Chowdhury T T, Arghandawi S, Brand J, Akanji O O, Bader D L, Salter D M, Lee D A
School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK.
Arthritis Res Ther. 2008;10(2):R35. doi: 10.1186/ar2389. Epub 2008 Mar 18.
Nitric oxide and prostaglandin E2 (PGE2play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1beta and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.
Chondrocyte/agarose constructs were cultured under free-swelling conditions with or without IL-1beta and/or SB203580 (inhibitor of p38 MAPK) for up to 48 hours. Using a fully characterized bioreactor system, constructs were subjected to dynamic compression for 6, 12 and 48 hours under similar treatments. The activation or inhibition of p38 MAPK by IL-1beta and/or SB203580 was analyzed by western blotting. iNOS, COX-2, aggrecan and collagen type II signals were assessed utilizing real-time quantitative PCR coupled with molecular beacons. Release of nitrite and PGE2 was quantified using biochemical assays. Two-way analysis of variance and the post hoc Bonferroni-corrected t-test were used to examine data.
IL-1beta activated the phosphorylation of p38 MAPK and this effect was abolished by SB203580. IL-1beta induced a transient increase in iNOS expression and stimulated the production of nitrite release. Stimulation by either dynamic compression or SB203580 in isolation reduced the IL-1beta induced iNOS expression and nitrite production. However, co-stimulation with both dynamic compression and SB203580 inhibited the expression levels of iNOS and production of nitrite induced by the cytokine. IL-1beta induced a transient increase in COX-2 expression and stimulated the cumulative production of PGE2 release. These effects were inhibited by dynamic compression or SB203580. Co-stimulation with both dynamic compression and SB203580 restored cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which we observed no response with the cytokine and/or SB203580.
These data suggest that dynamic compression directly influences the expression levels of iNOS and COX-2. These molecules are current targets for pharmacological intervention, raising the possibility for integrated pharmacological and biophysical therapies for the treatment of cartilage joint disorders.
一氧化氮和前列腺素E2（PGE2）在骨关节炎的发病机制和关节软骨的分解代谢过程中均起关键作用。这些介质受白细胞介素-1β（IL-1β）和机械负荷的影响，并涉及诱导型一氧化氮合酶（iNOS）和环氧化酶（COX）-2的改变。为了确定两种刺激所激活的特定相互作用，我们研究了动态压缩对iNOS和COX-2表达水平的影响以及p38丝裂原活化蛋白激酶（MAPK）途径的参与情况。
软骨细胞/琼脂糖构建体在有无IL-1β和/或SB203580（p38 MAPK抑制剂）的自由肿胀条件下培养长达48小时。使用一个特征充分的生物反应器系统，在类似处理下对构建体进行6、12和48小时的动态压缩。通过蛋白质印迹法分析IL-1β和/或SB203580对p38 MAPK的激活或抑制作用。利用实时定量PCR结合分子信标评估iNOS、COX-2、聚集蛋白聚糖和II型胶原蛋白信号。使用生化测定法定量亚硝酸盐和PGE2的释放。采用双向方差分析和事后Bonferroni校正t检验来检验数据。
IL-1β激活了p38 MAPK的磷酸化，而SB203580消除了这种作用。IL-1β诱导iNOS表达短暂增加并刺激亚硝酸盐释放。单独的动态压缩或SB203580刺激均降低了IL-1β诱导的iNOS表达和亚硝酸盐产生。然而，动态压缩和SB203580共同刺激抑制了细胞因子诱导的iNOS表达水平和亚硝酸盐产生。IL-1β诱导COX-2表达短暂增加并刺激PGE2释放的累积产生。这些作用被动态压缩或SB203580抑制。动态压缩和SB203580共同刺激恢复了细胞因子诱导的对聚集蛋白聚糖表达的抑制。这与II型胶原蛋白相反，在II型胶原蛋白中我们未观察到细胞因子和/或SB203580的反应。
这些数据表明动态压缩直接影响iNOS和COX-2的表达水平。这些分子是目前药物干预的靶点，这增加了综合药物和生物物理疗法治疗软骨关节疾病的可能性。
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