Local administration of transcription factor decoy oligonucleotides to nuclear factor-κB prevents carrageenin-induced inflammation in rat hind paw

Abstract

The transcription factor nuclear factor-κB (NF-κB) plays a key role in the expression of several genes involved in the inflammatory process. In the present study we investigated in an acute model of inflammation, the carrageenin-induced hind paw edema, the anti-inflammatory effect of double stranded oligodeoxynucleotides (ODN) with consensus nuclear factor-κB (NF-κB) sequence as transcription factor decoys (TFD) to inhibit NF-κB binding to native DNA sites. Local administration of wild-type, but not mutant-ODN decoy, dose-dependently inhibited edema formation induced by carrageenin in rat paw. Molecular analysis performed on soft tissue obtained from inflamed paw demonstrated: (1) an inhibition of NF-κB DNA binding activity; (2) a decreased nuclear level of p50 and p65 NF-κB subunits; (3) an inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expression, two inflammatory enzymes transcriptionally controlled by NF-κB. Furthermore, SN-50, a cell-permeable peptide capable of inhibiting the nuclear translocation of NF-κB complexes, exhibited a similar profile of activity of ODN decoy. Our results indicate for the first time that ODN decoy, acting as an in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent a novel strategy to modulate acute inflammation.

Introduction

Acute inflammation depends on the release of several mediators, which bring about edema formation as a result of extravasation of fluid and proteins from the local microvasculature and accumulation of polymorphonuclear leukocytes (PMNs) at the inflammatory site.1 Rat paw edema induced by carrageenin, an experimental model of acute inflammation, is characterized by an early phase (0–1 h) brought about by the release of histamine, 5-hydroxytriptamine and bradykinin followed by a late phase (1–6 h) mainly sustained by prostaglandins (PGs) and nitric oxide (NO).234 It has been demonstrated that both PGs and NO released at inflammatory site are generated by the inducible isoforms of cyclooxygenase (COX-2) and nitric oxide synthase (iNOS), respectively.34567891011 The promoter region of COX-2121314 and iNOS genes151617 has been cloned and sequenced. These promoter regions contain at least one putative NF-κB consensus sequence that has been shown to act as positive regulatory element for both COX-218 and iNOS19 transcription. NF-κB is a member of the Rel family proteins and is typically a heterodimer of p50 and p65 subunit.2021 Each member of this family contains a conserved N-terminal region called the Rel-homology domain (RHD) within which lies the DNA-binding and dimerization domains and the nuclear localisation sequences (NLS). In quiescent cells, NF-κB resides in the cytosol in latent form bound to inhibitory proteins, IκBs. These proteins also comprise a structurally and functionally related family of molecules all containing multiple copies of a 30–33 amino acid sequence, called ankyrin repeats. The specific interaction between the ankyrin repeats and the RHD is the defining feature of the association between NF-κB and IκB by which, IκB molecules, mask the NLS of NF-κB and prevent its nuclear translocation.2223 Stimulation of different types of cells with lipopolysaccharide, cytokines or oxidants triggers a series of signalling events that ultimately converge to the activation of one or more redox-sensitive kinases which specifically phosphorylate IκB, resulting in IκB polyubiquinitation and subsequent degradation.24 Once activated, the liberated NF-κB translocates into the nucleus and stimulates transcription by binding to cognate κB sites in the promoter regions of various target genes such as cytokines, chemokines, cell adhesion molecules and inflammatory enzymes involved in the immune and inflammatory response by controlling leukocyte trafficking and activation.25 We have recently shown that NF-κB is activated in carrageenin-induced rat pleurisy and its inhibition by pyrrolidine dithiocarbamate, an antioxidant inhibitor of NF-κB.26 was associated with a reduction of both exudate formation and leukocyte infiltration.2728 Several studies on the activation pathway of NF-κB led to the discovery of new molecules capable of blocking the transcriptional activity of this transcription factor.29 The decoy strategy has recently been developed30 and is considered a useful tool for analyzing the blockade of the expression of a wide variety of NF-κB-dependent pro-inflammatory mediators.3132 Recent evidence has demonstrated that synthetic double stranded ODN as decoy cis elements block the binding of NF-κB to promoter regions of its targeted genes, resulting in the inhibition of gene transactivation in vitro333435 and in vivo.3132 An alternative approach utilizes a cell-permeable peptide, called SN-50, carrying a functional domain NLS capable of inhibiting the nuclear translocation of NF-κB complexes.36 In the present study we investigated the anti-inflammatory effect of double-stranded ODN decoy to NF-κB on paw edema formation induced by carrageenin in the rat. We provide evidence that ODN decoy to NF-κB inhibits inflammatory reaction and reduces both COX-2 and iNOS protein expression.

Results

Effect of ODN decoy and SN50 on carrageenin-induced paw edema

In preliminary experiments we established that injection into the rat paw of test agents at doses used in this study did not produce any detectable edema (data not shown). Carrageenin injection caused a time-dependent increase of paw volume with a peak occurring at 4 h (Figure 1a). In carrageenin-treated animals (control group) the average of paw volume was 1.59 ± 0.11 ml (n = 8), whereas in saline-treated rats was 0.12 ± 0.04 ml (n = 8). Coinjection of wild-type ODN decoy (3, 10 and 30 μg per paw) inhibited edema formation significantly and in a dose-related fashion (by 8%, n = 5; 40%, P < 0.0001, n = 6; and 60%, P < 0.0001, n = 7, respectively). In contrast, mutant-ODN decoy (30 μg per paw; n = 6) did not show any effect (Figure 1a). SN-50 (10 μg per paw), but not mutant peptide (SN-50M, 10 μg per paw) significantly inhibited edema formation (by 56%, P < 0.0001, n = 7) (Figure 1b).

Figure 1
figure1

Effect of ODN decoy on rat carrageenin oedema. (a) Effect of wild-type ODN decoy 3 μg per paw (), 10 μg per paw () and 30 μg per paw (▪) or mutant-ODN decoy 30 μg per paw (□) on paw oedema induced by carrageenin. The oedema induced by carrageenin alone (control group) is shown by solid circles (). (b) Effect of SN50 10 μg per paw () or SN50 M 10μg per paw (□) on paw oedema induced by carrageenin. The oedema induced by carrageenin alone is shown by solid circles (•). The results are expressed as mean ± s.e.m. where n = 5–8 rats. **P < 0.001, ***P < 0.0001, versus control group (carrageenin alone).

Effect of ODN decoy and SN-50 on NF-κB activation in inflamed paw tissue

To detect NF-κB/DNA binding activity, nuclear extracts from tissue of each rat hind paw injected with carrageenin or saline were analyzed by EMSA. A low basal level of NF-κB/DNA binding activity was detected in nuclear proteins from tissue of saline-treated rats. The DNA binding activity significantly increased in nuclear extracts obtained from inflamed paw tissue of control animals after 4 h from carrageenin injection. Treatment of rats with wild-type ODN decoy (3, 10 and 30 μg per paw) caused a significant dose-dependent inhibition of carrageenin-induced NF-κB/DNA binding activity (by 12%, n = 5; 35%, P < 0.001, n = 5; and 54%, P < 0.0001, n = 6, respectively) (Figure 2). The administration of SN-50 (10 μg per paw) also significantly suppressed NF-κB/DNA binding activity (by 34%, P < 0.001, n = 5). In contrast, both mutant-ODN decoy (30 μg per paw, n = 6) and SN-50M (10 μg per paw, n = 6) did not modify carrageenin-induced NF-κB/DNA binding activity compared with control animals (Figure 2).

Figure 2
figure2

Effect of ODN decoy and SN 50 on NF-κB/DNA binding activity in rat carrageenin paw oedema. Representative EMSA of NF-κB (a) as well as the densitometric analysis (b) show the effect of wild-type (wt) (3, 10 and 30 μg per paw) or mutant (mut) (30 μg per paw) ODN decoy and SN50 or SN50M (10 μg per paw) on NF-κB/DNA binding activity evaluated in soft tissue from rat hind paw 4 h after injection of carrageenin. EMSA in panel a is representative of one paw out of 5–6 analyzed. The results in panel b are expressed as mean ± s.e.m. from 5–6 paws. **P < 0.001; ***P < 0.0001 versus control group (carrageenin alone).

Characterization of NF-κB complex induced by carrageenin in rat paw

The composition of the NF-κB complex activated by carrageenin was determined by competition and supershift experiments (Figure 3). The specificity of NF-κB/DNA binding complex was demonstrated by the complete displacement of the NF-κB/DNA binding in the presence of a 50-fold molar excess of unlabeled NF-κB probe (WT 50 ×) in the competition reaction. In contrast a 50-fold molar excess of unlabeled mutated NF-κB probe (Mut 50 ×) or Sp-1 oligonucleotide (Sp-1 50 ×) had no effect on this DNA-binding activity. The composition of the NF-κB complex activated by carrageenin was determined by using specific antibodies against p50 (p50), p65 (p65) and c-Rel (c-Rel) subunits of NF-κB proteins. Addition of either anti-p50 or anti-p65 or anti-p50 + anti-p65, but not anti-c-Rel to the binding reaction resulted in a marked reduction of NF-κB band intensity.

Figure 3
figure3

Identification of carrageenin-induced NF-κB/DNA binding proteins in rat paw oedema. Nuclear extracts from carrageenin-injected rat hind paw were prepared as described in Materials and methods and incubated with 32P-labeled NF-κB probe. In competition reaction nuclear extracts were incubated with radiolabeled NF-κB probe in the absence or presence of identical but unlabeled oligonucleotides (WT 50 ×), mutated non-functional κB probe (Mut 50 ×) or unlabeled oligonucleotide containing the consensus sequence for Sp-1 (Sp-1 50 ×). In supershift experiments nuclear extracts were incubated with antibodies against p50 (p50), p65 (p65), p50 + p65 (p50 + p65) or c-Rel (c-Rel) 30 min before incubation with radiolabeled NF-κB probe. Data illustrated are from a single experiment and are representative of three separate experiments.

Effect of ODN decoy and SN-50 on p50 and p65 nuclear level in inflamed paw tissue

The level of p50 and p65 in nuclear extracts from tissue of carrageenin or saline- treated rats was examined by immunoblotting analysis. In carrageenin-treated animals nuclear level of p50 and p65 NF-κB subunits were increased as compared with saline-treated rats (Figures 4 and 5, respectively). Administration of wild-type ODN decoy (3, 10 and 30 μg per paw) reduced both p50 and p65 band intensity in a dose-dependent manner (by 7%, n = 5; 40%, P < 0.001, n = 5; and 70%, P < 0.0001, n = 6 for p50 and by 6%, n = 5; 38%, P < 0.001, n = 5; and 72%, P < 0.0001, n = 6 for p65, respectively). SN-50 (10 μg per paw) also caused a significant decrease of both p50 and p65 nuclear level (by 60%, P < 0.0001, n = 5). In contrast, both mutant-ODN decoy (30 μg per paw; n = 6) and SN-50M (10 μg per paw; n = 6) had no effect on carrageenin-induced increase of p50 and p65 nuclear level as compared with that of the control group.

Figure 4
figure4

Effect of ODN decoy and SN50 on p50 nuclear translocation in rat carrageenin paw oedema. Representative Western blot of p50 (a) as well as the densitometric analysis (b) shows the effect of wild-type (wt) (3, 10 and 30 μg per paw) or mutant (mut) (30 μg per paw) ODN decoy and SN50 or SN50M (10μg per paw) on p50 nuclear level evaluated in soft tissue from rat hind paw 4 h after injection of carrageenin. Immunoblotting in panel a is representative of one paw out of 5–6 analyzed. The results in panel b are expressed as mean ± s.e.m. from 5–6 paws. **P < 0.001, ***P < 0.0001 versus control group (carrageenin alone).

Figure 5
figure5

Effect of ODN decoy and SN50 on p65 nuclear translocation in rat carrageenin paw oedema. Representative Western blot of p65 (a) as well as the densitometric analysis (b) shows the effect of wild-type (wt) (3, 10 and 30 μg per paw) or mutant (mut) (30 μg per paw) ODN decoy and SN50 or SN50M (10 μg per paw) on p65 nuclear level evaluated in soft tissue from rat hind paw 4 h after injection of carrageenin. Immunoblotting in panel a is representative of one paw out of 5–6 analyzed. The results in panel b are expressed as mean ± s.e.m. from 5–6 paws. **P < 0.001, ***P < 0.0001 versus control group (carrageenin alone).

Effect of ODN decoy and SN-50 on COX-2 and iNOS protein expression in inflamed paw tissue

COX-2 and iNOS protein levels in cytosolic extracts from rat paw tissue were determined by immunoprecipitation and Western blot analysis. As shown in Figures 6 and 7 a low basal level of COX-2 and iNOS protein was detected in cytosolic extracts from tissue of saline-treated rats. Conversely, carrageenin injection caused the appearance of both COX-2 and iNOS bands, the intensity of which was reduced in a dose-dependent manner by treatment with wild-type ODN decoy (3, 10 and 30 μg per paw) (by 4%, n = 5; 33%, P < 0.05, n = 5; and 64%, P < 0.0001, n = 6 for COX-2 and by 3%, n = 5; 24%, P < 0.05, n = 5; and 49%, P < 0.0001, n = 6 for iNOS, respectively). SN-50 (10 μg per paw) also determined a significant decrease of COX-2 and iNOS protein expression (by 64% and 39%, P < 0.0001, n = 6 respectively). Both mutant-ODN decoy (30 μg per paw) and SN-50M (10 μg per paw) failed to inhibit carrageenin-induced COX-2 and iNOS protein expression compared with control animals.

Figure 6
figure6

Effect of ODN decoy and SN50 on COX-2 protein expression in rat carrageenin paw oedema. Representative Western blot of COX-2 (a) as well as the densitometric analysis (b) shows the effect of wild-type (wt) (3, 10 and 30 μg per paw) or mutant (mut) (30 μg per paw) ODN decoy and SN50 or SN50M (10 μg per paw) on COX-2 protein expression evaluated in soft tissue from rat hind paw 4 h after injection of carrageenin. Immunoblotting in panel a is representative of one paw out of 5–6 analyzed. The results in panel b are expressed as mean ± s.e.m. from 5–6 paws. *P < 0.05, ***P < 0.0001 versus control group (carrageenin alone).

Figure 7
figure7

Effect of ODN decoy and SN50 on iNOS protein expression in rat carrageenin paw oedema. Representative Western blot of iNOS (a) as well as the densitometric analysis (b) shows the effect of wild-type (wt) (3, 10 and 30 μg per paw) or mutant (mut) (30 μg per paw) ODN decoy and SN50 or SN50M (10 μg per paw) on iNOS protein expression evaluated in soft tissue from rat hind paw 4 h after injection of carrageenin. Immunoblotting in panel a is representative of one paw out of 5–6 analyzed. The results in panel b are expressed as mean ± s.e.m. from 5–6 paws. *P < 0.05, ***P < 0.0001 versus control group (carrageenin alone).

Discussion

Synthetic double-stranded oligodeoxynucleotides as ‘decoy’ cis elements block the binding of transcription factors to promoter regions of target genes, resulting in the inhibition of gene transactivation in vitro and in vivo.30 Recently, a few studies have described application of decoy strategy as in vivo gene therapy.313237 It has also been shown that administration of NF-κB ODNs reduced the severity of chronic inflammatory reactions such as streptococcal cell wall- and collagen-induced arthritis in rats.3839 In the present study we report a novel experimental approach to reduce the acute inflammatory response induced by carrageenin in rat paw by using in vivo administration of a decoy cis element to bind the transcription factor NF-κB. We have previously shown NF-κB activation in a model of acute inflammation, the rat carrageenin-induced pleurisy.2728 Here, we demonstrate that carrageenin injection into rat hind paw determines the appearance of NF-κB proteins in the nuclear extracts of paw homogenates as demonstrated by EMSA and Western blot studies. Coinjection of double stranded ODN as decoy, by competitively inhibiting binding of NF-κB to native DNA sequence, reduces carrageenin-induced paw edema in a dose-dependent manner. The specificity of the anti-inflammatory effect of ODN decoy to NF-κB is supported by the following evidence: (1) the reduction of paw swelling is strictly dependent on the reduction of NF-κB/DNA binding activity suggesting a tight correlation between edema formation and DNA binding activity; (2) ODN decoy with a mutated NF-κB consensus sequence does not affect carrageenin-induced edema formation; (3) SN50, a peptide capable of inhibiting NF-κB nuclear translocation,36 shows the same profile of activity of ODN decoy confirming the hypothesis that these compounds may act as anti-inflammatory agents by blocking NF-κB nuclear translocation. Carrageenin-induced paw edema is a model of non-immune acute inflammatory reaction.40 The initial phase of edema (0–1 h) has been attributed to the release of histamine, 5-hydroxytriptamine and bradykinin.2 In contrast, the second phase of swelling (1–6 h), which is inhibited by nonsteroidal anti-inflammatory drugs, has been correlated with the elevated production of PGs and NO following the induction of COX-2 and iNOS protein expression in activated leukocytes infiltrated into the carrageenin-injected rat paw.23456 Since the discovery of the involvement of iNOS and COX-2 in inflammation,345678910 molecules endowed with the ability of interfering with the expression of these two enzymes have attracted great interest. Moreover, a growing body of evidence has demonstrated that many of the clinically important anti-inflammatory agents, including salicylates41 and glucocorticoids,424344 share the ability to inhibit NF-κB activation and therefore a large variety of inflammatory genes, amongst these COX-24546 and iNOS.4748 Recent studies have demonstrated in vitro the efficacy of TFD strategy in suppressing iNOS48 and COX-2.3435 expression. In this study we demonstrate, for the first time, that in vivo ODNs decoy to NF-κB reduce the expression of iNOS and COX-2 and, consequently, the excessive production of NO and PGs in an acute inflammatory model. The mechanism of NF-κB activation may depend on the release of several mediators, including reactive oxygen species which appear to play a central role in the inflammatory response.49 Recent study has shown that the removal of O2, by recombinant human Cu/Zn superoxide dismutase coupled to polyethyleneglycol, significantly inhibited carrageenin-induced rat paw edema.4 Such reactive oxygen species may act as intracellular second messengers via activation of the inactive cytoplasmic form of NF-κB by the release of the inhibitory subunit IκBα.50 N-acetyl-L-cysteine and pyrrolidine dithiocarbamate have been shown to inhibit NF-κB activation in vitro and in vivo and subsequently the induction of pro-inflammatory cytokines and intracellular adhesion molecule-1.5152 Therefore, the inhibition of carrageenin-induced edema formation by ODN decoy may also depend on the reduced expression of cell adhesion molecules or cytokines through a NF-κB-dependent mechanism. Although a number of important issues, such as safety and side-effects, has not been addressed in this study, the decoy strategy against NF-κB may provide a new approach alternative to the classical nonsteroidal anti-inflammatory drugs since these compounds, acting as NF-κB ‘trap’ molecules, are able to suppress COX-2 and iNOS expression simultaneously. These results suggest that transcription factor decoy strategy, by blocking NF-κB activation and expression of NF-κB-dependent pro-inflammatory genes, may be useful for controlling the inflammatory process.

Materials and methods

Animals

Male Wistar rats (Harlan, Correzzana, Italy), weighing 140–160 g, were used in all experiments. Animals were provided with food and water ad libitum. The light cycle was automatically controlled (on 07 h 00 min; off 19 h 00 min) and the room temperature thermostatically regulated to 22 ± 1°C. Before the experiments, animals were housed in these conditions for 3–4 days to become acclimatised. Animal care was in accordance with Italian and European regulations on protection of animals used for experimental and other scientific purposes.

Transcription factors decoy oligonucleotides

Plain double-stranded ODN decoy to NF-κB was prepared by annealing of sense and antisense oligonucleotides in vitro in 1x annealing buffer (20 mM Tris-HCl, pH 7.5, 20 mM MgCl2 and 50 mM NaCl). The mixture was heated at 100°C for 12 min and allowed to cool to room temperature slowly over 18 h.

The sequence of ODN decoy to NF-κB used was:

  1. 1

    Wild-type NF-κB consensus sequence: 5′-GAT CGA GGG GAC TTT CCC TAG C-3′; 3′-CTA GCT CCC CTG AAA GGG ATC G-5′.

  2. 2

    Mutant-NF-κB consensus sequence with a mutation of the bolded bases (GGAC to AAGC) of wild-type NF-κB consensus sequence: 5′-GAT CGA GGA AGC TTT CCC TAG C-3′; 3′-CTA GCT CCT TCG AAA GGG ATC G-5′.

Paw edema

Paw edema was induced by subplantar injection into the right hind paw of 0.1 ml sterile saline containing 1% λ-carrageenin in the presence or absence (control group) of wild-type ODN decoy (3–10–30 μg per paw), mutant-ODN decoy (30 μg per paw), SN-50 (10 μg per paw) and SN-50M (10 μg per paw). The volume of the paw was measured by a plethysmometer (Basile, Milan, Italy) immediately after the injection as previously described.2 Subsequent readings of the same paw were carried out at 1 h intervals up to 6 h and compared with the initial readings. The increase in paw volume was taken as edema volume. In some experiments the rats were killed in an atmosphere of CO2 immediately after the readings at 4 h and soft tissue from each inflamed paws was recovered by scalpel, immediately and separately processed to obtain cytosolic and nuclear extracts (see below).

Cytosolic and nuclear extracts

Soft tissue from each paw removed 4 h after carrageenin or saline injections, was cut and processed as previously described53 with some modification. Briefly, inflamed tissue was frozen in liquid nitrogen, immediately suspended in 6 ml of ice-cold ipotonic lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulphonylfluoride, 1.5 μg/ml soybean trypsin inhibitor, 7 μg/ml pepstatin A, 5 μg/ml leupeptin, 0.1 mM benzamidine, 0.5 mM dithiothreitol) and homogenised at the highest setting for 2 min in a Polytron PT 300 tissue homogenizer. The homogenates were divided in three aliquots of 2 ml, chilled on ice for 15 min and then vigorously shaken for another 15 min in the presence of 20 μl of 10% Nonidet P-40. The nuclear fraction was precipitated by centrifugation at 1500 g for 5 min, the supernatant containing the cytosolic fraction was removed and stored at −80°C. The nuclear pellet was resuspended in 700 μl of high salt extraction buffer (20 mM pH 7.9 HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% v/v glycerol, 0.5 mM phenylmethylsulphonylfluoride, 1.5 μg/ml soybean trypsin inhibitor, 7 μg/ml pepstatin A, 5 μg/ml leupeptin, 0.1 mM benzamidine, 0.5 mM dithiothreitol) and incubated with shaking at 4°C for 30 min. The nuclear extract was then centrifuged for 15 min at 13000 g and supernatant was aliquoted and stored at −80°C. Protein concentration was determined by the BioRad (Milan, Italy) protein assay kit.

Electrophoretic mobility shift assay (EMSA)

A double-stranded oligonucleotides containing the NF-κB recognition sequence (5′-CCA ACT GGG GAC TCT CCC TTT G-3′) were end-labeled with 32P-γ-ATP. Nuclear extracts (40 μg) from each paw were incubated for 30 min with radiolabeled oligonucleotides (2.5–5.0 × 104 c.p.m.) in 20 μl reaction buffer containing 2 μg poly dI-dC, 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin, 10% (v/v) glycerol. The specificity of the DNA/protein binding was determined by competition reaction in which a 50-fold molar excess of unlabeled wild-type, mutant or Sp-1 oligonucleotide was added to the binding reaction 10 min before addition of radiolabeled probe. In supershift assay, antibodies reactive to p50, p65 or c-Rel proteins were added to the reaction mixture 30 min before the addition of radiolabeled NF-κB probe. Nuclear protein–oligonucleotide complexes were resolved by electrophoresis on a 6% nondenaturing polyacrylamide gel in 1 × Tris borate EDTA buffer at 150 V for 2 h at 4°C. The gel was dried and autoradiographed with an intensifying screen at −80°C for 20 h. Subsequently, the relative bands were quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (BioRad) and a computer program (Molecular analyst, IBM, Milan, Italy).

Immunoprecipitation and Western blot analysis

The level of p50 and p65 and the expression of COX-2 and iNOS were quantified in nuclear and cytosolic extracts, respectively, by immunoprecipitation followed by Western blot analysis according to the manufacturer's instructions (Santa Cruz, Milan, Italy). Briefly, protein concentration was determined and equivalent amounts (200 μg) for each sample were mixed 40 μl of protein A-sepharose and 2 μl of anti-p50, anti-p65, anti-COX-2 or anti-iNOS polyclonal antibodies and left overnight at 4°C with continuous shaking. Immunocomplexes were washed three times with 500 μl of buffer A (10 mM TRIS-HCl pH 7.5, 1 M NaCl, 0.2% Triton-X 100 and 2 mM EDTA), mixed with 40 μl of gel loading buffer (50 mM Tris/10% SDS/10% glycerol/10% 2-mercaptoetanol/2 mg of bromophenol per ml) and then boiled for 3 min. Samples obtained were electrophoresed in a 12% discontinuous polyacrylamide minigel. The proteins were transferred on to nitrocellulose membranes, according to the manufacturer's instructions (BioRad). The membranes were saturated by incubation at 4°C overnight with 10% non-fat dry milk in PBS and then incubated with anti-p50, anti-p65, anti-COX-2 or anti-iNOS antibodies for 1 h at room temperature. The membranes were washed three times with 1% Triton-X 100 in PBS and then incubated with anti-rabbit or anti-goat immunoglobulins coupled to peroxidase. The immunocomplexes were visualized by the ECL chemiluminescence method (Amersham, Milan, Italy). Subsequently, the relative bands were quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (BioRad) and a computer program (Molecular analyst, IBM).

Statistics

Data are expressed as mean ± s.e.m. of n rats. Statistical significance was calculated by one way analysis of variance (ANOVA) and Bonferroni-corrected P value for multiple comparison test. The level of statistically significant difference was defined as P < 0.05.

Reagents

Phosphate-buffered saline was from Celbio (Milan, Italy). DL-dithiothreitol, phenylmethylsulfonilfluoride, soybean trypsin inhibitor, pepstatin A, leupeptin and benzamidine were from Calbiochem (Milan, Italy). 32P-γ-ATP was from ICN Biomedicals (Milan, Italy). Poly dI-dC was from Boehringer Mannheim (Milan, Italy). p50, p65, c-Rel, anti-COX-2 or anti-iNOS-specific anti-peptide antibodies were from Santa Cruz. Non-fat dry milk was from BioRad. Oligonucleotide synthesis was performed to our specifications by Tib Molbiol, Boehringer Mannheim (Genova, Italy). All other reagents were from Sigma (Milan, Italy).

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Acknowledgements

This work was supported in part by MURST 40% and 60% research grant 1997 and 1998.

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Correspondence to R Carnuccio.

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D'Acquisto, F., Ialenti, A., Ianaro, A. et al. Local administration of transcription factor decoy oligonucleotides to nuclear factor-κB prevents carrageenin-induced inflammation in rat hind paw. Gene Ther 7, 1731–1737 (2000). https://doi.org/10.1038/sj.gt.3301295

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Keywords

  • acute inflammation
  • cyclooxygenase-2
  • inducible nitric oxide synthase
  • transcription factor decoy
  • nuclear factor-κB

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