Rofecoxib modulates multiple gene expression pathways in a clinical model of acute inflammatory pain

Abstract

New insights into the biological properties of cyclooxygenase-2 (COX-2) and its response pathway challenge the hypothesis that COX-2 is simply pro-inflammatory and inhibition of COX-2 solely prevents the development of inflammation and ameliorates inflammatory pain. The present study performed a comprehensive analysis of gene/protein expression induced by a selective inhibitor of COX-2, rofecoxib, compared with a non-selective COX inhibitor, ibuprofen, and placebo in a clinical model of acute inflammatory pain (the surgical extraction of impacted third molars) using microarray analysis followed by quantitative RT-PCR verification and Western blotting. Inhibition of COX-2 modulated gene expression related to inflammation and pain, the arachidonic acid pathway, apoptosis/angiogenesis, cell adhesion and signal transduction. Compared to placebo, rofecoxib treatment increased the gene expression of ANXA3 (annexin 3), SOD2 (superoxide dismutase 2), SOCS3 (suppressor of cytokine signaling 3) and IL1RN (IL1 receptor antagonist) which are associated with inhibition of phospholipase A(2) and suppression of cytokine signaling cascades, respectively. Both rofecoxib and ibuprofen treatment increased the gene expression of the pro-inflammatory mediators, IL6 and CCL2 (chemokine C-C motif ligand 2), following tissue injury compared to the placebo treatment. These results indicate a complex role for COX-2 in the inflammatory cascade in addition to the well-characterized COX-dependent pathway, as multiple pathways are also involved in rofecoxib-induced anti-inflammatory and analgesic effects at the gene expression level. These findings may also suggest an alternative hypothesis for the adverse effects attributed to selective inhibition of COX-2.

Figure 1

Gene expression profile induced by acute inflammation and inhibition of cyclooxygenase in a clinical model of tissue injury. A, change in percentage of transcripts with significant up- or down-regulation following acute inflammation (placebo) and treatments by ibuprofen or rofecoxib (paired t-test, GCOS software). B, distribution and intersection of up-regulated transcripts (over 3-fold, p < 0.05, n = 6 per treatment) following tissue injury in each treatment group; C, distribution and intersection of down-regulated transcripts (over 3-fold, p < 0.05, n = 6 per treatment) following tissue injury in each treatment groups.

Figure 2

Comparison of changes in gene expression analyzed by microarray and qRT-PCR. The data are expressed as mean ± SEM. *, p < 0.05, compared between pre- and post-surgery tissue (paired t-test); +, p < 0.05, compared between place and rofecoxib treatments (F-test, df = 2); #, p < 0.05 compared between rofecoxib and ibuprofen treatments (F-test, df = 2).

Figure 3

The changes in gene expression following acute inflammation and drug treatments as assessed by qRT-PCR. The gene expression level is expressed as the average threshold cycle after normalization using 18 rRNA expression (Average Delt Ct). The open symbols represent the gene expression level from each subject in normal pre-surgery tissue and after tissue injury. The solid symbols represent mean ± SEM. *, p < 0.05 significant difference from the placebo group (one-way ANOVA followed by Tukey test).

Figure 4

COX-2 inhibition increased the protein expression of IL6 and SOCS3 determined by immunoblot analysis. Representative western blots for specific IL6 and SOCS3 expression were from independent samples (n = 3 per treatment). Positive controls as marked (+) were determined using Hela and BJAB cell lysates for IL6 and SOCS3, respectively. Data are represented as mean ± SEM. *, p < 0.05 ANOVA followed by post hoc pairwise t-test.

Figure 5

Inhibition of COX-2 modulates multiple functional pathways during acute inflammation and pain. Theoretical schematic diagram depicts the genes and their pathway modulated by rofecoxib treatment during acute inflammation. Besides inhibiting COX-2 activity, rofecoxib may also produce its anti-inflammatory effect through (1) up-regulating annexins and SOD2 expression, which directly suppress the activity of phospholipase A2; (2) up-regulating SOCS3, which inhibit cytokine signaling by negative feedback regulation of JAK/STAT cascade; (3) down-regulation of the gene expression IL1 and C3. However, by shunting down the cyclooxygenase pathway and reducing COX-2-derived PGs production, rofecoxib may induce the over-expression of the pro-inflammatory mediators (4) leukotrienes (indicated by the thicker solid lines), and (5) IL6 and CCL2 (MCP-1). The latter may play a critical role in the development of the adverse effects attributed to COX-2 inhibitors. The solid lines indicate facilitation and the dashed lines indicate inhibition.