The inflammatory bowel diseases (IBDs) have a worldwide distribution; its pathogenesis is not clearly understood (1). A major advance in the study of IBD and one that provides strong support for the previously discussed concept has been the discovery and subsequent analysis of a number of models of mucosal inflammation that resemble IBD (2). Recently, Blumberg et al. (3) have indicated that these models fall into four main categories (spontaneous, administration of exogenous agents, gene-targeted knockout or transgenic, and transfer of cells into immunodeficient animals), and each provides unique opportunities to discover insights into the nature of the pathogenesis of IBD. Dinitrobenzene sulfonic acid (DNBS)-induced colitis in experimental animals (e.g. mouse and rats) has proven to be a useful model of IBD because it possesses many of the cell and humoral immunity characteristics found in human IBD (4).
Glucocorticoids (GCs), one of the most used drugs to treat IBD patients, exert their anti-inflammatory action through the inhibition of lymphocyte proliferation and synthesis of proinflammatory cytokines and by down-regulating specific adhesion molecules, resulting in redistribution of lymphocyte traffic (5). These effects are generally mediated through binding to cytoplasmic receptors (GRs) (6). However, although GCs are potent anti-inflammatory drugs, their clinical effects are often transitory, disease recurs on tapering the drug, and chronic use of glucocorticoids is accompanied by serious side effects and dependence. Peroxisome proliferator-activated receptor α (PPAR-α) is an intracellular transcription factor activated by fatty acids that plays a role in inflammation (7).
Peroxisome proliferator-activated receptors are expressed in the intestine at various levels (8). Recently, it has been demonstrated that PPAR-α is also expressed in the digestive tract mainly localized in the intestinal mucosa in the small intestine and in the colon (8). In particular, it has been demonstrated that there is a higher expression of PPAR in the more differentiated colonic epithelial cells facing the intestinal lumen as compared with cells in the lower parts of the crypts (9).
Previous studies indicate PPAR-α expression is induced by GCs and can mediate some of the GC's effects, such as modulation of insulin sensitivity and resistance, and can contribute to GC-induced hyperglycemia and blood pressure increase (10). In addition, it has been reported that PPAR-α activation can result in inhibition of nuclear factor (NF)-κB activation and inflammatory gene expression (11). Our recent results in disease models of colitis and pleurisy show that mice lacking PPAR-α (PPAR-αKO) develop an increased inflammation as compared with wild-type (WT) mice. Moreover, treatment with appropriate doses of PPAR-α agonists can inhibit inflammatory diseases development (12). In as much, we have recently demonstrated that PPAR-α modulate the anti-inflammatory property of dexamethasone (DEX) in a mouse model of acute inflammation (13).
With the aim to characterize the role of PPAR-α in GC-mediated anti-inflammatory activity, we tested the efficacy of DEX, a synthetic GC specific for GR, in an experimental model of IBD induced by DNBS, comparing PPAR-αKO and WT mice. Results indicate that in a chronic situation such as IBD, DEX-mediated anti-inflammatory activity is weakened in PPAR-αKO mice as compared with WT controls.
MATERIALS AND METHODS
Mice (4-5 weeks old; 20-22 g) with a targeted disruption of the PPAR-α gene (PPAR-αKO) and littermate WT controls (PPAR-αWT) were purchased from Jackson Laboratories (Harlan Nossan, Italy). Mice homozygous for the PparatniJGonz targeted mutation mice are viable, fertile, and seem normal in appearance and behavior. Exon eight, encoding the ligand-binding domain, was disrupted by the insertion of a 1.14-kb neomycin resistance gene in the opposite transcriptional direction. After electroporation of the targeting construct into J1 ES cells, the ES cells were injected into C57BL/6N blastocysts. This stain was created on B6,129S4 background and is maintained as a homozygote on a 129S4/SvJae background by brother-sister matings. The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care was performed in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) and with the European Economic Community regulations (O.J. of E.C. L 358/1 12/18/1986).
Mice will be randomly allocated into the following groups: 1) for the PPAR-αWT DNBS + saline group, PPAR-αWT will be subjected to DNBS-induced colitis plus administration of saline (n = 10); 2) for the PPAR-αKO DNBS + saline group, PPAR-αKO will be subjected to DNBS-induced colitis plus administration of saline (n = 10); 3) the PPAR-αWT DEX group is identical to the PPAR-αWT DNBS + saline group but was administered DEX (2 mg/kg dissolved in saline, i.p. bolus), which was given daily as an intraperitoneal injection starting 1 h after the administration of DNBS (n = 10); 4) the PPAR-αKO DEX group is identical to the PPAR-αKO DNBS + saline group but was administered DEX (2 mg/kg dissolved in saline, i.p. bolus), which was given daily as an intraperitoneal injection starting 1 h after the administration of DNBS (n = 10); 5) for the PPAR-αWT sham + saline group, PPAR-αWT mice were subjected to the surgical procedures as the previously discussed groups except instead of DNBS, 100 μL of 50% ethanol was administered to the mice (n = 10); 6) for the PPAR-αKO sham + saline group, PPAR-αKO mice were subjected to the surgical procedures as the previously discussed groups except that instead of DNBS, 100 μL of 50% ethanol was administered to the mice (n = 10); 7) the PPAR-αWT sham + DEX group is identical to the PPAR-αWT sham + saline group except for the administration of DEX group. 8) and the PPAR-αKO sham + DEX group is identical to the PPAR-αKO Sham + saline group except for the administration of DEX.
Induction of experimental colitis
Colitis was induced with a very low dose of DNBS (4 mg per mouse) by using a modification of the method first described in rats (14). In preliminary experiments, this dose of DNBS was found to induce reproducible colitis without mortality. Mice were anesthetized by enflurane. Dinitrobenzene sulfonic acid (4 mg in 100 μL of 50% ethanol) was injected into the rectum through a catheter inserted 4.5 cm proximally to the anus. Carrier alone (100 μL of 50% ethanol) was administered in control experiments. Thereafter, the animals were kept for 15 min in a Trendelenburg position to avoid reflux. After colitis and sham-colitis induction, the animals were observed for 3 days. On day 4, the animals were weighed and anesthetized with chloral hydrate, and the abdomen was opened by a midline incision. The colon was removed, freed from surrounding tissues, opened along the antimesenteric border, rinsed, weighed, and processed for histology and immunohistochemistry. Colon damage (macroscopic damage score) was evaluated and scored by two independent observers according to the following criteria: 0, no damage; 1, localized hyperemia without ulcers; 2, linear ulcers with no significant inflammation; 3, linear ulcers with inflammation at one site; 4, two or more major sites of inflammation and ulceration extending more than 1 cm along the length of the colon; and 5 to 8, one point is added for each centimeter of ulceration beyond an initial 2 cm.
After fixation for 1 week at room temperature in Dietrich solution (14.25% ethanol, 1.85% formaldehyde, 1% acetic acid), samples were dehydrated in graded ethanol and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Thereafter, 7-μm sections were deparaffinized with xylene, stained with hematoxylin-eosin and trichromic van Giesson stain, and observed in a Dialux 22 Leitz (Wetziar, Germany) microscope. To have a quantitative estimation of colon damage, sections (n = 6 for each animals) were scored by two independent observers blinded to the experimental protocol. The following morphological criteria were considered: score 0, no damage; score 1 (mild), focal epithelial edema and necrosis; score 2 (moderate), diffuse swelling and necrosis of the villi; score 3 (severe), necrosis with presence of neutrophil infiltrate in the submucosa; and score 4 (highly severe), widespread necrosis with massive neutrophil infiltrate and hemorrhage.
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA assay
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling of fragmented DNA (TUNEL) assay was conducted by using a TUNEL detection kit according to the manufacturer's instruction (Apotag, horseradish peroxidase kit; DBA, Milan, Italy). Briefly, sections were incubated with 15 μg/mL proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase buffer containing deoxynucleotidyl transferase and biotinylated dUTP in terminal deoxynucleotidyltransferase buffer, incubated in a humid atmosphere at 37°C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with antihorseradish peroxidase-conjugated antibody, and the signals were visualized with diaminobenzidine. The number of TUNEL-positive cells per high-power field was counted in 5 to 10 fields for each coded slide.
Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as previously described (15). At 4 days after intracolonic injection of DNBS, the colon was removed and weighed. The colon was homogenized in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 × g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 μmol of peroxide per min at 37°C and was expressed in milliunits per gram weight of wet tissue.
Measurement of cytokines
Portions of terminal colon, collected at 4 days after intracolonic injection of DNBS, were homogenized as previously described (16) in PBS containing 2 mM of phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, Mo), and tissue levels of TNF-α and IL-1β were evaluated. The assay was performed by using a colorimetric commercial kit (Calbiochem-Novabiochem Corporation, San Diego, Calif) according to the manufacturer's instructions. All cytokine determinations were performed in duplicate serial dilutions.
Localization of nitrotyrosine, TNF-α, Bax, Bcl-2, FasL, nitrotyrosine, glucocorticoid-induced leucine zipper by immunohistochemistry
The tissues were fixed in 10% PBS-buffered formaldehyde 4 days after the administration of DNBS, and 8-μm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin-binding sites were blocked by sequential incubation for 15 min with avidin and biotin (DBA). Sections were incubated overnight with 1) antinitrotyrosine antibody (1:500 in PBS), anti-Bax antibody (1:500 in PBS; vol/vol; DBA), anti-TNF-α polyclonal antibody (1:100 in PBS; vol/vol) or anti-Bcl-2 antibody (1:100 in PBS; vol/vol; Santa Cruz, DBA;), anti-glucocorticoid-induced leucine zipper (GILZ) rabbit polyclonal antibody (1:100 vol/vol), and anti-FasL antibody (1:100 vol/vol). Specific labeling was detected with a biotin-conjugated goat antirabbit, donkey antigoat, or goat antimouse immunoglobulin (Ig)G and avidin-biotin peroxidase complex (DBA). To verify the binding specificity for Bax, Bcl-2, Fas ligand (FasL), GILZ, TNF-α, or nitrotyrosine, some sections were also incubated with primary antibody only (no secondary antibody) or with secondary antibody only (no primary antibody). In these situations, no positive staining was found in the sections, indicating that the immunoreactions were positive in all the experiments performed.
Immunocytochemistry photographs (n = 5) were assessed by densitometry by using Optilab Graftek software on a Macintosh personal computer and evaluated by two independent observers blinded to the experimental protocol.
Protein extraction and Western blot analysis
Tissue samples from colon were homogenized with a Polytron homogenizer in a buffer containing 0.32 M sucrose, 10 mM Tris-HCl, pH 7,4, 1 mM ethylene glycol tetraacetic acid, 2 mM ethylenediaminetetraacetic acid, 5 mM NaN3, 10 mM β-mercaptoethanol, 20 μM leupeptin, 0.15 μM pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 1 mM sodium orthovanadate, and 0.4 nM microcystin. The homogenates were centrifuged (1,000 × g, 10 min), and the supernatant was collected to evaluate contents of Bax and Bcl-2, IκB-α, and phospho-nuclear factor (NF)-κB p65 (ser536). Protein concentration was determined by the Bio-Rad protein assay using bovine serum albumin as standard. Equal amounts of protein (50 μg) were dissolved in Laemmli sample buffer, boiled, and run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then transferred to Hybond polyvinylidene difluoride membrane. Membranes were blocked for 60 min in Tris-buffered saline and 5% (wt/vol) nonfat milk and subsequently probed overnight at 4°C with anti-Bax antibody (1:500 in PBS; vol/vol; DBA), anti-Bcl2 antibody (1:500 in PBS; vol/vol; DBA), anti-IκB-α (Santa Cruz; 1:1,000), or anti-phospho-NF-κB p65 (ser536; 1:1,000; Cell Signaling Technology, Danvers, Mass) antibodies (in Tris-buffered saline; 5% wt/vol nonfat milk and 0.1% Tween-20). Blots were then incubated with horseradish peroxidase-conjugated goat antimouse or antirabbit IgG (1:2,000; Jackson ImmunoResearch Laboratories, West Grove, Pa) for 1 h at room temperature. Immunoreactive bands were detected by SuperSignal West Pico Chemiluminescent (Pierce, Rockford, Ill). Bands were quantified by densitometric analysis performed with a quantitative imaging system.
Biotin blocking kit, biotin-conjugated goat antirabbit IgG, and avidin-biotin peroxidase complex were obtained from Vector Laboratories (Burlingame, Calif). Primary antinitrotyrosine antibody was purchased from Upstate Biotech (Saranac Lake, NY). Primary intercellular adhesion molecule 1 (CD54) for immunohistochemistry was purchases by Pharmingen (San Diego, Calif). Reagents and secondary and nonspecific IgG antibody for immunohistochemical analysis were from Vector Laboratories. All other reagents and compounds used were obtained from Sigma.
All values in the figures and text are expressed as mean ± SEM of n observations, where n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. Data sets were examined by one- and two-way ANOVA, and individual group means were then compared with Student unpaired t test. Nonparametric data were analyzed with the Fisher exact test. A P value less than 0.05 was considered significant.
Role of functional PPAR-α gene in the anti-inflammatory property of DEX on the degree of colitis (histology and general assessment)
The colon seemed flaccid and filled with liquid stool 4 days after intracolonic administration of DNBS. The macroscopic inspection of cecum, colon, and rectum showed presence of mucosal congestion, erosion, and hemorrhagic ulcerations (Fig. 1B; see macroscopic score f). The histopathological features included a transmural necrosis and edema and a diffuse leukocyte cellular infiltrate in the submucosa of colon section from DNBS-treated PPAR-αWT mice (Fig. 2B; see histological score f). The observed inflammatory changes of the large intestine were associated with an increase in the weight of the colon (Table 1). The absence of PPAR-α gene significantly increases the extent and severity of the macroscopic signs (Fig. 1C; see macroscopic score f), histological signs of colon injury (Fig. 2C; see histological score f), and the colon weight (Table 1). All mice had diarrhea and a significant reduction in body weight (compared with the control groups of mice) 4 days after colitis induced by DNBS treatment (Fig. 1G). Absence of a functional PPAR-α gene in PPAR-αKO mice resulted in a significant augmentation of loss of body weight (Fig. 1G). No macroscopic (Fig. 1A; see macroscopic score f) or histological alteration (Fig. 2B; see histological score f) was observed in the colon tissue from vehicle-treated PPAR-αWT and PPAR-αKO mice (data not shown). On the other hand, the treatment of PPAR-αWT with DEX resulted in a significant decrease in the extent and severity of the macroscopic signs (Fig. 1D; see macroscopic score f), histological signs of colon injury (Fig. 2D; see histological score f), the colon weight (Table 1), and the loss of body weight (Fig. 1G). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX treatment (Figs. 1, E-G and 2, E and F).
Role of PPAR-α in DEX-induced inhibition of TNF-α and IL-1β expression after DNBS administration
Release of proinflammatory cytokines is an important mechanism responsible for inflammatory process, including IBD. A substantial increase in TNF-α and IL-1β formation was found in colon samples collected from PPAR-αWT mice at 4 days after DNBS administration (Table 1). Colon levels of TNF-α and IL-1β were significantly higher in PPAR-α-deficient mice in comparison with those of PPAR-αWT animals (Table 1). In contrast, a significant inhibition of TNF-α and IL-1β levels was observed in the colon tissues collected from PPAR-αWT treated with DEX (Table 1). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX on the production of proinflammatory cytokines (Table 1). In addition, tissue sections obtained from PPAR-αWT animals after DNBS administration demonstrate positive staining for TNF-α (Table 2) mainly localized in the infiltrated inflammatory cells in damaged mucosa. In DNBS-treated PPAR-αKO mice, the staining for TNF-α (Table 2) was visibly and significantly increased in comparison with the PPAR-αWT mice. Section from DEX-treated PPAR-αWT mice did not reveal positive staining TNF-α (Table 2). The genetic absence of the PPAR-α receptor significantly blocked the effect of DEX on the TNF-α expression (Table 2). Sections of colon from sham-administered PPAR-αKO mice did not stain for TNF-α (data not shown).
Role of PPAR-α in DEX-induced inhibition of PMN infiltration
The colitis caused by DNBS was also characterized by an increase in MPO activity, an indicator of the neutrophils accumulation in the colon (Table 1). This finding is consistent with the observation made with light microscopy that the colon of vehicle-treated DBNS rats contained a large number of neutrophils. In PPAR-αKO mice, colon MPO activity was markedly increased in comparison with those of PPAR-αWT animals (Table 1). On the contrary, DEX significantly reduced the degree of PMN infiltration (determined as increase in MPO activity) in inflamed colon (Table 1). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX on the neutrophils infiltration (Table 1).
Role of PPAR-α in DEX-induced inhibition of on nitrotyrosine formation
To determine the localization of peroxynitrite formation and/or other nitrogen derivatives produced during colitis, nitrotyrosine, a specific marker of nitrosative stress, was measured by immunohistochemical analysis in the distal colon. Sections of colon from sham-administered PPAR-αKO mice did not stain for nitrotyrosine (data not shown). Colon sections obtained from vehicle-treated DNBS-PPAR-αWT mice exhibited positive staining for nitrotyrosine (Table 2) localized in inflammatory cells and in disrupted epithelial cells. In DNBS-treated PPAR-αKO mice, the staining for nitrotyrosine was visibly and significantly increased (Table 2) in comparison with the PPAR-αWT mice. Section from DEX-treated PPAR-αWT mice did not reveal any positive staining for nitrotyrosine (Table 2). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX on nitrotyrosine formation (Table 2).
Role of PPAR-α in DEX-induced inhibition of on FasL expression
Sections of colon from sham-administered PPAR-αKO mice did not stain for FasL (data not shown). Colon sections obtained from vehicle-treated DNBS-PPAR-αWT mice exhibited positive staining for FasL (Table 2) localized in inflammatory cells and in disrupted epithelial cells. In DNBS-treated PPAR-αKO mice, the staining for FasL was visibly and significantly increased (Table 2) in comparison with the PPAR-αWT mice. Section from DEX-treated PPAR-αWT mice did not reveal any positive staining for FasL (Table 2). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX on FasL expression (Table 2).
Role of PPAR-α in DEX-induced inhibition of apoptosis
To test whether DNBS-induced colon injury was associated to cell death by apoptosis, we measured TUNEL-like staining in the injured tissue. Almost no apoptotic cells were detected in the sections of colon from sham-administered PPAR-αKO mice (data not shown). In addition, tissue sections obtained from PPAR-αWT animals after DNBS administration demonstrate a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments (Table 2). In DNBS-treated PPAR-αKO mice, the presence of apoptotic cell (Table 2) was visibly and significantly increased in comparison with the PPAR-αWT mice. No apoptotic cells were observed in the section from DEX-treated PPAR-αWT mice (Table 2). The genetic absence of the PPAR-α receptor significantly blocked the effect of DEX (Table 2).
Western blot analysis and immunohistochemistry for Bax and Bcl-2 expression
At 4 days after DNBS, the appearance of Bax in colon homogenates was investigated by Western blot. No Bax expression was detected in the colon from sham-treated animals (Fig. 3, A and A1). Bax levels were appreciably increased in the colon from DEX-treated PPAR-αWT mice (Fig. 3, A and A1). In DNBS-treated PPAR-αKO mice, the Bax expression was visibly and significantly increased (Fig. 3, A and A1) in comparison with the PPAR-αWT mice. On the contrary, a significant reduction in Bax expression was observed in the colon from DEX-treated PPAR-αWT (Fig. 3, A and A1). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX on Bax expression (Fig. 3, A and A1).
Western blot analyses also analyzed whole extracts from the colon of each mice to detect Bcl-2 expression. A basal level of Bcl-2 expression was detected in the colon tissues from sham-treated PPAR-αWT and PPAR-αKO mice, whereas in DNBS-PPAR-αWT mice, Bcl-2 levels were substantially reduced (Fig. 3, B and B1). In DNBS-treated PPAR-αKO mice, the inhibition of Bcl-2 expression was visibly and significantly increased (Fig. 3, B and B1) in comparison with the PPAR-αWT mice. On the contrary, a significant reduction in the DNBS-induced inhibition of Bcl-2 expression was observed in the colon from DEX-treated PPAR-αWT (Fig. 3, B and B1). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX on Bcl-2 expression (Fig. 3, B and B1). Moreover, samples of colon tissue were taken at 4 days after DNBS to determine the immunohistological staining for Bax and Bcl-2. Sections of colon from sham-administered PPAR-αKO mice did not stain for Bax (figure data not shown), whereas colon tissue sections obtained from DNBS-PPAR-αWT mice exhibited a positive staining for Bax (Table 2). In DNBS-treated PPAR-αKO mice, the staining for Bax was visibly and significantly increased (Table 2) in comparison with the PPAR-αWT mice. Section from DEX-treated PPAR-αWT mice did not reveal any positive staining for Bax (Table 2). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX on Bax expression (Table 2).
In addition, colon sections from sham-administered PPAR-αKO mice demonstrated Bcl-2-positive staining (data not shown), whereas in colon tissue sections obtained from DNBS-PPAR-αWT mice, the staining for Bcl-2 was significantly reduced (Table 2). In DNBS-treated PPAR-αKO mice, the loss of staining for Bcl-2 was visibly and significantly increased (Table 2) in comparison with the PPAR-αWT mice. The loss of positive staining for Bcl-2 was significant attenuated in the colon tissues from DEX-treated PPAR-αWT mice (Table 2). The genetic absence of the PPAR-α receptor significantly blocked the effect of the DEX on the loss of Bcl-2 staining (Table 2).
Role of PPAR-α in DEX-mediated inhibition of DNBS-induced NF-κB activation
Most inflammatory mediators, including iNOS, cyclooxygenase 2, IL-1β, and TNF-α, are controlled by NF-κB, a transcription factor important in inflammatory process, which is kept inactive by IκB and GILZ (17). Moreover, activation of NF-κB transactivation potential is increased by phosphorylation of the p65 subunit. We performed experiments to evaluate the possible effect of DEX on DNBS-induced NF-κB activation in WT and PPAR-αKO mice.
A basal level of IκB-α was detected in the colon tissues from sham WT mice and from sham PPAR-αKO mice. After DNBS administration, IκB-α levels were substantially reduced (Fig. 4, A and A1). This reduction was countered by DEX treatment (P < 0.01). However, the DEX effect was significantly more evident in WT than in PPAR-αKO mice (Fig. 4, A and A1). Furthermore, DNBS administration caused a significant increase in p65 phosphorylation at ser536 in the colon tissues from WT and PPAR-αKO mice, and DEX inhibited p65 phosphorylation of WT but not of PPAR-αKO mice (Fig. 4, B and B1). Of note, DNBS-induced increase in p65 phosphorylation in the lung tissues of PPAR-αKO was slightly more marked than that of WT mice (Fig. 4, B and B1).
Moreover, this difference was even enhanced in DNBS-treated cells, thus indicating that PPAR-α contributes to DEX-induced GILZ expression. We then evaluated the GILZ protein expression in colon tissues by immunohistochemical assay. No positive staining for GILZ was observed in the colon tissues collected from sham WT mice and PPAR-αKO mice (Fig. 5, A and B). On the contrary, tissue sections obtained from WT and KO animals at 4 days after DNBS administration showed few positive staining for GILZ localized in the infiltrating inflammatory cells (Fig. 5, C and D).
In DNBS-treated WT mice that have been treated with DEX, the staining for GILZ was visibly and significantly increased, mainly localizing in infiltrating macrophages, PMNs, and lymphocytes (Fig. 5, E, E1, and E2). On the contrary, the absence of a functional PPAR-α gene in PPAR-αKO mice resulted in a clear reduction of this DEX-induced GILZ expression (Fig. 5F).
In the present article, we show that the absence of PPAR-α in PPAR-αKO mice results in a reduced anti-inflammatory response to DEX treatment in an IBD model. These results are in agreement with our previous observations indicating that PPAR-αKO mice are more susceptible to induction of IBD, possibly due to a less efficient physiological anti-inflammatory control exerted by endogenous GCs (11).
Glucocorticoids, including DEX, are potent anti-inflammatory agents and, for that reason, are used in therapy of a number of human diseases. Peroxisome proliferator-activated receptor α itself is also able to directly mediate some anti-inflammatory effects, and it has been shown that its agonist-induced activation inhibits a number of inflammatory mechanisms, including TNF-α production, iNOS, cyclooxygenase 2, and adhesion molecule expression, as well as cell infiltration in the tissues (7).
Based on these observations, we performed studies in an attempt to determine whether the presence and/or the stimulation of PPAR-α could enhance the GCs anti-inflammatory efficacy. For that purpose, we used an experimental model of IBD induced by DNBS performed in WT and PPAR-αKO mice.
There is a large body of evidence showing that the production of reactive oxygen and nitrogen species play key roles in IBD (18). Several studies have also clearly demonstrated the pathogenic role of nitrogen-derived species such as peroxynitrite (19) in IBD; this is further supported by the fact that intracolonic administration of exogenous peroxynitrite induces a severe colonic inflammation that mimics the features of both ulcerative colitis and Crohn disease (18). In the present study, we clearly demonstrate that when WT and PPAR-αKO mice were treated with DEX, a significant inhibition of nitrotyrosine formation was observed in WT but not in PPAR-αKO mice. Recent evidence suggests that the activation of NF-κB may also be under the control of oxidant/antioxidant balance (20). It is known that NF-κB activation is central in inflammation, and that GCs can counter its activity by different mechanisms, including the increase in IκB expression (21). Moreover, we have shown that GCs rapidly induce expression of GILZ, another protein able to bind and inhibit NF-κB (22). Results here indicated that DEX-induced GILZ overexpression is clearly detected in colonic tissues of DNBS-treated WT but not of DNBS-treated PPAR-αKO mice. There is evidence that production of proinflammatory cytokines such as, for example, TNF-α and IL-1β, is important to induce local and systemic inflammation, and that production of this cytokine can be inhibited by treatment with GCs (23). When WT and PPAR-αKO mice were treated with DEX, a significant inhibition of TNF-α and IL-1β level was measured in WT but not in PPAR-αKO mice.
Activation of NF-κB is crucially involved in FasL expression induced by DNA-damaging agents such as genotoxic drugs and ultraviolet radiation (24). Recently, it has been pointed out that FasL signaling plays a central role in colitis (25). Furthermore, cell death induced by reactive oxygen species depends on FasL expression mediated by redox-sensitive activation of NF-κB (26). We confirm here that DNBS-induced colitis leads to a substantial activation of FasL in the colon tissues, which likely contributes in different capacities to the evolution of tissues injury. In the present study, we found that when WT and PPAR-αKO mice were treated with DEX, a significant inhibition of FasL activation was evaluated in WT but not in PPAR-αKO mice. Recent studies have also demonstrated the induction of apoptosis in different cell lines in response to reactive oxygen species, peroxynitrite, and NO (27). Apoptosis may occur within several hours to several days after injury in some locations; thus, the suppression of cell death is clinically relevant. The role of apoptotic cell death in the inflamed intestine has not been clarified. In ulcerative colitis, the frequency of apoptosis is considerably increased, and loss of epithelial cells seems to occur mainly by apoptosis (28). These findings have suggested that epithelial apoptosis may lead to an alteration of the epithelial barrier function leading to pathogenic microorganism infiltration. On the other hand, apoptosis regulates the lymphocyte population and may inhibit immune response at the inflammatory site. Therefore, it has been proposed that an increased T-cell resistance to apoptosis may contribute to disease progression and mucosal alterations in patients with Crohn disease (29). In this regard, Zingarelli et al. (30) have clearly demonstrated that inhibition of poly(adenosine diphosphate- ribose) polymerase 1 may reduce the apoptotic process by shifting the ratio of apoptotic regulators toward Bcl-2 along with reduction of c-Jun N-terminal kinase activity and the activator protein 1-dependent proinflammatory mediators.
In the present study, we clearly demonstrate that when WT mice were treated with DEX, a significant inhibition of apoptotic process by reducing the expression of proapoptotic genes such as BAX and preventing the loss of antiapoptotic gene Bcl-2. Notably, the absence of functional PPAR-α receptor in PPAR-αKO mice inhibits the effect of DEX treatment on the apoptotic process.
The results described here clearly indicate that the anti-inflammatory efficacy of DEX treatment is favored by the presence of PPAR-α. Recently, we have demonstrated that DEX regulates GR and PPAR-α expression, but their levels are similar in healthy or mice subjected to experimental lung injury, either WT or PPAR-αKO, thus indicating that impaired DEX anti-inflammatory activity in PPAR-αKO mice is not due to modulation of GR or PPAR-α expression (13). Moreover, previous studies showed that PPAR-α agonists exert some anti-inflammatory activity (31). The efficacy of GC treatment in inflammatory and autoimmune diseases is an important therapeutic subject and, whereas some patients obtain clinical benefits from treatment, others are not responsive or even resistant to therapy. As an example, it is known that only a certain percentage of patients affected by IBDs are cured by GCs treatment, whereas others are not (32). The reasons of response or lack of response to therapy are not fully understood, and molecular mechanisms such as, for example, change in the ratio between different GR isoforms, preexisting levels of NF-κB, and heat-shock proteins, have been drawn out in an attempt to explain and predict sensitivity and resistance (33). Results discussed here suggest a new mechanism contributing to determine the full GC efficacy and suggest future studies aimed to analyze the possible relevance of PPAR-α in other human inflammatory disease models such as sepsis.
In conclusion, our results clearly indicate that PPAR-α can contribute by enhancing the anti-inflammatory activity of DEX in DNBS-induced IBD model. These observations could suggest new therapeutic approaches of combination therapy with GCs and PPAR-α agonists in inflammatory diseases.
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