶類(MMPs)的生合成。我們置換的方式是從第二型蛋白酶活化胜肽(由六個胺基酸組成) ，,藉由胺基酸的結構和特性來作變換。我們將IIe換成Ala，形成SLAGKV後觀察對於第二型蛋白酶受體活化的影響。第二型蛋白酶活化受體的抑制胜肽對於滑膜瘤細胞和人類滑膜液細胞的COX-2生成以及NF-κB，利用西方墨點法測定。本篇研究初步證實了胰蛋白酶在人類滑膜液細胞中可誘導環氧化酶以及基質金屬蛋白酶發炎因子產生;並利用第二型活化蛋白酶抑制胜肽之後除了發現降低了COX-2生成，並可直接間影響了胰蛋白酶去進一步活化第二型蛋白酶。另外,我們發現胰蛋白酶以及第二型蛋白酶會在短時間磷酸化NF-κB的訊息傳遞路徑來調控這些發炎物質的產生。第二型活化蛋白酶抑制胜肽並可影響了胰蛋白酶去進一步活化NF-κB。這種利用第二型蛋白酶活化受體的抑制胜肽來結合到受體後，以至於干擾了蛋白酶調控下游蛋白的功能,而達到降低發炎反應的產生。有鑑於此，在未來理想的第二型活化抑制胜肽可當做治療退化性關節炎病程進展的方法之一 。

關鍵字

骨關節炎 ； 第二型蛋白酶活化受體 ； 環氧化酶-2 ； 基質金屬蛋白酶類

並列摘要

Proteinase-activated receptor 2 (PAR-2) is activated by trypsin-like serine proteinases and PAR-2 was shown to be involved in inflammatory pathways. The level of PAR-2 in OA chondrocytes was much higher than that in normal chondrocytes. Previous studies have investigated that PAR-2 mRNA and protein level was induced by IL-1β in OA synovial cells. These results suggest PAR-2 as an potential new therapeutic target for the treatment of OA. The aim of this study is to design a effective proteinase-activated receptor 2 inhibition peptide (PAR-2 IP) as an antagonists to achieve inhibition of inflammatory response, including metalloproteinase (MMP) and inflammatory mediator cyclooxygenase 2 (COX-2). We replace similar aminio acid based on chemical and character from PAR-2 activating peptide(SLIGKV) . When Ala substitution of IIe from PAR-2 activating peptide, giving SLAGKV. The effect of PAR-2 IP on COX-2 production and NF-κB were measured on stimulated SW982 cell lines and human synoviocytes by Western blotting. In this study, we first demonstrated that the expression of COX-2 and MMP-1 were up-regulation by trypsin. Further examination showed that PAR-2 IP not only couldn’t activate COX-2 expression but also block trypsin to activate PAR-2. Disrupting proteolytic activation beause of using PAR-2 IP directed to bind the receptor activation site, so that could suppress inflammatory response. In addition, we find that trypsin and PAR-2 activating peptide induce IκBα-dependent NF-κB activation. PAR-2 IP directed to bind the receptor activation site, so that could suppress NF-κB activation. In conclusion,prolonged therapy with PAR-2 IP decreases COX-2 production by trypsin active PAR-2. In the future,optimal PAR-2 inhibition peptide composition can be defined as a novel target for the treatment of OA.

並列關鍵字

OA ； PAR2 ； COX-2 ； MMP

參考文獻

1. Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum 2001; 44: 1237-47.

2. Goldring MB. The role of the chondrocyte in osteoarthritis. Arthritis Rheum 2000; 43: 1916-26.

3. Goldring MB. Osteoarthritis and cartilage: the role of cytokines. Curr Rheumatol Rep 2000; 2: 459-65.

4. Goldring MB, Birkhead J, Sandell LJ, Kimura T, Krane SM. Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes. J Clin Invest 1988; 82: 2026-37.

5. Aigner T, Dudhia J. Phenotypic modulation of chondrocytes as a potential therapeutic target in osteoarthritis: a hypothesis. Ann Rheum Dis 1997; 56: 287-91.