The progression of the events implicated in soft oral tissue repair following injury involves a diverse array of signaling cues exerting their influence on mucosal inflammatory responses and the processes that control cellular proliferation, differentiation and migration to the site of injury (1 - 3). The soft tissue injury and oral mucosal ulcer onset are accompanied by upregulation in nitric oxide and prostaglandin production, massive enhancement in epithelial cell apoptosis, and the induction of proinflammatory TNF-alpha that triggers trancriptional factor NF-kappaB activation (3 - 5). The process initiated by the engagement of cell surface TNFR1 by soluble TNF-alpha, results in intracellular signal transduction cascade that leads to activation of IkappaB proteins (inhibitors of nuclear factor kappaB) (6). Upon activation, the IkappaB undergoes critical phosphorylation by the family of activated MAP kinases, which targets IkappaBs for degradation by the ubiquitin-proteosome pathway and leads to translocation of NF-kappaB to the nucleus where it activates genes mediating various aspects of inflammatory responses, incuding the induction of inducible cyclooxygenase (COX-2) and NOS-2 (6 - 9).

While generation of nitric oxide and prostaglandins is controlled by sets of constitutive and inducible enzymes, there are strong indications that enzyme compartmentalization and substrate availability determines the segregated utilization of the respective products in the physiological and pathophysiological processes (9 - 11). Thus, while prostaglandins and nitric oxide produced by COX-1 and cNOS enzymes play housekeeping functions and are recognized as an essential element of the mucosal defense mechanism, the induction of COX-2 and NOS-2 occurs rapidly in response to a variety of stimuli associated with inflammatory processes, and the resulting overproduction of nitric oxide and prostanoids have been intimately implicated as the promoting event for the development of alimentary tract metaplasia and cancer (12 - 15). Moreover, the prostanoid products of COX-2 gene induction have been found to serve as modulators of inflammation through activation of ligand -dependent transcriptional factor, termed peroxisome proliferator-activated receptor-gamma (PPARgamma) (16 - 19). The factor, along with its two other closely related subtypes (alpha and ß), belongs to the steroid hormone receptor superfamily which act by altering the transcription of genes with which they associate (8, 16, 19). Indeed, activation of PPARgamma has been linked to the induction of apoptosis, regulation of cell differentiation, and resolution of inflammation by the inhibition of NFkappaB transactivation of COX-2 and NOS-2 target genes (16, 18). Therefore, pharmacological manipulation of PPARgamma activation might provide therapeutic benefits in preventing the potentiation of TNF-induced proinflammatory events during ulcer healing.

Accordingly, in this study, using the animal model of acetic acid-induced buccal mucosal ulcer model (4), we investigated the effect of a specific synthetic agonist of PPARgamma, ciglitazone (17), on the course of ulcer healing by analyzing mucosal expression and activity of inducible nittric oxide synthase, and cyclooxygenases responsible for prostaglandin generation.


MATERIALS AND METHODS
Animals

The study was conducted with 180 to 200 g Sprague-Dawley rats in compliance with the experimental protocols approved by the Institutional Animal Care and Use Committee. The animals were deprived food and water 2 h before the procedure. Under ether anesthesia, the buccal surfaces of the animals were exposed for 20 s to contact with glacial acetic acid, using a plastic tube of 4 mm in diameter. This produced an immediate mucosal necrosis within affected area followed 2 days latter by the development of chronic ulcer with a well-defined crater, which normally healed within 10 days (4). On the second day after the procedure (designated as ulceration day 0), the animals were divided into groups and subjected twice daily for 10 days to intragastric administration of a specific PPARgamma activator, ciglitazone (Calbiochem, La Jolla, CA) at 5, 10, and 15 mg/kg or the vehicle consisting 5% gum arabic in saline. The dose range of ciglitazone used in the experiments was chosen based on the data as to the effectiveness of the related thiazolidinedione agents in inhibiting gastric mucosal injury in rats (20). The animals were killed at different intervals of ulcer healing for up to 10 days, and the buccal mucosa from the ulcer area together with its margin excised and used for biochemical measurements. The rate of ulcer healing was assessed by measuring the ulcer crater by planimetry (2). The protein content of samples was measured with the BCA protein assay kit (Pierce, Rockford, IL).

NOS-2 activity assay

Buccal mucosal activity of NOS-2 was measured with a NOS-Detect Assay Kit (Stratagene, La Jolla, CA). The individual specimens of buccal mucosa were homogenized in a sample buffer containing 10 mM EDTA and centrifuged at 800g for 10 min (4). The aliquots of the resulting supernatants were incubated for 30 min at 25°C in the presence of L-[2,3,4,5-3H] arginine (50 µCi/µl), 10 mM NAPDH, 5µM tetrahydrobiopterin, and 50 mM Tris-HCl buffer, pH 7.4, in a final volume of 250 µl. (4). Following addition of stop buffer and DOWEX-50W (Na+) resin, the mixtures were transferred to spin cups, centrifuged and the formed L[3H]citrulline contained in the flow through was quantified by scintillation counting.

Mucosal PGE2 generation assay

The individual specimens of the freshly excised buccal mucosal tissue were rinsed in ice-cold buffer consisting of 50 mM Tris-HCl, pH 8.5, placed in 1 ml of the buffer and thoroughly minced with scissors. The mixture of each sample was vortexed at room temperature for 1 min, centrifuged at 10,000g for 15 min at 4°C, and the supernatants used for PGE2 determination with a PGE2 EIA kit, according to the manufacturer’s (Cayman, Ann Arbor, MI) instruction. The mucosal capacity for PGE2 synthesis was expressed in pg/mg wet tissue (21).

Western blot analysis

The buccal mucosal tissue specimens were suspended in ice-cold lysis buffer, consisting of 50 mM HEPES, pH 7.5, 250 mM NaCl, 0.3% Triton X-100, 1 mM EDTA, 100 mM DTT,1 mM PMSF, 100 mM orthovanadate, 20 µM pestatin and 20 µM leupeptin, and homogenized for 1 min in a Polytron tissuemizer. The homogenates were centrifuged at 15,000g for 20 min at 4°C, and the resulting supernatants were collected and normalized with respect to protein content (BCA protein assay, Pierce, Rockford, IL) to ensure equal loading on SDS-polyacrylamide gel electrophoresis. The protein extracts (20 - 30 µg) were electrophoresed through an 8.5% reducing SDS-polyacrylamide gels under standard conditions (22), and electroblotted to polyvinylidene difluoride membranes. The membranes were blocked for 16 h at 4°C with 1% nonfat milk in 20 µM Tris-Tris-HCl, pH 7.4, 0.13 M NaCl and 0.02% Tween 20, and then probed with polyclonal rabbit antibodies (Calbiochem, La Jolla, CA) for COX-1, COX-2 and iNOS. Polyclonal anti-ß-actin antibody (Sigma, St. Louis, MO) was used as a control probe for protein integrity. Following washing, the membranes were incubated with anti- rabbit IgG conjugated to horseradish peroxidase and the protein bands were revealed using an enhaced chemiluminescence (Amersham Pharmacia Biotech) system.

Data analysis

All experiments were carried out in duplicate, and the results are expressed as the means ± SD. Analysis of variance (ANOVA) was used to determine significance, and the significance level was set at p < 0.05.


RESULTS
The acetic acid-induced buccal mucosal ulcer model was used to investigate the effect of activation of PPARgamma on the course of events associated with soft oral tissue repair. The repair process was assessed with respect to the rate of ulcer healing, changes in the mucosal capacity for prostaglandin generation and NOS-2 activity, and the expression of COX-1, COX-2 and NOS-2 proteins, using rats subjected to intragastric administration of ciglitazone, a specific synthetic agonist of PPARgamma. As depicted in Fig. 1, the ulcer crater at the onset of the experiments (day, 0) averaged 12.4 mm2, which, in the control group, decreased to 8.5mm2 by the second day and to 0.9mm2 by the sixth day, and virtually healed by the tenth day. Compared with that of normal mucosa, the ulcer onset was characterized by a 6.7-fold increase in PGE2 production (Fig. 2), massive (86-fold) induction in NOS-2 activity (Fig.3), and a marked up-regulation in COX-2 and NOS-2 protein expression, while the expression of COX-1 protein remained unchanged (Fig. 4). The healing was accompanied by a gradual reduction in buccal mucosal NOS-2 activity, decline in PGE2 production, and a decrease in COX-2 and NOS-2 protein expression, but the NOS-2 activity and the production of PGE2 at the end of ten day of healing still remained significantly higher than that of normal mucosa.

Fig. 1. Effect of PPARgamma activator, ciglitazone, on the rate of buccal mucosal ulcer healing. Intragastric administration (twice daily for 10 days) of ciglitazone (at 5, 10, and 15 mg/kg) was commenced on the day of ulcer onset (day, 0). Values represent the means ±SD obtained with 8 animals in each group. *P < 0.05 compared with that of the control.

Fig. 2. Effect of ciglitazone administration, twice daily for 10 days, on the mucosal generation of PGE2 during buccal mucosal ulcer healing. Values represent the means ± SD of duplicate analyses performed with 8 animals in each group. *P < 0.05 compared with that of the control.

Fig. 3. Effect of ciglitazone administration, twice daily for 10 days, on the mucosal expression of NOS-2 activity during buccal mucosal ulcer healing. Values represent the means ± SD of duplicate analyses performed with 8 animals in each group. *P < 0.05 compared with that of the control.

Fig. 4. Western blot analysis of COX-1, COX-2, NOS-2 and b-actin protein expression during buccal mucosal ulcer healing in the presence of ciglitazone (10 mg/kg) administration. Lane 1, normal mucosa. Lane 2 and 4, buccal mucosa at day 6 and 10 of ulcer healing in the absence of ciglitazone. Lane 3 and 5, buccal mucosa at day 6 and 10 of ulcer healing in the presence of ciglitazone.

The effect of intragastric administration of PPARgamma activator, ciglitazone, on the rate of buccal mucosal ulcer healing is presented in Fig. 1. While the course of buccal mucosal tissue repair was not affected by ciglitazone administration, the treatment led to a dose-dependent reduction in the ulcer-induced mucosal NOS-2 activity and the capacity for PGE2 generation. The pattern of changes in buccal mucosal PGE2 generation during ulcer healing in the presence of ciglitazone administration is shown in Fig. 2, and the data on the mucosal NOS-2 activity are summarized in Fig. 3. A 23.4% reduction in PGE2 production and a 58.6% reduction in NOS-2 activity was attained at the end of ten days of ulcer healing with ciglitazone at 5 mg/kg, while a 45.4% reduction in PGE2 and a 76.5% reduction in NOS-2 occurred with the agent at 10 mg/kg. Increasing the dose of ciglitazone to 15 mg/kg produced only negligible additional effect.

The influence of ciglitazone administration on the mucosal expression of prostaglandin and inducible nitric oxide syntase enzyme proteins during ulcer healing was assessed by Western blot analyses (Fig. 4). The results revealed that whilst the mucosal expression of COX-1 protein did not change with ulcer development or during healing, the ulcer onset was reflected in a massive induction of COX-2 and NOS-2 proteins. The ulcer healing in the presence of ciglitazone administration produced accelerated suppression of COX-2 and NOS-2 proteins but had no apparent effect on the mucosal expression of COX-1 protein.


DISCUSSION
Peroxisome proliferator-activated receptors (PPARs) are members of the steroid receptor superfamily of ligand-dependent nuclear transcription factors that function as mediators of the target gene expression (8, 16, 17). The PPARs family consists of three members, PPARa, PPARb and PPARgamma, exhibiting specific patterns of tissue distribution and mediating transcriptional regulation by their central DNA binding domain that recognizes response elements in the promoters of specific genes (19). Indeed, PPARa and PPARb expression predominates in tissues exhibiting high triglyceride and fatty acid catabolism, such as liver, while PPARgamma expression has been linked to adipocytes differentiation, regulation of glucose homeostasis, macrophage activation, and tissue responses to proinflammatory cytokines (19, 23, 24). The indigenous activators of PPARs are mostly derived from arachidonic acid metabolism associated with upregulation of COX-2 expression, and their level appears to reflect the extent of inflammatory involvement. The most potent ligand for the nuclear receptor PPARgamma is a cyclopentanone product of COX-2 induction, 15-deoxy-delta12,14-PGJ2 (15d-PGJ2), which acting as a ligand for PPARgamma activation, exerts a negative influence on proinflammatory stimuli by causing inhibition of transactivation of NFkappaB target genes, including COX-2 and NOS-2 (16 - 19).

As the onset of oral mucosal ulcer is manifested by a marked enhancement in the mucosal level of proinflammatory TNF-alpha and the induction of prostaglandin and nitric oxide production (1, 4, 5), in the study presented herein we investigated the effect of a specific synthetic activator of PPARgamma, ciglitazone (17, 25), on the course of buccal mucosal ulcer healing and the mucosal expression and activity of NOS-2, COX-1, and COX-2 enzymes. The results obtained revealed that while the course of ulcer healing was not affected by ciglitazone administration, the agent evoked a dose-dependent reduction in the ulcer-induced mucosal NOS-2 activity and the capacity for PGE2 generation, which by end of ten days of healing was reflected in a 76.5% reduction in NOS-2 and a 45.4% decline in PGE2. Moreover, the ulcer healing in the presence of ciglitazone administration led to the accelerated suppression of COX-2 and NOS-2 protein expression, but had no apparent effect on the mucosal expression of COX-1 protein. The finding that the course of ulcer healing was not affected by the reduction in buccal mucosal expression of COX-2 and NOS-2 activity brought about by ciglitazone administration provides a strong indication that up-regulation of COX-2 and NOS-2 expression associated with the ulcer onset does not exert discernible effect on the rate of buccal mucosal repair, and hence may reflect only a general pattern of mucosal inflammatory responses to injury. This interpretation is supported by our earlier findings with whole animal studies indicating that the expression of constitutive NOS activity plays a vital role in the maintenance of oral mucosal integrity (26), and the literature data from in vitro setting demonstrating that the induction of NOS-2 leads to the formation of NO-related species that evoke transcriptional disturbances, cause alterations in prostaglandin formation and lead to up-regulation of proinflammatory cytokine production (27 - 29). Furthermore, the results of pharmacological analyses clearly established that nonsteroid anti-inflammatory drug selectivities for COX-1 rather than COX-2 are associated with mucosal injury and gastrointestinal complications, and that a selective COX-1 inhibitors exert only marginal anti-inflammatory or analgesic effect (30, 31).

The complexity of molecular mechanism controlling the segregated utilization of inducible and constitutive products of COX and NOS enzymes is not well understood, but there are strong indications that enzyme compartmentalization and substrate availability are the main controlling factors (9, 32, 33). The constitutive enzymes, which are localized primarily in the endoplasmic reticulum and cytosol, aside for the higher concentration of substrate requirement, appear to access different pool of substrates than more distant inducible enzymes localized in the perinuclear envelope (10, 33, 34). A growing body of evidence also suggests the cross-talk between NO synthesis and prostaglandin generation, and the products of COX-2 and NOS-2 induction have been implicated in tissue integrity maintenance (29, 35 - 37), but our knowledge about the role of these factors in oral mucosal repair remains obscure. However, considerable literature data exist on healing of gastric mucosal injury (38 - 40). The results, however, vary depending on the type of used inhibitor and the experimental setting. Thus, while some reports indicate that the products of COX-2 and NOS-2 induction appear to exert beneficiary effect on gastric mucosal ulcer healing (36, 37), other data, acquired with the use of selective inhibitors, suggest that the inhibition of COX-2 and NOS-2 leads to the impairment in healing (38 - 40). Moreover, studies with NOS-2 knockout mice imply a functional relationship between nitric oxide biosynthesis and prostaglandin generation, and nitric oxide donors as well as up-regulation of NO production through NOS-2 induction inhibit COX-2-derived prostaglandin production (27,35, 41, 42).

Induced expression of COX-2 is also viewed as a promoting event for colorectal cancer, atrophic gastritis and intestinal metaplasia, and the use of nonsteroidal anti-inflammatory drugs that inhibit COX-2 expression decreases the risk for gastrointestinal cancer (43, 44). Moreover, in addition to our current results, pharmacologic activation of PPARgamma has been shown to reduce the extent of inflammation in murine model of colitis and intestinal ischemia-reperfusion injury (13, 14). Hence, our findings with ciglitazone, a specific synthetic PPARgamma activator, provide a clear indication that the products of induced COX-2 and NOS-2 enzymes, associated with oral mucosal inflammatory responses to injury, do not play a significant role in ulcer healing.


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