TY - JOUR
AU - PhD, Fievos L. Christofi,
AB - Abstract Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine-based drugs in experimental inflammatory bowel disease. High-density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS-treated (7 days), and oral IB-MECA-treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin-trap monitoring of free radicals. Remarkable colitis-induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis-related dysregulation. Downregulation occurs in membrane transporter, mitogen-activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox-sensitive genes. Oral IB-MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation (≤20-fold) of Bzrp, P2X1R, P2X4R, P2X7R, P2Y2R, P2Y6R, and A2aR/A2bR but not A1R or A3R genes or downregulated P2X2R, P2Y1R, and P2Y4R. Real-time SYBR green polymerase chain reaction validated gene chip data for both induction of colitis and treatment with IB-MECA for >90% of genes tested (33 of 37 genes). We conclude that our validated high-density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine-based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease. TNBS colitis, microarray, adenosine A3 receptors, antioxidant effects, IB-MECA, gene dysregulation, purine receptor gene dysregulation Many substances have been used to prevent the development of experimental colitis and inflammatory bowel disease (IBD), but to date, available treatments for Crohn's disease (CD) and ulcerative colitis (UC) are limited. Models of IBD used to test new therapeutic agents include chemical, microbial, or polymer induction; genetic models; and spontaneous colitis models. The rat 2,4,6-trinitrobenzene sulfonic acid (TNBS) model is one of the most frequently used models of experimental colitis to test new therapeutic strategies, and it has attributes of both UC and CD.1 Adenosine (ADO) receptors may offer a novel therapeutic target in gut inflammation, IBD,2 and other diseases.3,–6 Endogenous ADO (eADO) is a metabolite of adenosine triphosphate (ATP) that acts at ADOA1R, ADOA2aR, ADOA2bR, or ADOA3Rs to influence mucosal secretion and neural sensory motor reflexes. ATP itself or other related purinergic mediators act at additional P2YRs and P2XRs to influence gut reflexes and function.7,–9 In general, ADO may exert anti-inflammatory effects by inhibiting synthesis of TH1 cytokines (i.e., interferon-γ [INF-γ], tumor necrosis factor-α [TNF-α]) and suppressing neutrophil functions, including degranulation, superoxide production, and adhesion to and damage to the endothelium.10,–13 Clinical use of ADO, ADOR agonists, or certain modulators of ADO degradation may be complicated by undesirable pharmacokinetics, side effects, or progressive desensitization of certain ADORs14,15 that may limit their usefulness in IBD2 or other diseases.2,9,16,–21 Increasing the concentration of eADO is one mechanism by which several drugs may reduce gut inflammation.20,22,–24 An ADO kinase inhibitor (used in dextran sulfate [DDS] colitis) or an ADOA3R agonist N6-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) may be beneficial in murine models of colitis,16,17 but the mechanisms involved remain poorly understood. ADOA3Rs in particular have a wide range of physiological and disease-related effects,5,9,25,26 with promise for treatment of a variety of heart disease, uveitis, colorectal cancer, and inflammation.19 IB-MECA is in phase I and II clinical trials for a chronic inflammatory disease, rheumatoid arthritis, and is apparently without toxicity (www.canfite.com/develop.html). In the digestive tract, ADOA3Rs seem to play an important role in the function of the distal colon that is the primary site of inflammation in rat TNBS colitis. ADOA3Rs are expressed throughout the human, rat, guinea pig, and mouse colon.9,27 A3R immunoreactivity is differentially expressed, with the highest expression being in the distal colon of the rat. Our initial studies also identified an inhibitory limb of the motor reflex that may be activated by putative A3Rs in rat distal colon.9 ADOA3Rs modulate the neural reflexes involved in the coordinated motility and secretory responses in rodent colon (F.L.C., unpublished observations). We were interested in probing further the complex mechanisms that may be involved in any beneficial effects of the ADOA3R agonist IB-MECA in the rat TNBS colitis model and whether such treatment can prevent or attenuate alterations in gene expression and dysregulation profiles. No attempts have been made to establish gene expression profiles in experimental models of colitis or IBD such as in TNBS colitis using high-density oligonucleotide microarray analysis. Validation of microarray data is necessary through other approaches, including real-time quantitative polymerase chain reaction (PCR) and parallel analysis of histopathology and function. A recent microarray study in human IBD has compared and contrasted the patterns of gene expression and dysregulation in UC, CD, and other chronic inflammatory conditions.28 Activation of ADOA3Rs in various cell types leads to an increase in activity of the cellular antioxidant enzymes,29 but it remains unknown whether ADOA3R agonists have antioxidant effects in a model like TNBS colitis, which represents a suitable model to study oxidative stress.1 Our approach is to study the effects of an ADOA3R agonist on oxidative stress genes or free radical generation in colitis tissues by electron paramagnetic resonance imaging (EPRI). Expression of purine-related genes is modulated by inflammatory or other diseases or drug treatments.30,31 Therefore, it was also of interest to establish whether purine receptor gene dysregulation occurs in TNBS colitis for adenosine, P2X, and P2Y gene receptor families. This is critical in the assessment and interpretation of purine-based therapies in experimental models of IBD. In particular, oral IB-MECA is presumed to be highly selective for A3Rs in the treatment of inflammatory diseases such as rheumatoid arthritis in humans, but definitive proof is lacking. It remains possible that in vivo activity of IB-MECA is not restricted to ADOA3R, and it could act on other ADO receptors such as A1R, A2aR, or A2bR. The ADOA3R, a potential therapeutic target, may itself be subject to dysregulation by inflammation.30,31 Our goal was to assess the protective effect of the ADOA3R agonist IB-MECA on gene dysregulation and injury in a rat chronic model of TNBS-induced colitis. It was necessary to develop and validate a microarray technique to test the protective effects of purine-based drugs in experimental IBD. We applied high-density oligonucleotide microarray analysis using a rat RNU34 neurobiology chip of 724 genes to identify gene expression patterns of dysregulation and to assess the prophylactic effect of the ADOA3R agonist IB-MECA on gene dysregulation and gut injury. Immune, cytokine, chemokine, inflammatory, epithelial transporter, growth factor, and oxidant/antioxidant genes that are relevant in IBD were examined using the RNU34 chip, and the findings validated by real-time SYBR green PCR for selected clusters of genes. Tissue injury and inflammation were confirmed by histological and clinical evaluations and weight loss. The antioxidant potential of IB-MECA also was assessed ex vivo by spin-trap/EPRI of free radicals and oxidant enzyme gene expression profiles. Validated gene array analysis revealed useful cluster information on neural and nonneural genes that can be targeted easily in drug treatment studies. We present novel data that oral IB-MECA nearly prevents rat TNBS colitis and gene dysregulation, suggesting that ADOA3R is a new potential therapeutic target in IBD. Materials and Methods Induction of Inflammation and IB-MECA Treatment Harlan Sprague-Dawley Lewis rats (Harlan, Indianapolis, Ind) were randomized to receive enemas with saline (0.9% NaCl) or TNBS (60 mg in 33% ethanol, 1.0 mL total volume). Some animals with TNBS received IB-MECA (1.5 mg/kg b.i.d.) by oral gavage, and age-matched controls received saline/vehicle (dimethylsulfoxide) starting 12 h after induction on day 1. To induce colitis in the distal colon, rats were anesthetized with isofluorane (induced at 4%), and TNBS was delivered into the lumen of the colon through a polyethylene catheter inserted rectally 6 cm proximal to the anus.32 Animals were maintained in a control environment for 7 days after TNBS, saline, IB-MECA, or other treatments. On day 7, animals were killed, and tissue samples were collected for analysis. The severity of colitis was assessed by weight loss and reduction in food intake over 7 days, histology, and clinical score. Each colon was removed and opened, and damage was scored as described by Peterson et al.33 All animal handling and histological, biochemical, and clinical assessments were performed as treatment-blinded assessments. Criteria of macroscopic scoring of colonic damage were as follows: ulceration, normal appearance (score = 1); focal hyperemia, no ulcers (score = 2); ulceration without hyperemia or bowel wall thickening (score = 3); ulceration with inflammation at 1 side (score = 4); >2 sites of ulceration and inflammation (score = 5); and major sites of damage extending >1 cm along colon (score = 6). When an area of damage extended >2 cm along the length of the colon, the score was increased by 1 for each additional centimeter of involvement (score = 7–11). The criteria for adhesions were as follows: no adhesions (score = 0), minor adhesions (score = 1), and major adhesions (score = 2). For diarrhea, the scoring was as follows: no (score = 0) and yes (score = 2). The total score for each animal was calculated by adding up the scores for each criterion. Whole-thickness tissue samples or microdissected mucosa, submucosa, or longitudinal muscle-myenteric plexus8 were immediately frozen in liquid nitrogen for later analysis by microarray or SYBR green PCR. Animal protocols were approved by the institutional Laboratory Animal Care and Use Committee at Ohio State University. RNA Collection and Amplification Frozen tissue samples were collected and total cellular RNA was isolated from each sample with the TRIzol RNA isolation reagent. The total RNA was then cleaned with Qiagen RNeasy Mini columns (catalog ID 74104). Samples were then run on RNA 6000 Nano Chips (Agilent, catalog ID 5065–4476) and analyzed for integrity with an Agilent Bioanalyzer 2100 system and Degradometer software.34 cDNA was synthesized with the following T7-(dT)24 primer: 5-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3. The ds cDNA was cleaned with Qiagen cDNA spin columns (Affymetrix, catalog ID 900371). After cleanup, in vitro transcription labeling was performed with the Enzo BioArray Kit (Enzo, catalog ID 42655). The biotinylated cRNA was purified with Qiagen cRNA spin columns (Affymetrix, catalog ID 900371). The cRNA was then fragmented and hybridized to RNU34 GeneChip Arrays (Affymetrix, catalog ID 900283), according to the manufacturer's recommendations.8 The RNU34 array contains 1322 transcripts representing, among others, cell surface receptors, cytokines, chemokines, growth factors, oxidant/antioxidant genes, neural genes, and oncogenes34 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx, http://www.affymetrix.com/support/technical/datasheets/rgu34_arrays_datasheet.pdf). Real-time SYBR Green PCR Forward and reverse primers used in real-time SYBR green PCR are listed in Table 1. SYBR green PCR was performed by taking 1 μg total RNA and reverse transcribing with Superscript II (Invitrogen, Palo Alto, Calif) and oligo(dT) priming in 20 μL. Then, 0.5-μL reverse transcription reaction was used per PCR reaction. Real-time PCR was performed in triplicate for each sample with the iQ-Cycler (Biorad, Hercules, Calif). Amplification products were detected with SYBR Green Supermix (Biorad). Expression levels were estimated relative to the expression levels of the housekeeping gene β-actin (ACTB). Differential gene expression was calculated from mean values of expression for technical replicates and biological triplicates. Table 1. Primers Used for SYBR Green PCR in Colonic Tissues from Age-matched Control and TNBS-induced Colitis Rats     View Large Table 1. Primers Used for SYBR Green PCR in Colonic Tissues from Age-matched Control and TNBS-induced Colitis Rats     View Large Table 1. Primers Used for SYBR Green PCR in Colonic Tissues from Age-matched Control and TNBS-induced Colitis Rats (Cont.)     View Large Table 1. Primers Used for SYBR Green PCR in Colonic Tissues from Age-matched Control and TNBS-induced Colitis Rats (Cont.)     View Large Microarray Analysis Gene expression levels are estimated from GeneChip perfect match (PM) probe intensities by means of an enhanced version of the Li-Wong PM-only algorithm (http://genomebiology.com/2001/2/8/RESEARCH/0032). Results were expressed as fold-change value (baseline = 1) or log2 fold-change values (baseline = 0). The fold-change is reported as the ratio of TNBS to control or TNBS plus IB-MECA to control. Results were then assessed with the PM-only model using software developed by Kornacker.34 The enhanced algorithm (1) scales all PM and MM probe intensities to minimize between-array differences in the scaled MM probe intensity distributions; (2) applies between-array regression analysis to the scaled PM probe intensities to estimate PM-specific sensitivities, excluding any PM probes that fail to show significantly positive sensitivities; (3) estimates gene expression levels by regressing scaled PM probe intensities on estimated PM probe sensitivities within each probe set, excluding any PM probes that show significant nonmonospecificity; and (4) tests a probe-level general linear model within each probe set to estimate the probability values for between-array differential gene expression35 (http://www.pnas.org/cgi/content/full/98/26/15044). The estimated probability values can be several orders of magnitude lower than 0.05 (P < 0.000001) as required by the Bonferroni correction, which applies when simultaneously testing thousands of genes for significant differential expression. Coexpressed genes are identified by means of a custom “agglomerative partitioning” algorithm that successively partitions gene expression profiles into a hierarchy of typical and exceptional subgroups (KTree). Graphs of selected KTree nodes are created using Eisen's TreeView software, following the usual color scheme that represents log-fold changes as red (positive), green (negative), or black (zero), with color saturation representing at least a 2-fold change in gene expression, unless otherwise noted. Electron Paramagnetic Resonance Spectroscopy and Spin Trapping in Ex Vivo Bowel Segments Spin-trapping measurements of reactive oxygen species (ROS) generation were made with a Bruker ER 300 spectrometer. To detect O2−· and OH·, 50 mmol/L of the spin trap DMPO was used. The distal colon was removed, cut open along the mesenteric border, and pinned out (with ≈20% stretch), and 1-cm square segments of whole-thickness specimens were incubated in Krebs buffer (in millimoles per liter: NaCl 120, KCl 6.0, MgCl2 1.2, NaH2PO4 1.35, NaHCO3 14.4, CaCl2 2.5, glucose 12.7) maintained for 20 min at 37°C and pH 7.4 by gassing with 95% O2/5% CO2. The spin trap was added, and ROS was monitored for 10 min by EPRI. The incubation buffer was loaded into a quart flat cell and measured at X-band in a TM-110 cavity. Additional control groups included superoxide dismutase (SOD), catalase, and flavoprotein to verify the nature of the signals observed. Quantification of oxygen radical signals was performed by comparing the double integral of the observed signal with that of a known concentration of the 2,2,6,6,-tetramethylpiperidinoxy free radical in solution.36 IB-MECA or other adenosinergic compounds were preincubated for 15 min with tissue before assessing their effect on ROS generation. Results A total of 30 rats were used in our studies. A large data set is included in the analysis for gene dysregulation in TNBS colitis for both microarray and SYBR green PCR. Data of >90% of genes showing significant dysregulation are included in the results, but detailed descriptions and analyses of genes are limited to those that are known to be implicated in human IBD and those that display remarkable upregulation or downregulation with TNBS or sensitivity to IB-MECA treatment. Microarray Analysis of the Effect of IB-MECA on Colitis-related Strong Gene Dysregulation (Up- or Downregulation) A rat neurobiochip (RNU34) with a gene microarray for ≈1322 gene transcripts representing 724 genes was used successfully to identify various genes that were upregulated or downregulated by TNBS/ethanol-induced colitis in the rat. On the basis of our exclusion criteria for analysis, TNBS induction caused significant dysregulation in 198 genes representing 27% of the gene pool in RNU34, including neural, immune, proinflammatory, cytokine, chemokine, growth factor, antioxidant/oxidant, receptor, epithelial membrane ATPase transporter, voltage-gated channel, or other cell signaling genes. Figure 1 is a histogram representation of the changes in genes that displayed significant colitis-related dysregulation in TNBS colitis. These genes were clustered according to their reported functions. Significant differences in gene expression between age-matched vehicle controls and TNBS colitis occurred with a value of P = 0.000001 or greater. Figures 1, 2, and 4 represent genes with the most exceptional level of dysregulation or illustrate a unique pattern of dysregulation with or without treatment with IB-MECA. Figure 1. View largeDownload slide Functional analysis of colitis-related gene dysregulation profiles in rat TNBS-induced colitis using the RNU34 neurobiochip with 724 genes. Microarray analysis revealed that significant upregulation or downregulation occurred in 198 genes representing a wide variety of different functional classes of genes that are implicated in IBD. Alterations occurred in (A) genes representing oxidant/antioxidant, cytokine, inflammation, chemokine, complement, platelet, protease, growth factor, heat shock protein, channels, and signal transduction/kinase genes, as well as (B) membrane transporter, receptor, neural, early-onset/transcription factor/oncogene, retinoid metabolism, and adhesion/chemotaxis gene clusters. (The most dysregulated genes in colitis are displayed in Fig. 2.) Figure 1. View largeDownload slide Functional analysis of colitis-related gene dysregulation profiles in rat TNBS-induced colitis using the RNU34 neurobiochip with 724 genes. Microarray analysis revealed that significant upregulation or downregulation occurred in 198 genes representing a wide variety of different functional classes of genes that are implicated in IBD. Alterations occurred in (A) genes representing oxidant/antioxidant, cytokine, inflammation, chemokine, complement, platelet, protease, growth factor, heat shock protein, channels, and signal transduction/kinase genes, as well as (B) membrane transporter, receptor, neural, early-onset/transcription factor/oncogene, retinoid metabolism, and adhesion/chemotaxis gene clusters. (The most dysregulated genes in colitis are displayed in Fig. 2.) Figure 2. View largeDownload slide Oral IB-MECA administration prevents colitis-induced alterations in gene expression patterns in the most exceptional clusters of genes. Two modes of colitis-related gene dysregulation profiles are depicted. A, Hierarchical gene cluster analysis of genes 1 through 7 associated with the strongest colitis-related downregulation; B, histogram representation of the fold changes in genes 1 through 7; C, hierarchical gene cluster representation of genes 9 through 49 associated with the strongest colitis-related upregulation; and D, histogram representation of the effect of IB-MECA in preventing gene upregulation in genes 9 through 49. Thirty-nine different genes are represented. n = 3 animals/group; P < 0.00001 for differences. Pseudocolor scale: green denotes log2-fold downregulation compared with age-matched controls; red, ≥2-fold upregulation compared with control; and black, no difference between groups. Lanes 1 and 2 depict cluster data according to calculated mean ratios of 1, IBMECA plus TNBS to control, and 2, TNBS to control. Figure 2. View largeDownload slide Oral IB-MECA administration prevents colitis-induced alterations in gene expression patterns in the most exceptional clusters of genes. Two modes of colitis-related gene dysregulation profiles are depicted. A, Hierarchical gene cluster analysis of genes 1 through 7 associated with the strongest colitis-related downregulation; B, histogram representation of the fold changes in genes 1 through 7; C, hierarchical gene cluster representation of genes 9 through 49 associated with the strongest colitis-related upregulation; and D, histogram representation of the effect of IB-MECA in preventing gene upregulation in genes 9 through 49. Thirty-nine different genes are represented. n = 3 animals/group; P < 0.00001 for differences. Pseudocolor scale: green denotes log2-fold downregulation compared with age-matched controls; red, ≥2-fold upregulation compared with control; and black, no difference between groups. Lanes 1 and 2 depict cluster data according to calculated mean ratios of 1, IBMECA plus TNBS to control, and 2, TNBS to control. Gene association profiles of microclusters were obtained with EASE analysis (http://david.niaid.nih.gov/david). The EASE-corrected probability value (SD Sidak) identified significant functional groups for colitis-related dysregulation. EASE identified 3 associated downregulated genes involved in ATPase activity coupled to transmembrane movement of ions. For upregulation, response to biotic stimulus and response to stress revealed an association for 20 genes. Gene association profiles were also obtained with GeneMerge version 1.1 software and revealed upregulated genes involved in the inflammatory response and retinoid metabolism or downregulated genes involved in amino acid metabolism, potassium ion transport, and sodium ion transport. Data are summarized in Tables 2 and 3. Table 2. EASE Analysis of Exceptional Microclusters     View Large Table 2. EASE Analysis of Exceptional Microclusters     View Large Table 3. Gene Merge Analysis     View Large Table 3. Gene Merge Analysis     View Large Figure 2 represents the most exceptional cluster of 49 genes/probe sets and 39 different genes that make up 5.7% of the gene pool in RNU34 and illustrate 2 distinct modes of colitis-related dysregulation. The first mode produced strong colitis-related downregulation in 5 genes that was nearly prevented by IB-MECA treatment. They represent 3 epithelial membrane transporters/ATPases for H+/K+ transport (nongastric, α-polypeptide), Na+/K+ transport (α1), or Ca2+ transport (plasma membrane 1). The other 2 genes are sterol-C4-methyl oxidase-like and mitogen-activated protein kinase 6 (MapK6; note that log2-fold changes are shown). The second mode generally produced strong colitis-related upregulated in the remaining 34 genes ranging from 2- to 12.5-fold upregulation (a ratio of 1.0 means no difference from control; net fold changes are described with 1.0 subtracted for control). IB-MECA nearly prevented the strong upregulation in >90% of the 34 genes. Upregulation of solute carrier family 1, member 3 (Slc1a3) was prevented by IB-MECA. Other upregulated genes included retinol binding proteins 1 and 2 (rbp1, 2), but rbp1 was upregulated by 10-fold compared to 1.6-fold for rbp2. Four chemokine-related genes that were significantly upregulated include MIP-1α receptor gene, chemokine (C-X-C motif) ligand 2, chemokine (C-C motif) ligand 3, and C-X-C chemokine LIX. Upregulated cytokines were IL-1β and small inducible cytokine A2. Two probe sets gave 3- and 5.5-fold changes in the small inducible cytokine; for IL-1β, different probes yielded 2.5- and 6-fold changes. Probe sets for complement component 3 (C3) revealed 5.2- and 4.0-fold upregulation; C4 was upregulated by 2.4-fold in TNBS colitis. Antioxidant enzymes/related genes that were upregulated by TNBS colitis are SOD2, inducible nitric oxide synthase (iNOS/NOS2), heme oxygenase-1 (Hmox1), and transferrin. Two different probe sets for Hmox1 gave the same 4-fold upregulation. Neural/related genes that were upregulated included neural cell adhesion molecule (Ncam1), activity and neurotransmitter-induced early gene protein 4 (Ania4), tachykinin gene encoding SP, neurokinin A, neurokinin K, neuropeptide γ, neuropilin, and vimentin. The cluster includes 4 growth factors and related genes that were upregulated, namely insulin-like growth factor binding protein 5 (Igfbp5), early growth response 1 (Egr1, 2.7- or 3.7-fold upregulation by 2 different probes), insulin-like growth factor 1 (Igf1), and platelet-derived growth factor receptor α (Pdgfrα). Other genes are platelet selectin, C/EBPβ (3.5- or 5-fold upregulation), the acute-phase reactant apolipoprotein E (ApoE), the benzodiazepine receptor (Bzrp, peripheral benzodiazepine receptor), and several heat shock protein genes (HSP 70, 70-kDa protein 1A, 27-kDa protein 1). Real-time SYBR Green PCR Validation of Selected Transcripts to Assess the Effect of IB-MECA on Colitis-related Gene Dysregulation In general, real-time SYBR green PCR analysis (Fig. 3) confirmed the relative changes in upregulation or downregulation of >90% of the genes depicted in Figure 2 with gene chip microarray analysis. These genes represent a heterogeneous set of genes with distinct and unrelated functions (see below). Several points deserve further consideration. In the cluster in Figure 3 in which we have analyzed all known genes by PCR, Igfbp5 is missing because the PCR conditions did not work. MapK6 did not show significant regulation, and the Na+/K+ transporting ATPase (α1) (Atp1a1) showed upregulation on TNBS treatment (in contrast to the chip results). Sc4mol,sterol-C4-methyl oxidase-like gene, and 3 ATPase transporters were downregulated by TNBS and showed the same tendency as the chip results with IB-MECA treatment, although Sc4mol dysregulation was not significantly affected by IB-MECA (unlike chip data). All of the other genes were strongly upregulated, typically in the range of 5- to 1000-fold, and oral IB-MECA treatment reduced or nearly prevented dysregulation; receptor genes were upregulated by 2.5- to 20-fold. The absolute magnitudes of the change in gene expression varied tremendously as expected between gene microarray and SYBR green PCR; PCR is much more sensitive than microarray. Notably, upregulation of rbp1 exceeded rbp2 as occurred with gene chip analysis. The highest upregulation occurred in IL-1β, C3, chemokines, ApoE, benzodiazepine receptor, platelet selectin, insulin-like growth factor 1, and small inducible cytokine (ranging from 20- to 500-fold upregulation); NOS2 (i.e., iNOS) was the most upregulated gene at >1000-fold. The tendency for C3 upregulation to be greater than C4 upregulation is consistent with gene chip data. Note that IB-MECA treatment caused significant reduction or attenuation or nearly prevented gene dysregulation at values between P < 10−6 and 10−11 in most cases. TNBS caused significant dysregulation in all genes at values in the range of P < 0.002 to P < 10−9 (with noted exceptions earlier). Overall, 34 of 37 genes were confirmed by microarray and SYBR green PCR for both induction with TNBS and treatment with oral IB-MECA, yielding a 92% confidence in the microarray analysis for those genes. Figure 3. View largeDownload slide SYBR green PCR validation analysis of genes represented in the most exceptional hierarchical clusters of colitis-related gene dysregulation profiles. A-C, SYBR green PCR analysis illustrating the alteration of gene expression between normal colon, TNBS treatment, and IB-MECA plus TNBS treatment in individual samples. A, ACTB, housekeeping gene β-actin. B, Effect of IB-MECA on Atp1b1, Na+/K+ transporting β1 polypeptide dysregulation. C, Partial inhibition of the upregulation of the chemokine ligand 2 (C-X-C motif, Ccl3) by IB-MECA. Graphs show relative fluorescence units (y axis) vs cycle numbers (x axis); orange lines show cycle threshold used for calculation of gene expression; measurements are averages from data in 3 samples/animal for each treatment group and triplicate PCR determinations for each animal. Graphs are shown for β-actin (A1), a housekeeping gene used as loading control; Atp1b1 (A2), a gene that is repressed by TNBS treatment (green, blue, and red lines) and derepressed by IB-MECA treatment (blue, purple, and pink lines); and Ccl3 (C), a gene induced by TNBS treatment (green, purple, and pink lines) and partially repressed by IB-MECA treatment (blue, purple, and pink lines). D, Real-time SYBR green PCR histogram analysis of the effect of IB-MECA on a selected set of 39 genes that showed the greatest colitis-related dysregulation. SYBR green PCR analysis also revealed purine receptor gene dysregulation in TNBS-induced colitis. A1R or A3R gene dysregulation is not significantly altered by oral IB-MECA treatment. A2bR was not affected by TNBS induction or IB-MECA treatment. Significant levels of upregulation of P2X1, P2X4, P2X7, P2Y2, and P2Y6 purinoceptor mRNA levels ranging from 5- to 20-fold upregulation were either prevented or mostly blocked by IB-MECA treatment. Relative expression in normal colon is defined as 1. Fold changes are calculated as ratios of values of TNBS treated to control and TNBS plus IB-MECA to control. Probability values of significant differences between TNBS treatment and IB-MECA are shown on the histogram for each gene. Figure 3. View largeDownload slide SYBR green PCR validation analysis of genes represented in the most exceptional hierarchical clusters of colitis-related gene dysregulation profiles. A-C, SYBR green PCR analysis illustrating the alteration of gene expression between normal colon, TNBS treatment, and IB-MECA plus TNBS treatment in individual samples. A, ACTB, housekeeping gene β-actin. B, Effect of IB-MECA on Atp1b1, Na+/K+ transporting β1 polypeptide dysregulation. C, Partial inhibition of the upregulation of the chemokine ligand 2 (C-X-C motif, Ccl3) by IB-MECA. Graphs show relative fluorescence units (y axis) vs cycle numbers (x axis); orange lines show cycle threshold used for calculation of gene expression; measurements are averages from data in 3 samples/animal for each treatment group and triplicate PCR determinations for each animal. Graphs are shown for β-actin (A1), a housekeeping gene used as loading control; Atp1b1 (A2), a gene that is repressed by TNBS treatment (green, blue, and red lines) and derepressed by IB-MECA treatment (blue, purple, and pink lines); and Ccl3 (C), a gene induced by TNBS treatment (green, purple, and pink lines) and partially repressed by IB-MECA treatment (blue, purple, and pink lines). D, Real-time SYBR green PCR histogram analysis of the effect of IB-MECA on a selected set of 39 genes that showed the greatest colitis-related dysregulation. SYBR green PCR analysis also revealed purine receptor gene dysregulation in TNBS-induced colitis. A1R or A3R gene dysregulation is not significantly altered by oral IB-MECA treatment. A2bR was not affected by TNBS induction or IB-MECA treatment. Significant levels of upregulation of P2X1, P2X4, P2X7, P2Y2, and P2Y6 purinoceptor mRNA levels ranging from 5- to 20-fold upregulation were either prevented or mostly blocked by IB-MECA treatment. Relative expression in normal colon is defined as 1. Fold changes are calculated as ratios of values of TNBS treated to control and TNBS plus IB-MECA to control. Probability values of significant differences between TNBS treatment and IB-MECA are shown on the histogram for each gene. In contrast to the previous 39 genes, SYBR green PCR analysis of purine genes (Fig. 4) revealed some differences in gene dysregulation from those detected with gene chip microarray analysis. It is notable that purine genes did not cluster in microarray analysis indicative for variable and heterogeneous dysregulation profiles. Therefore, PCR revealed upregulation of A1R gene mRNA expression of ≈6-fold that was not affected by IB-MECA treatment. Adenosine A3R expression was upregulated by 2-fold but was not significantly altered by IB-MECA. A2aRs were undetectable by SYBR green PCR using 2 different sets of primers. A2bR was not affected by TNBS induction or IB-MECA treatment. In contrast, a 4-fold P2X4R mRNA upregulation in TNBS colitis was nearly prevented by IB-MECA. Significant levels of upregulation of P2X1, P2X4, P2X7, P2Y2, and P2Y6 purinoceptor mRNA levels ranging from 5- to 20-fold upregulation was either prevented or mostly blocked by IB-MECA treatment. It is noteworthy that P2YR upregulation was much greater than that of P2XRs. Figure 4. View largeDownload slide Gene cluster of colitis-dysregulated genes with moderate sensitivity to IB-MECA treatment. For instance, 19 of the genes that displayed colitis-related downregulation were moderately or not affected by oral IB-MECA treatment. Pseudocolor scale: green denotes log2-fold downregulation compared with age-matched controls; red, ≥2-fold upregulation compared with control; and black, no difference between groups. Lanes 1 and 2 depict cluster data according to calculated mean ratios of 1, IBMECA plus TNBS to control, and 2, TNBS to control. Figure 4. View largeDownload slide Gene cluster of colitis-dysregulated genes with moderate sensitivity to IB-MECA treatment. For instance, 19 of the genes that displayed colitis-related downregulation were moderately or not affected by oral IB-MECA treatment. Pseudocolor scale: green denotes log2-fold downregulation compared with age-matched controls; red, ≥2-fold upregulation compared with control; and black, no difference between groups. Lanes 1 and 2 depict cluster data according to calculated mean ratios of 1, IBMECA plus TNBS to control, and 2, TNBS to control. Gene chip analysis with adenosine receptor probes for A1, A2a, A2b, and A3Rs on the chip revealed that only A2a and A2bR genes showed statistically significant differential expression in TNBS colitis. Therefore, downregulation of A2bR gene mRNA expression was significantly blocked by IB-MECA treatment but not prevented. Upregulation of A2a was prevented by IB-MECA. The associated probability values are <0.000000001. Among all of the P2 purinergic receptors on the chip (including P2X, P2Y, P2Y4, P2Y1, P2X1, P2X2, P2X3, P2X4, P2X7), only P2X4 receptors (P2rx4) showed statistically significant differential expression. The log2 ratios showed approximately equal upregulation in inflamed (0.43) and treated (0.36); therefore, IB-MECA did not reveal any blockade of P2X4R gene mRNA expression. Both log2 ratios are highly significant (P < 0.000000001). It is interesting that the benzodiazepine receptor (Bzrp) gene, unlike the purinoceptor genes, was shown to be regulated in a similar fashion by both techniques. However, Bzrp was strongly upregulated by gene chip analysis, unlike purine receptor genes. Microarray Analysis of Genes Modestly Dysregulated by TNBS Figures 4 and 5 depict additional clusters of genes with moderate colitis-related downregulation or downregulation and upregulation, respectively. In general, all genes were significantly dysregulated by colitis (P < 0.000000001), but oral IB-MECA treatment blocked dysregulation in some of the genes. In the remainder, IB-MECA had a modest effect on dysregulated genes. In some genes, IB-MECA converted a downregulation to an upregulation or the reverse. For instance, in the gene clusters depicted in Fig. 4, TNBS caused moderate downregulation of IL-18 and cathepsin E, but IB-MECA prevented and strongly upregulated these genes. CD48 antigen was upregulated by IB-MECA. Additional examples are shown in Fig. 5. In addition, 19 genes that displayed colitis-related downregulation were moderately or not affected by oral IB-MECA treatment. Examples of these genes are neurodap 1, MAP kinase 3, glutamine synthase 1, glutamate dehydrogenase 1, chloride channel 2, and PKC ζ (Fig. 4). Figure 5. View largeDownload slide Another gene cluster moderately dysregulated by TNBS colitis with strong sensitivity to oral IB-MECA treatment. Some upregulated genes converted to downregulation after oral IB-MECA treatment. Pseudocolor scale: green denotes log2-fold downregulation compared with age-matched controls; red, ≥2-fold upregulation compared with control; and black, no difference between groups. Lanes 1 and 2 depict cluster data according to calculated mean ratios of 1, IBMECA plus TNBS to control, and 2, TNBS to control. Figure 5. View largeDownload slide Another gene cluster moderately dysregulated by TNBS colitis with strong sensitivity to oral IB-MECA treatment. Some upregulated genes converted to downregulation after oral IB-MECA treatment. Pseudocolor scale: green denotes log2-fold downregulation compared with age-matched controls; red, ≥2-fold upregulation compared with control; and black, no difference between groups. Lanes 1 and 2 depict cluster data according to calculated mean ratios of 1, IBMECA plus TNBS to control, and 2, TNBS to control. Effect of Oral IB-MECA Treatment on Colonic Histopathology, Clinical Score, and Progression of Inflammation TNBS-induced colitis leads to macroscopic and microscopic signs of inflammation (see Fig. 6A-I) compared with age-matched vehicle/control animals (Fig. 6A-C). TNBS/ethanol induction caused variable regions of necrosis in mucosa (arrows) with regions of hemorrhage in both mucosa and submucosa (arrowhead) and extensive infiltration of inflammatory cells (Fig. 6D). Injury to the mucosa leads to ulceration, villus atrophy, and dense transmural infiltration of inflammatory cells (Fig. 6E). Major adhesions were visible, and ulcerations occurred at ≥1 sites in association with hyperemia and bowel wall thickening. Oral administration of IB-MECA (1.5 mg/kg weight b.i.d. for 6 days) nearly prevented all visible signs of inflammation and colitis, and the gut wall appeared normal except for a few inflammatory cells still present in the submucosa and circular muscle layers (Fig. 6G-I) (n = 4 animals/group). Figure 6. View largeDownload slide IB-MECA prevents cell and tissue injury in TNBS-induced colitis in rats. Colon sections from age-matched control (A-C), TNBS-treated (D-F), and IB-MECA-treated TNBS rats (G-I). A-C, Normal histological profile. D, TNBS administration caused areas of necrosis in mucosa (M; arrows) with evident regions of hemorrhage in both mucosa and submucosa (SM; arrowhead) and infiltration of inflammatory cells (curved, hollow arrow). E, Injured mucosa resulting in ulceration (arrows), atrophy of villi (arrowheads), edema, fibrosis, and inflammatory cell infiltration (hollow arrows). F, Atrophy of villi (arrowhead) and dense infiltration of all of the layers (arrows). G-I, Oral administration of IB-MECA (1.5 mg/kg b.i.d. for 6 days) nearly prevented the colitis. A few immune cells more than in age-matched controls are still visible in submucosa (arrows, G-I) and circular muscle (CM) layer (arrowhead, G). Hematoxylin and eosin (H & E) staining. Scale bar = 50 μm. J, Clinical scoring confirms effects of IB-MECA in protecting against histopathological damage (K). Food intake is nearly normal in IB-MECA-treated rats (P < 0.05). L, Total food intake (7 days) is much greater in IB-MECA-treated TNBS animals than in TNBS colitis animals. M, IB-MECA treatment prevented weight loss that occurs as a result of TNBS colitis. Figure 6. View largeDownload slide IB-MECA prevents cell and tissue injury in TNBS-induced colitis in rats. Colon sections from age-matched control (A-C), TNBS-treated (D-F), and IB-MECA-treated TNBS rats (G-I). A-C, Normal histological profile. D, TNBS administration caused areas of necrosis in mucosa (M; arrows) with evident regions of hemorrhage in both mucosa and submucosa (SM; arrowhead) and infiltration of inflammatory cells (curved, hollow arrow). E, Injured mucosa resulting in ulceration (arrows), atrophy of villi (arrowheads), edema, fibrosis, and inflammatory cell infiltration (hollow arrows). F, Atrophy of villi (arrowhead) and dense infiltration of all of the layers (arrows). G-I, Oral administration of IB-MECA (1.5 mg/kg b.i.d. for 6 days) nearly prevented the colitis. A few immune cells more than in age-matched controls are still visible in submucosa (arrows, G-I) and circular muscle (CM) layer (arrowhead, G). Hematoxylin and eosin (H & E) staining. Scale bar = 50 μm. J, Clinical scoring confirms effects of IB-MECA in protecting against histopathological damage (K). Food intake is nearly normal in IB-MECA-treated rats (P < 0.05). L, Total food intake (7 days) is much greater in IB-MECA-treated TNBS animals than in TNBS colitis animals. M, IB-MECA treatment prevented weight loss that occurs as a result of TNBS colitis. IB-MECA treatment improved the clinical score, appetite, and weight gain in TNBS-treated rats (Fig. 6J-M). IB-MECA nearly prevented the colitis because the clinical score remained modestly but significantly higher than age-matched controls (Fig. 6J), and food intake fully recovered after 72 to 48 h from TNBS induction (Fig. 6K). About 12 h after the first oral dose of IB-MECA (e.g., 1 day after TNBS induction), TNBS-induced rats receiving IB-MECA were eating very little food compared with controls, and their food intake was the same as that of TNBS-induced animals. Within 48 h, IB-MECA/TNBS-treated rats began to eat as much as age-matched controls and significantly more than TNBS rats (Fig. 6K). As a result, total food intake in IB-MECA-treated rats remained significantly lower than controls (Fig. 6L), but the weight loss profile during the 7-day period from induction indicates that IB-MECA treatment was effective in preventing the 15% body weight loss caused by TNBS induction (Fig. 6M). Effect of Acute IB-MECA Treatment on Free Radical Production in TNBS-Inflamed Colon The antioxidant role of A3Rs is a target for its beneficial effect that was explored directly by EPRI of superoxide anions. EPRI analysis using DMPO to spin trap hydroxyl free radicals (R-OH) showed that TNBS-induced colitis leads to a several-fold increase in R-OH (Fig. 7A, B); R-OH is low in normal colon. Acute exposure to IB-MECA (0.1–10 μmol/L) for 20 min significantly reduces the R-OH by 30% to 70% in whole-thickness gut (shown) or mucosa (not shown) in inflamed gut (Figs. 7A, B). The source of the R-OH is superoxide anions because SOD abolishes it. Exogenous application of adenosine (100 μmol/L) or a cocktail of EHNA plus NBTI (1 μmol/L) that preserves endogenous adenosine released caused a similar inhibition of R-OH. Figure 7. View largeDownload slide Acute ex vivo IB-MECA/adenosine treatment reduces superoxide anion/hydroxyl radical production. A, Riboflavin generates hydroxyl radicals. A small but detectable hydroxyl radical response occurs in normal gut. In inflamed gut, there is a 3-fold increase in free radical production. IB-MECA (10 μmol/L) treatment (20 min of acute exposure) reduces the response. Adenosine suppresses the response. A cocktail of EHNA plus NBTI that blocks breakdown and reuptake of eADO, respectively, mimics the effects of adenosine or IB-MECA. The hydroxyl radicals are derived from superoxide anions because SOD (1 μmol/L) blocks them in inflamed gut. EPRI is used to determine hydroxyl free radical production using DMPO spin trapping. Tissues were preincubated for 20 min in Krebs' oxygenated buffer at 37°C and then preincubated with vehicle or drugs for 20 min. EPRI of DMPO spin trapping was done for 10 min. B, Hydroxyl radical generation. Pooled data show that an increase in radical production occurs in colitis and is reduced ≈50% by IB-MECA (P < 0.01). n = 3 to 4 tissues/condition. A.U. indicates arbitrary units. Figure 7. View largeDownload slide Acute ex vivo IB-MECA/adenosine treatment reduces superoxide anion/hydroxyl radical production. A, Riboflavin generates hydroxyl radicals. A small but detectable hydroxyl radical response occurs in normal gut. In inflamed gut, there is a 3-fold increase in free radical production. IB-MECA (10 μmol/L) treatment (20 min of acute exposure) reduces the response. Adenosine suppresses the response. A cocktail of EHNA plus NBTI that blocks breakdown and reuptake of eADO, respectively, mimics the effects of adenosine or IB-MECA. The hydroxyl radicals are derived from superoxide anions because SOD (1 μmol/L) blocks them in inflamed gut. EPRI is used to determine hydroxyl free radical production using DMPO spin trapping. Tissues were preincubated for 20 min in Krebs' oxygenated buffer at 37°C and then preincubated with vehicle or drugs for 20 min. EPRI of DMPO spin trapping was done for 10 min. B, Hydroxyl radical generation. Pooled data show that an increase in radical production occurs in colitis and is reduced ≈50% by IB-MECA (P < 0.01). n = 3 to 4 tissues/condition. A.U. indicates arbitrary units. SYBR Green PCR Analysis of Free Radical Enzyme/Pathways Two important antioxidant enzymes in the gut mucosa with overlapping activities are glutathione peroxidase 1 and 2 (GPx1 and GPx2). SYBR green PCR revealed a 7-fold upregulation (Fig. 3). Gene chip microarray analysis and/SYBR green PCR revealed strong colitis-related upregulation of the various antioxidant/oxidant enzymes SOD1, SOD2, iNOS, and heme oxygenase (Hmox1) and iron-metabolic pathways (transferrin, transferrin receptor, iron-responsive element binding protein). Oral IB-MECA treatment blocked or prevented such upregulation in these genes. Differential Dysregulation in Neural and Nonneural Layers SOD1 and SOD2 are important for EPRI analysis of R-OH because we are trapping extracellular superoxide with DMPO that is dismutated by SOD2 to H2O2. We show that SOD was differentially upregulated in neural and nonneural muscle layers: it was upregulated 0.52-fold in longitudinal muscle-myenteric plexus, 0.21-fold in mucosa, and 2.16-fold in submucosa (all changes, P < 0.000001; n = 3 rats). Gene chip analysis indicated that inducible nitric oxide synthase mRNA is elevated in colitis by 2-fold in mucosa, 0.30-fold in longitudinal muscle-mysenteric plexus, and 4-fold in SMP (P < 0.000001), suggesting a putative role for nitric oxide in ONOO- and nitrotyrosine-induced transmural damage. The increase in iNOS mRNA was similar to previous estimates from PCR.37 NO derived from iNOS mediates, in part, inflammation-induced epithelial transport dysfunction.37 Genes Induced by NF-κB Genes that are reported to be regulated by the transcription factor are summarized in Figure 8. TNBS colitis caused upregulation in these genes, including chemokines, complement, cytokines, adhesion molecules, heme oxygenase, and SOD2. IB-MECA blocked such dysregulation. Figure 8. View largeDownload slide Summary of TNBS colitis-induced genes that were reported to be regulated by the redox-sensitive transcription factor NF-κB. Upregulation of redox-sensitive genes is blocked by oral IB-MECA treatment. Figure 8. View largeDownload slide Summary of TNBS colitis-induced genes that were reported to be regulated by the redox-sensitive transcription factor NF-κB. Upregulation of redox-sensitive genes is blocked by oral IB-MECA treatment. Neural Gene Dysregulation Neural genes that are dysregulated by TNBS colitis are illustrated in Figure 9. IB-MECA prevented such dysregulation. Figure 9. View largeDownload slide Neural genes that are dysregulated by TNBS colitis are sensitive to oral IB-MECA treatment. Neuromedin and benzodiezepine receptors are the most dysregulated neural genes. Their dysregulation is highly sensitive to oral IB-MECA treatment. Figure 9. View largeDownload slide Neural genes that are dysregulated by TNBS colitis are sensitive to oral IB-MECA treatment. Neuromedin and benzodiezepine receptors are the most dysregulated neural genes. Their dysregulation is highly sensitive to oral IB-MECA treatment. Discussion To the best of our knowledge, this is the first study to examine the prophylactic effect of ADOA3Rs in experimental colitis using high-density oligonucleotide microarray analysis of gene expression profiles. Overall, 5.4% of the gene pool in the RNU34 gene chip represent the most exceptional gene cluster, revealing 2 modes of colitis-related gene dysregulation (upregulation or downregulation). These findings, validated by real-time quantitative SYBR green PCR for both induction of colitis and treatment with oral IB-MECA, are consistent with histopathology and clinical observations of the severity of inflammation and tissue injury. Certain types of genes were strongly upregulated and others were downregulated by colitis. A distinguishing feature of a subset of genes (rbp1, IL-1β,C3, chemokines, iNOS, Igf1, platelet selectin, Hmox1, Bzrp, and the small inducible cytokine) is the strong 50- to 1000-fold gene upregulation by SYBR green PCR. Despite obvious differences in etiology between chemical (TNBS) induction of experimental colitis and immune-based IBD in humans, a remarkable number of different functional gene families that are dysregulated in rat colitis resemble those in human IBD.28 This suggests that the TNBS colitis model is a suitable IBD model for molecular gene dysregulation studies on chemokine/related genes (i.e., MIP-1α receptor gene, chemokine C-C motif), cytokine (IL-1β), complement (C3, C4a), growth factor (Igfbp5, Egf1, Pdgfrα), receptor (i.e., Bzrp, purine receptors), HSP (HSP70, HSP27), retinoid metabolism (rbp1), oxidant/antioxidant pathway (SOD2, Hmox1, GPX1, iNOS, ferritin), neural and remodeling genes, and mucosal transporters (i.e., Atp2b1, Ca2+transporter). Some upregulated genes are involved in the inflammatory response or are a response to a biotic stimulus or stress (Tables 2 and 3). Our study on gene dysregulation provides convincing data that an ADOA3R agonist can prevent development of chronic inflammation and most gene dysregulation, histopathology, clinical signs of damage, and weight loss in TNBS-induced colitis. Three days after TNBS induction, animals treated with IB-MECA were eating as much as TNBS-induced animals, and they did not lose any weight, in contrast to TNBS animals. This indirectly suggests that IB-MECA also may block the development of the acute phase and its progression to chronic inflammation. At the dose given, IB-MECA could not prevent all gene dysregulation induced by TNBS colitis by day 7. In other studies, limited marker analysis suggested that IB-MECA may be beneficial in DDS or IL-10 spontaneous models of colitis or other models. An ADO kinase inhibitor also was shown to be of benefit in DDS colitis.16,17 The potential of ADO receptor agonists such as IB-MECA in the treatment of active chronic inflammation once established remains unknown in experimental colitis or IBD. IB-MECA was more effective in protecting against rat TNBS-induced colitis than in preventing murine colitis in previous studies on DDS or IL-10 knockout mice. Differences in susceptibility to IB-MECA may reflect differences in mechanism of induction, species differences, and the primary role of oxidative stress in the injury and colitis induced by TNBS in contrast to DDS or IL-10 knockout mice. An important finding is that a cluster of genes that were strongly downregulated by colitis were Na+, amino acid, and K+ and Ca2+ transporters (i.e., H+/K+ transporter, nongastric; Na+/K+ transporter, α1, α3 probes; Ca2+ transporter, plasma membrane 1; Na+/K+ transporter, β1 polypeptide) that are also indicative of the extent of mucosal barrier injury and loss of epithelial functional integrity. These genes can be targeted for therapy in studies focused on mucosal barrier function, integrity, and transport in the rat TNBS colitis model. These transporters are important in the normal function and maintenance of electrolyte and water balance across the epithelial membranes. IB-MECA almost prevented downregulation in these transport mechanisms and protected against mucosal and transmural cell injury, reflected in the lack of histopathology and normal clinical scores/profiles. It should be noted, however, that downregulation of the Na+/K+ transporter 1α was only modestly affected by IB-MECA treatment. Upregulation of Na+/K+ transporter also is prevented by IB-MECA. Our study did not delineate which receptor is involved in the protective action of IB-MECA. However, the therapeutic oral dose used in rats (1.5 mg/kg) is likely sufficient to activate other ADORs that have a higher affinity than ADOA3R.5,6 All 4 ADOR subtypes are expressed (differentially) in immune cells and neutrophils38,39 and in the intestinal tract in neural and nonneural elements,8 and their expression is similar in rats and humans (F.L.C., unpublished observations). However, ADOA3Rs are a more likely target for the effects of oral IB-MECA administration than ADOA1Rs, although A2aRs also have potential. Activation of A1Rs by IB-MECA would not be beneficial because chronic A1R activation could lead to rapid desensitization/internalization of A1Rs and disinhibition in the enteric nervous system, adding to neural dysfunction.8,40 In addition, neutrophils contain A1Rs, A2Rs, and A3Rs that produce opposite effects. A1Rs cause chemotaxis and phagocytosis,38,39 and these proinflammatory actions limit the benefits of targeting A1Rs in IBD and certainly argue against the involvement of A1Rs in the benefits of oral IB-MECA. Interestingly, ADOA1R/mRNAs are upregulated by a variety of conditions, including glucocorticoid treatment, ethanol withdrawal, and oxidative stress, rabbit ileitis,30 and TNBS colitis. In contrast, activation of the upregulated ADOA3R or A2aR activation by eADO exerts anti-inflammatory effects39,41 in human neutrophils, inhibits free radicals, stimulates antioxidant enzymes, and/or has putative neuroprotective effects42 that may contribute to the effects of IB-MECA in vivo. Neuronal plasticity in the enteric nervous system is a prominent feature of the rat colitis model.32 Hyperexcitability in gut sensory afferent AH neurons and facilitation of synaptic transmission occurs during chronic TNBS colitis.32 Activity-dependent signaling pathways lead to neuronal induction of early genes encoding various proteins, including transcription factors involved in neuronal functional plasticity.43,44 Dysregulation (upregulation or downregulation) occurred in a variety of neural genes encoding neuropeptides (Tac1, for substance P, neurokinin A, neuropeptide K, and neuropeptide γ; Penk-rs-pre-pro-enkephalin), neural receptors (Bzrp, galanin, GABA-A, bradykinin-B, neuropilin, purine receptors), neural kinases (Ania4, Ania2), neural cell adhesion (Ncam1), and neuronal development (Nnat, neuronatin; Nrp, neuropilin). Inflammatory mediators are known to affect all components of gut neural reflexes and to influence receptor expression and neuronal phenotypic changes that may affect release of cytokines such as IL-1. IL-1β inhibits acetylcholine release and increases iNOS expression/activity. In TNBS colitis, the activity of myenteric neurons in the inflamed gut may be under the ongoing influence of IL-1β that is strongly upregulated and is implicated in colonic dysmotility in experimental colitis.45 Oral IB-MECA was effective against neural gene dysregulation in TNBS colitis. The neuroprotective effects of ADOA3R agonists have been demonstrated after chronic administration46 that may be, in part, indirect by inhibiting the production of the proinflammatory cytokines MIP-1α, TNF-α, or IFN-γ in macrophages or monocytes, known to express ADOA3Rs.5,6,47 These inflammatory cytokines play a key role in IBD,1 and IB-MECA was effective against the production of these cytokines in other colitis models.16 This study showed that gene induction of various cytokine/chemokine/proinflammatory genes was blocked by IB-MECA. Our study revealed novel information on dysregulation of 19 receptor genes. We can predict that because all of these genes are important in the neurophysiology of the gut, their dysregulation would impair normal gut function. Significant purine receptor gene upregulation occurs in purinergic ADOA1, A3, A2a, P2X1, P2X3, P2X7, P2Y2, and P2Y6 receptor genes. Such widespread dysregulation would result in significant disruption in normal gut functions, motility, secretion, neural reflexes, and coordination reflexes known to be modulated by such receptors.7,–9 These receptors are present in the human digestive tract and/or in various rodent species studied to date7 (F.L.C., unpublished observations). Except for putative A3Rs targeted by oral IB-MECA, it remains unknown whether other purine receptors serve any beneficial role in colitis. Expression of ADOA1R or ADOA3R or receptor mRNA has been shown to be sensitive to intestinal inflammation in a rabbit ileitis/CD model,30 and ADOA1R downregulation leads to disruption of neurotransmission.31 IB-MECA partially blocked or prevented dysregulation of selected P2X and P2Y receptor subtypes without affecting ADORA1R or ADOA3R or downregulated P2Y1R, P2Y4R, or P2X2R gene products. P2Y nucleotide receptor gene dysregulation was much more sensitive to IB-MECA treatment than P2X receptor genes. Therefore, there is selective influence of the orally active ADOA3R agonist IB-MECA on preventing dysregulation of subsets of purine genes in TNBS-induced colitis. This study revealed novel complex purine-purine receptor interactions in chronic gut inflammation that were not previously reported in other tissues. Strong upregulation of the peripheral benzodiazepine receptor (Bzrp) gene occurs in the gut of colitis animals. Bzrp is involved in oxidative metabolism and steroidogenesis48 and is localized to surface epithelium, absorptive goblet cells, and enteric neurons of rat colon.49 Upregulation of Bzrp occurs in human neurodegenerative/inflammatory diseases and monocytes.50,51 Expression of Bzrp also is subject to regulation by chronic stress such as TNBS colitis (i.e., EASE analysis). A protective role is suggested for Bzrp in TNBS colitis that may be related, in part, to its anti-inflammatory actions.52 Its expression and functional importance in IBD remain unknown. TNBS may be converted to ROS,53 which can contribute to injury and inflammation of the colon. In the inflamed mucosa, the main sources of free radicals are activated mucosal leukocytes (neutrophils and macrophages), xanthine oxidase during ischemia/reperfusion, and arachidonic acid metabolites. Oxidative stress may lead to tissue injury by ROS interacting with proteins, lipids, carbohydrates, and DNA and stimulation of neutrophil chemotaxis, 5-lipoxygenase, and phospholipase A2.37 Finally, ROS can activate transcription factors such as NF-κB, c-fos, c-myc, and c-jun37 involved in cytokine release, hypertrophic signaling, and cell growth. Superoxide generation may play a role in IBD.1 In rat TNBS colitis, SOD2 was strongly elevated in the colonic tissues in a failed attempt to protect the gut against ROS injury, whereas a decrease in SOD was reported in patients with IBD,54 which would increase tissue injury. Species differences may be the result of the longer duration of the oxidative stress period in IBD, leading to further compromises in antioxidant defense mechanisms. SOD2 is induced by IL1-β (and TNF-α) and serves a protective function against oxidative damage. IBD in humans are immune-mediated diseases, with dysregulated T cell activity1 leading to inflammation and ROS production. An SOD mimetic reduced ICAM-1 upregulation, expression of P-selectin, and lipid peroxidation products in TNBS colitis;55 these genes were upregulated in our study. ICAM-1 and P-selectin were shown to be expressed in endothelial and epithelial cells and neutrophils in the rat TNBS colon.55 Leukocyte-endothelial interactions and neutrophil migration from the vessel require P-selectin, β2 integrins, and ICAM-1.56 IB-MECA was effective in suppressing these oxidative stress and chemotactic mechanisms in TNBS colitis. Thirty-three upregulated genes are targets for redox-sensitive transcriptional regulation through NF-κB (i.e., chemokines, cytokines, complement, SOD2, heme oxygenase, iNOS, NCAM 1, ICAM 1, selectin, Bzrp, IL-1β, Jun B proto-oncogene, ADOA1R), and others for heat shock factor 1 (HSF1). Virtually all of these genes were upregulated by colitis, and their sensitivity to oral IB-MECA is consistent with the hypothesis that activation of ADOA3R prevents colitis by suppressing free radical production and redox-sensitive transcriptional regulation through NF-κB, HSF1, and other factors involved in the induction of a wide variety of genes including proinflammatory genes. Antisense oligonucleotide to NF-κB p65 treatment was shown to ameliorate inflammation even after induction of colitis.1 eADO release into the extracellular environment under conditions of oxidative stress is believed to play a cytoprotective role.21 Neurons/nerve terminals, neutrophils, and endothelial cells have been reported to release high levels of ADO at sites of inflammation, infection, and metabolic stress. During inflammation, eADO may become high enough to exert immunomodulatory, neuroprotective, immunosuppressive, and antioxidant effects through putative low-affinity ADOA3Rs. With respect to the last effect, free radicals are implicated in IBD and TNBS colitis, and activation of ADOA3Rs is known to stimulate the antioxidant defense mechanisms.29 SOD or GPX knockout mice are used to study free radicals in colitis.57,58 SODs convert superoxide radicals (O2−) to H2O2 and then to water by GPX or catalase. SOD2 is inducible by oxidative insults and cytokines. It is not surprising that in TNBS colitis SOD2 is tremendously upregulated, given the 3-fold elevation in free radical generation in the inflamed gut. An increase in peroxidation of membrane lipids also suggests the involvement of oxidative stress-mediated mechanisms of cell/tissue injury in both rat TNBS colitis and IBD.30 The transmural damage and gene dysregulation seen in rat TNBS colitis also provide another link with human CD/IBD. The ability of IB-MECA or ADO to suppress free radicals in ex vivo inflamed colon indicates that ADO has direct antioxidant effects in inflamed gut. This may be one mechanism by which it exerts protection. The inhibitory effect of IB-MECA on GPX1, GPX2, Hmox1, SOD2, iNOS, and iron-handling genes supports its antioxidant mode of action in TNBS colitis and mimics the effects of eADO at putative ADOA3Rs. ROS regulates the antioxidant genes heme oxygenase (Hmox1) and GSH.59 The expression of the small heat shock protein (HSP) Hmox1 was strongly upregulated in the inflamed intestine, and it may afford neuroprotection.60 HSP27, which was elevated 5-fold by TNBS, also has been suggested to have neuroprotective effects60 and anti-inflammatory effects in pancreatitis.61 Autoantibodies against various HSPs exist in autoimmune diseases and IBD.13 Ferritin, the major intracellular iron storage protein, is responsive to intracellular oxidative stress and ROS generated during gut inflammation. Related proteins, transferrin, the transferrin receptor, and iron-responsive element-binding protein, are downregulated in TNBS colitis.62 These genes are restored by oral IB-MECA treatment, indicating that IB-MECA was able to restore normal gut function. Overall, IB-MECA seems to be highly effective in protecting against the deleterious effects of proinflammatory mediators, including free radicals, restoring oxidant/antioxidant balance in experimental colitis, and potentially having some efficacy in IBD. Lamina propria mononuclear cells express higher levels of IL-1β in IBD (both UC and CD). IL-1β release is sustained throughout the clinical course, with a prominent neutrophil infiltrate in IBD patients (CD or UC).1 In TNBS colitis, IL-1β was one of the genes with the strongest upregulation. IL-1 was a sensitive indicator of mucosal inflammation, and its values correlated well with myeloperoxidase activity in TNBS/ethanol-induced colitis in the rat.1 Caspase-1 is an IL-1β-converting enzyme (ICE) that cleaves and activates IL-18 and IL-1β. ICE expression and release of these cytokines contribute to gut inflammation. Manipulations that interfere with ICE expression or effect protect against inflammation.63 The strong upregulation of the IL-1β gene is probably in part a consequence of ICE downregulation in TNBS colitis. In IBD, there appears to be a disturbed balance between proinflammatory and anti-inflammatory cytokines. Higher levels of proinflammatory cytokines occur in IBD (IL-1, IL-8, TNF-α, IL-6) from macrophages, lymphocytes, and polymorphonuclear leukocytes. Their synthesis is induced by the activation of NF-κB.64 IL-1 is produced primarily by macrophages, and its expression is increased in inflammatory lesions of patients with IBD. An imbalance between IL-1 and IL-1R antagonist occurs in inflamed mucosa.64 Data indicate that complement activation also contributes to the pathogenesis of IBD. Serum concentrations of C3 were higher in CD than in UC, which can be used in the differential diagnosis to distinguish patients with CD and UC.65 Our findings with upregulation in C3 and C4 in TNBS colitis are consistent with IBD. Overall, the ability of IB-MECA to block IL-1β, ICE, IL-18, C3, and C4a gene dysregulation contributes to its anti-inflammatory effects and protection against tissue injury. The expression of colonic chemokines is nonselectively upregulated in IBD. Chemokines promote leukocyte migration to areas of inflammation and initiate cell activation events. Macrophage inflammatory proteins 1α and 1β (MIP-1α, MIP-1β) are β chemokines that attract monocytes and T lymphocytes and are involved in their activation.66 Epithelial cells, macrophages, T lymphocytes, neutrophils, and plasma cells in IBD mucosa produce chemokines. It has been suggested that the “degree of local inflammation and damage in IBD depends on local chemokine expression in IBD tissues.”66 Recent findings indicated that activated platelets may contribute to mucosal inflammation and pathogenesis of UC and CD by mediating the increase in the CD40-dependent expression of adhesion molecules (ICAM-1, VCAM- 1) and chemokine production by intestinal microvascular endothelial cells, which is followed by T cell adhesion to these cells.67 Abnormalities in platelet numbers and function occur in IBD patients,68 and activated platelets induce neutrophil activation and degranulation and discharge chemoattractant molecules such as MIP-1α (this study) that participate in leukocyte recruitment.68 In activated IBD platelets, there is upregulation of VCAM-1 and ICAM-1, 2 key leukocyte adhesion molecules (reviewed above). IB-MECA inhibits upregulation of these genes, an effect that may contribute to its protective effect. Various chemokine or platelet genes that are strongly upregulated in TNBS colitis are highly sensitive to IB-MECA treatment and are suitable therapeutic/screening targets. MAP kinase 6 was greatly downregulated compared to other kinases or other downregulated genes in TNBS colitis. The mitogen-activated protein kinase pathways regulate most cellular processes, including defense mechanisms such as stress reactions and inflammation in IBD. Several MAP kinase inhibitors reached clinical trials with promise in CD.69 They are key elements in the regulation of all stages of inflammation and are activated by G-protein coupled receptors or cytokine receptors (i.e., IL-1R, TNF-αR). Activation of MAP kinases can lead to subsequent activation of nuclear kinases and transcriptional regulation (AP-1, cAMP response element binding protein). All MAP kinase signaling cascades are key players in the initiation and propagation of inflammation, and considerable cross-talk occurs with other inflammatory pathways such as NF-κB.69 Downregulation of MapK6 in TNBS colitis is likely to represent a host-adoptive response during colitis in a failed attempt to limit or prevent the inflammatory response. IB-MECA protected against gut injury and prevented downregulation of MapK6. The iNOS/NO signaling pathway also could play a key role in the pathogenesis of IBD. In rat colitis, iNOS was the most upregulated gene product (>1000-fold by PCR). Increased iNOS mRNA or iNOS protein occurs in IBD and in human epithelial cells exposed to cytokines.70,71 In contrast, the inducible Hmox1 is believed to act as a protective antioxidant mechanism. Hmox1 converts heme into biliverdin, CO, free ferrous iron, and eventually to bilirubin and bile pigments. These actions provide a potent antioxidant system that limits both ROS- and lipid peroxidation-associated damage of tissues and anticomplement effects.72,73 Hmox1 is essential for iron homeostasis. Heme and NO donors are potent inducers of Hmox1 in the human intestinal epithelial cells,70 suggesting potential interactions in IBD. In rat colitis, the remarkable upregulation of Hmox1 may result from the upregulated iNOS expression and elevation of NO to activate the pathway. Ferritin sequestration of ferrous iron reduces its availability to catalyze lipid peroxidation and oxygen radical formation.74 Iron also regulates NO synthase gene expression.60 The overexpression of Hmox1 in TNBS colitis may be involved in immunomodulation, host defense, antioxidant, and neuroprotective mechanisms.60 Conclusions Validated gene microarray analysis established patterns of gene dysregulation in rat TNBS colitis that are largely consistent with IBD. We conclude that our validated high-density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine-based or other drugs in experimental colitis. The ADOA3R is a new potential therapeutic target for IBD that selectively modulates certain purine receptor genes altered by colitis. These included upregulations (≤20-fold in mRNA levels) in ADOA1R, ADOA3R, P2X1R, P2X4R, P2X7R, P2Y2R, and P2Y6R receptor mRNA with obvious implications for altering gut reflexes in colitis. Other purinergic receptors such as P2X2R, P2Y1R, and P2Y4R that were downregulated were insensitive to the ADOA3R agonist. Oral administration of an ADOA3R agonist is effective in protecting the intestine from experimental colitis. 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TI - ADOA3R as a Therapeutic Target in Experimental Colitis: Proof by Validated High-density Oligonucleotide Microarray Analysis
JF - Inflammatory Bowel Diseases
DO - 10.1097/00054725-200608000-00014
DA - 2006-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/adoa3r-as-a-therapeutic-target-in-experimental-colitis-proof-by-s03N0IL0QR
SP - 766
EP - 789
VL - 12
IS - 8
DP - DeepDyve
ER -