Protective effects of N-acetylcysteine on acetic acid-induced colitis in a porcine model
© Wang et al.; licensee BioMed Central Ltd. 2013
Received: 30 March 2013
Accepted: 24 August 2013
Published: 30 August 2013
Ulcerative colitis is a chronic inflammatory disease and involves multiple etiological factors. Acetic acid (AA)-induced colitis is a reproducible and simple model, sharing many characteristics with human colitis. N-acetylcysteine (NAC) has been widely used as an antioxidant in vivo and in vitro. NAC can affect several signaling pathways involving in apoptosis, angiogenesis, cell growth and arrest, redox-regulated gene expression, and inflammatory response. Therefore, NAC may not only protect against the direct injurious effects of oxidants, but also beneficially alter inflammatory events in colitis. This study was conducted to investigate whether NAC could alleviate the AA-induced colitis in a porcine model.
Weaned piglets were used to investigate the effects of NAC on AA-induced colitis. Severity of colitis was evaluated by colon histomorphology measurements, histopathology scores, tissue myeloperoxidase activity, as well as concentrations of malondialdehyde and pro-inflammatory mediators in the plasma and colon. The protective role of NAC was assessed by measurements of antioxidant status, growth modulator, cell apoptosis, and tight junction proteins. Abundances of caspase-3 and claudin-1 proteins in colonic mucosae were determined by the Western blot method. Epidermal growth factor receptor, amphiregulin, tumor necrosis factor-alpha (TNF-α), and toll-like receptor 4 (TLR4) mRNA levels in colonic mucosae were quantified using the real-time fluorescent quantitative PCR.
Compared with the control group, AA treatment increased (P < 0.05) the histopathology scores, intraepithelial lymphocyte (IEL) numbers and density in the colon, myeloperoxidase activity, the concentrations of malondialdehyde and pro-inflammatory mediators in the plasma and colon, while reducing (P < 0.05) goblet cell numbers and the protein/DNA ratio in the colonic mucosa. These adverse effects of AA were partially ameliorated (P < 0.05) by dietary supplementation with NAC. In addition, NAC prevented the AA-induced increase in caspase-3 protein, while stimulating claudin-1 protein expression in the colonic mucosa. Moreover, NAC enhanced mRNA levels for epidermal growth factor and amphiregulin in the colonic mucosa.
Dietary supplementation with NAC can alleviate AA-induced colitis in a porcine model through regulating anti-oxidative responses, cell apoptosis, and EGF gene expression.
KeywordsN-acetylcysteine Acetic acid Colon injury Claudin-1 Epidermal growth factor Amphiregulin
N-acetylcysteine (NAC), the precursor of L-cysteine and therefore reduced glutathione, has been widely used as an antioxidant in vivo and in vitro . NAC is rapidly metabolized by the small intestine to produce glutathione  and is usually not detectable in the plasma or tissues of pigs receiving no NAC supplementation . Previous studies have shown the protective effect of NAC against the toxicity of chemicals due to its dual role as a nucleophile and as a -SH donor . Specifically, NAC acts as a direct ROS scavenger to regulate the redox status and also affects several signaling pathways involved in apoptosis, angiogenesis, cell growth and arrest, redox-regulated gene expression, and inflammatory response [5, 6]. Moreover, NAC exerts an indirect antioxidant effect through the synthesis of glutathione, a primary intracellular factor against toxic agents . Therefore, NAC may not only protect against the direct injurious effects of oxidants, but also beneficially alter inflammatory events .
Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are complex disorders characterized by chronic, local, and systemic inflammation as well as a spontaneously relapsing course . Ulcerative colitis is a chronic inflammatory disease  and involves multiple etiological factors [11, 12]. A large body of evidence suggests that oxidant derivatives and reactive oxygen species (ROS) are produced in excess by the inflamed mucosa and may be pathogenic factors in IBD [13, 14]. Oxidative stress have an important bearing on inflammation via the activation of redox-sensitive transcriptional factors such as nuclear factor kB (NF-kB) and activator protein 1, which regulate expression of key genes encoding pro-inflammatory mediators and protective antioxidant proteins. In support of this view, pharmacological agents that lower the amounts of reactive oxygen metabolites may reduce inflammation . Many animal models of IBD have been developed to study its pathogenesis and therapeutic means . Acetic acid (AA)-induced colitis is a reproducible and simple model, sharing many characteristics with human colitis [11, 16].
In the intestinal tract, energy status is a fundamental regulator of epithelial cell metabolism . An energy deficit has been considered to be a pathogenic factor in ulcerative colitis, which is substantiated by the fact that the intestinal mucosa has a limited capacity for de novo synthesis of purine nucleotides  and is more prone to reduced ATP concentrations compared with the liver or muscle . On the other hand, IL-6 and tumor necrosis factor α (TNF-α) has been shown to play an important role in the pathogenesis of inflammatory bowel disease . These pro-inflammatory cytokines drive the activation and recruitment of inflammatory cells, amplify the production of other pro-inflammatory cytokines, and activate nuclear transcription factors, thereby promoting and maintaining the inflammatory response . Additionally, release of transforming growth factor-α (TGF-α) and expression of TGF-α mRNA are increased after acute gastric injury and in the colonic mucosa from patients with IBD [21, 22].
Neonates are prone to various stresses, such as early-weaning, inflammatory bowel disease, and infection, resulting in intestinal mucosal injury and absorptive dysfunction [23–25]. However, effective prevention and treatments are currently limited . Many nutrients (vitamin E, selenium and trimetazidine) have been investigated as possible agents to protect animals against the IBD. Dietary supplementation with vitamin E and selenium reduced both the severity of colonic lesions and the levels of malondialdehyde (MDA) [27, 28]. Likewise, intraperitoneal administration of trimetazidine improved macroscopic and microscopic scores and decreased colonic myeloperoxidase (MPO) activity in rats receiving administration of AA . In previous studies, we have reported that NAC reduced inflammation, alleviated oxidative stress, improved energy status, and ameliorate tissue damage in the small intestine of piglets [2, 30]. Thus, we postulated that dietary supplementation with NAC may alleviate the AA-induced colonic injury in piglets. The purpose of the present study was to test this hypothesis and to elucidate the underlying molecular mechanisms. As the piglet is a well-established animal model for studying human gastrointestinal disease, findings of this study will provide vital clues for prevention of human colitis.
Animal care and diets
The animal use protocol for this research was approved by the Animal Care and Use Committee of Hubei Province. Eighteen healthy crossbred female piglets (Duroc × Landrace × Yorkshire), which were reared by sows, were weaned at 21 days of age. After a 7-day period of adaptation, piglets (average body weight of 6.44 ± 0.39 kg) were housed individually in stainless steel metabolic cages (1.20 × 1.10 m2) and maintained in an environmentally controlled room (25°C) by air conditioning, with electric light being provided between 8:00 AM and 8:00 PM . Each cage was equipped with a feeder and a nipple waterer to allow piglets free access to food and drinking water [26, 31–33]. The corn- and soybean meal-based diet was formulated to meet National Research Council (NRC 1998) requirements for all nutrients .
In the first week, all weanling piglets had free access to the basal diet to help them adapt to solid food. Then, eighteen healthy piglets were allocated randomly into one of the three treatments: 1) control group (piglets fed the basal diet and receiving intrarectal administration of 10 mL of sterile saline); 2) AA group (piglets fed the basal diet and receiving intrarectal administration of 10 mL of 10% AA); 3) NAC group (piglets fed the basal diet supplemented with 500 mg/kg NAC and receiving intrarectal administration of 10 mL of 10% AA). NAC (powder) was well mixed with the basal diet. Diets for the control and AA groups were supplemented with 500 mg/kg cornstarch to obtain approximately isocaloric diets. The dosage of NAC was chosen according to the results of our previous study indicating that dietary supplementation with 500 mg/kg NAC could ameliorate growth depression and restore intestinal function in weanling piglets [2, 30]. It is unnecessary to use non-essential amino acids as an isonitrogenous control because the dietary supplementation with 500 mg/kg NAC only resulted in an increase of 0.0042% nitrogen. On day 15 of the trial, piglets in the AA and NAC groups received intrarectal administration of 10 mL of 10% AA, whereas the control group piglets received the same volume of saline. The dosage of AA was chosen according to the studies of Jurjus et al. . During days 0–15 of the trial (pre-challenge), all the piglets had free access to feed and drinking water. To exclude a possible effect of AA-induced reduction in food intake on the piglet intestine, the control and NAC piglets were pair-fed the same amount of feed per kg body weight as AA piglets during days 15–21 of the trial (post-challenge with AA). On day 22 of the trial, all piglets were sacrificed by injection of sodium pentobarbital (50 mg/kg BW) to obtain the colonic mucosa for the evaluation of intestinal morphology and biochemical analysis .
Blood sample collection
On day 22 of the trial, blood samples were collected from the anterior vena cava into heparinized vacuum tubes (Becton Dickinson Vacutainer System, Franklin Lake, NJ, USA), as we previously described . Blood samples were centrifuged at 3,000 rpm for 10 min at 4°C to obtain plasma [26, 35]. Plasma was stored at −80°C until analysis.
Intestinal sample collection
The piglet abdomen was surgically opened immediately from the sternum to the pubis, and then the whole gastrointestinal tract was immediately exposed [26, 36]. The large intestine, dissected free of the mesentery, was placed on a chilled stainless steel tray. Colon segments (5- to 10-cm) were obtained, flushed gently with ice-cold phosphate buffered saline (PBS, pH 7.4), and placed in 10% fresh chilled formalin solution for histological measurements [26, 34]. Additional colon segments were opened longitudinally and the contents were flushed with ice-cold PBS [26, 37]. Thereafter, the mucosa was collected by scraping using a sterile glass microscope slide at 4°C [26, 38], rapidly frozen in liquid nitrogen, and stored at −80°C until analysis. All samples were collected within 15 min after sacrifice.
Histologic assessments of colonic damage
Tissue samples for the morphometric study were dehydrated and embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin . Stained sections were determined for evidence of colonic injury using the following criteria: crypt lesion, bowel wall thickening, lymphocyte infiltration, goblet cell depletion, and denuded epithelium [9, 39, 40]. The degree of damage on microscopic cross-sections of the colon was graded semi-quantitatively using a score of 0 to 20. For example, grades of crypt lesion from 0 to 4 were as follows: 0: intact crypt, 1: loss of the one-third tissue, 2: loss of the two-third tissue, 3: loss of the entire crypt, 4: erosion . The total possible score was 20 (absence of any abnormality = 0 and most severe injury = 20) . Morphometric measurements were performed with a light microscope (American Optical Co., Scientific Instrument Div., Buffalo, NY, USA). Crypt depth (the distance from the crypt mouth to the base) was measured using a linear ocular micrometer with a computer-assisted morphometric system (BioScan Optimetric, BioScan Inc., Edmonds, WA, USA). Colonic intraepithelial lymphocyte (IEL) number and goblet cell number in crypts were measured. The variables were expressed per 100 enterocytes. Measurements were taken in 10 well-oriented crypts from each intestinal section of a study animal. On the basis of the cellular morphology, differences among the nuclei of enterocytes, goblet cells, and lymphocytes were clearly distinguishable at 400× magnification. Intra-villus lamina propria cell density was determined by counting total visibly stained nuclei and total lymphocytes in 8 fields from each section using a grid ocular (Olympus, Microplanet). Cell density was expressed as the number of total stained cells or the number of lymphocytes per 1,000 μm2. The number of lymphocytes in relation to the number of total cells was also calculated. All morphometric analysis was done by the same person, who was blinded to the treatments.
Measurement of mucosal DNA, RNA, and protein
DNA, RNA, and protein were extracted from the colonic mucosa, using TRI REAGENT-RNA/DNA/Protein isolation reagent and their concentrations were determined colorimetrically, as previously described . Mucosal DNA was analyzed fluorimetrically using the method of Prasad et al. . RNA was determined by spectrophotometry using a modified Schmidt-Tannhauser method as described by Munro and Fleck . Protein was analyzed according to the method of Lowry et al. . For measurement of colonic DNA and RNA levels, the mucosa was homogenized (~2 min) in a 100-fold volume of ice-cold saline (0.9%) and the homogenate was centrifuged at 1,800 × g for 10 min at 4°C to obtain the supernatant fluid for analysis. For measurement of mucosal protein, intestinal mucosal samples (~0.1 g) were homogenized using a tissue homogenizer in 1 mL of ice-cold PBS-EDTA buffer (0.05 mol/L Na3PO4, 2.0 mol/L NaCl, 2 mmol/L EDTA, pH 7.4) and the homogenates were centrifuged at 12,000 × g for 10 min at 4°C to obtain the supernatant fluid for assays.
Assessments of antioxidant status
The colonic mucosa (~200 mg), homogenized in a nine-fold volume of freezing saline, was centrifuged at 2,500 rpm for 10 min at 4°C to obtain the supernatant fluid used for assays. Myeloperoxidase, superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in the plasma and colonic mucosa were determined using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Determination of proinflammatory cytokines in the plasma and colonic mucosae
Frozen intestinal mucosal samples were powdered under liquid nitrogen, then homogenized in ice-cold 0.9% NaCl solution using a homogenizer (1 g sample/9 mL of 0.9% NaCl). The homogenates were centrifuged at 3,000 rpm for 15 min at 4°C to obtain the supernatant fluid .
Tumor necrosis factor-α (TNF-α) in plasma was analyzed using commercially available 125I RIA kits (Beijing North Institute of Biological Technology, Beijing, China). The detection limit was 0.3 ng/mL and the intra-and inter-assay coefficients of variation were 5% and 8%, respectively.
Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) in plasma and the supernatant fluid of colonic mucosae were analyzed using commercially available 125I RIA kits (Beijing Sino-UK institute of Biological Technology, Beijing, China). The detection limits for interleukin-6 and prostaglandin E2 analyses were 50 and 0.12 pg/mL, respectively. The coefficients of variation for intra-and inter-assays of interleukin-6 were < 7% and < 15%, respectively. The coefficients of variation for intra-and inter-assays of prostaglandin E2 were < 7.5% and < 10.5%, respectively.
Determination of EGF in plasma and TGF-α in colonic mucosae
Epidermal growth factor (EGF) in plasma and transforming growth factor-α (TGF-α) in colonic mucosae were analyzed using commercially available 125I RIA kit (Beijing Sino-UK Institute of Biological Technology, Beijing, China). The coefficients of variation for intra-and inter-assay of EGF were < 5% and < 10%, respectively. The coefficients of variation for intra-and inter-assay of TGF-α were 4.4% and 7.4%, respectively. The detection limit for EGF and TGF-α were 0.1 ng/mL and < 5 pg/mL, respectively.
Protein immunoblot analysis
Analysis of caspase-3 and claudin-1 proteins in colonic mucosae were performed by western blot as described by Hou et al. . Briefly, frozen samples were powdered under liquid nitrogen and homogenized in lysis buffer. The homogenates were centrifuged at 12,000 × g for 15 min at 4°C to get the supernatant fluid. A portion of this fluid is mixed with 2 × SDS sample buffer in a 1:1 ratio. The samples were boiled for 5 min and cooled on ice before western blot analysis. The proteins (60 μg/sample for caspase-3, claudin-1 and β-actin) were separated by electrophoresis on a 10% (for caspase-3) or 12% (for claudin-1) polyacrylamide gel. Proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. Non-fat dry milk in TBS-T (1 × Tris-buffered saline including 0.1% Tween 20) was used to block membranes for at least 1 h at room temperature . Membranes were then incubated with primary antibodies overnight at 4°C: caspase-3 (rabbit polyclonal antibodies from Cell Signaling Technology, Inc., Danvers, MA, USA; dilution 1:1000), claudin-1 (rabbit monoclonal antibodies from Invitrogen Technology, Inc., Danvers, MA, USA; dilution 1:1000), β-actin (mouse monoclonal antibody from Sigma Chemicals; dilution 1:5000). The membranes were washed three times for 10 min with TBS-T and incubated for 1 h at room temperature with anti-rabbit (mouse) immunoglobulin G horseradish peroxidase-conjugated secondary antibody (Beijing ZhongShan Golden Bridge Biological Technology Co., LTD, China; dilution 1:10000) . Incubation of the secondary antibodies was followed by five washes for 8 min. Blots were developed using an Enhanced Chemiluminescence western blotting kit (ECL-plus, Amersham Biosciences, Sweden), visualized and quantified using an imaging system (Alpha Innotech FluorChem FC2, CA, USA) [2, 30].
Determination of EGFR, AR, TNF-α and TLR4 mRNA levels using quantitative real-time polymerase-chain reaction (RT-PCR)
Epidermal growth factor receptor (EGFR), Amphiregulin (AR), tumor necrosis factor-alpha (TNF-α) and toll-like receptor 4 (TLR4) mRNA levels in colonic mucosae were quantified using quantitative RT-PCR as described by Hou et al. . Approximately 100 mg of a frozen mucosal sample, powdered under liquid nitrogen using a mortar and pestle, were homogenized in a buffer and total RNA was isolated using the TRIzol Reagent protocol (Invitrogen, Carlsbad, CA, USA). Total RNA was quantified using the NanoDrop® ND-2000 UV–VIS spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at an OD of 260 nm, and the purity was assessed by determining the OD260/OD280 ratio. All of the samples had an OD260/OD280 ratio above 1.8 corresponding to 90-100% pure nucleic acids. Meanwhile, the integrity of RNA in each sample was assessed using 1% denatured agarose gel electrophoresis. RNA was used for quantitative RT-PCR analysis when the sample had a 28 S/18 S rRNA ratio ≥ 1.8 .
Primers for RT-PCR analysis
5′- GGCCTCCATGCTTTTGAGAA -3′
5′- GACGCTATGTCCAGGCCAA -3′
5′- TCCAATGGCAGAGTGGGTATG -3′
5′- AGCTGGTTGTCTTTCAGCTTCAC -3′
5′- GCCTTTCTCTCCTGCCTGAG -3′
5′- AGCTCCATGCATTGGTAACTAATG -3′
5′- GAGAAACCGTCGCCGAAT -3′
5′- GCCCACCAGGAGCAAGTT -3′
Data, expressed as means ± SD, were analyzed by one-way analysis of variance. The normality and constant variance for experimental data were tested by the Levene’s test. If data did not have homogenous variance, they underwent logarithm transformation to meet the necessary assumptions of analysis of variance . Differences among treatment means were determined by Duncan’s multiple range tests. All statistical analyses were performed using SPSS 17.0 software (Chicago, IL, USA). P values < 0.05 were taken to indicate statistical significance .
Effects of NAC supplementation on the growth performance of AA-treated piglets (between days 15 and 21 of the trial)
Average daily feed intake (g/day)
575 ± 3
576 ± 2
572 ± 4
Average daily weight gain (g/day)
301 ± 48
266 ± 69
267 ± 41
F/G (feed: gain)
1.9 ± 0.3
2.3 ± 0.7
2.2 ± 0.3
Effects of NAC supplementation on the colonic mucosal morphology of AA-treated piglets
6.3 ± 1.5a
12.5 ± 2.6b
8.3 ± 1.8a
Crypt Depth1, μm
190 ± 2.2
200 ± 3.5
Goblet cells/100 enterocytes
10.3 ± 2.2b
1.6 ± 0.5a
2.7 ± 0.8b
1.8 ± 0.9a
1.3 ± 0.4
0.9 ± 0.3
1.0 ± 0.1
1.6 ± 0.2a
2.4 ± 0.7b
1.7 ± 0.3a
Concentrations of DNA, RNA and protein in the colonic mucosa
Effects of NAC supplementation on the colonic growth of AA-induced piglets
2.93 ± 0.19
3.20 ± 0.44
2.64 ± 0.38
4.55 ± 0.78
4.20 ± 1.44
4.45 ± 1.80
22.2 ± 3.8b
18.4 ± 1.6a
24.0 ± 3.2b
5.30 ± 0.64
4.55 ± 0.66
6.01 ± 1.92
Effects of NAC on redox status
Effects of NAC on redox status in the plasma and colonic mucosa of AA-induced piglets
MPO, U/ L
136 ± 12a
172 ± 24b
150 ± 24a
SOD, U/ mL
87.3 ± 30.2
82.0 ± 13.6
77.2 ± 11.8
CAT, U/ mL
4.58 ± 1.29
3.64 ± 1.03
6.50 ± 3.19
MDA, nmol/ mg protein
5.12 ± 0.51a
6.97 ± 1.24b
5.41 ± 1.02a
MPO, U/g wet mucosa
0.071 ± 0.003a
0.095 ± 0.018b
0.063 ± 0.016a
SOD, U/mg protein
20.9 ± 1.4
18.5 ± 4.2
20.5 ± 6.0
CAT, U/mg protein
1.16 ± 0.12b
0.99 ± 0.17a
0.87 ± 0.16a
MDA, nmol/mg protein
0.33 ± 0.04a
0.58 ± 0.16b
0.41 ± 0.15a
Concentrations of inflammatory mediators in plasma and colonic mucosae, EGF in plasma and TGF-α in the colonic mucosa
Effects of NAC on proinflammatory mediators and growth modulator in the plasma and colonic mucosa
0.61 ± 0.17a
0.84 ± 0.11b
0.49 ± 0.14a
106.4 ± 23.6
115.2 ± 34.2
113.6 ± 18.3
57.9 ± 11.5
55.1 ± 13.1
51.9 ± 11.8
0.65 ± 0.08ab
0.60 ± 0.07a
0.76 ± 0.10b
134.3 ± 12.7
133.6 ± 17.2
130.8 ± 11.3
74.5 ± 3.9a
96.0 ± 14.5b
90.5 ± 15.0b
3.50 ± 0.83a
4.28 ± 0.33b
2.56 ± 0.54c
Abundance of caspase-3 and claudin-1 proteins in the colon mucosa
EGFR, AR, TNF-α and TLR4 mRNA levels in the colonic mucosa
Effects of NAC on EGFR, AR, TNF-α and TLR4 mRNA levels in the colonic mucosa
1.00 ± 0.29b
0.82 ± 0.19ab
0.61 ± 0.12a
1.00 ± 0.17a
1.28 ± 0.20a
1.58 ± 0.17b
1.00 ± 0.16b
0.61 ± 0.16a
0.60 ± 0.11a
1.00 ± 0.04
0.86 ± 0.34
0.71 ± 0.10
Emerging evidence suggests that members of the claudin-family of proteins play a critical role in tight junction formation and also affect the permeability characteristics in the gut . Although the contribution of other tight junction proteins is less clear, up-regulation of claudin-1 appears to be a common mechanism by which colonic epithelial barrier function can be maintained and/or enhanced . To extend these observations, we analyzed the relative level of claudin-1 expression in the colon mucosa. The results (Figure 2) showed that the abundance of claudin-1 protein in AA-induced piglets was decreased, when compared with the control group. Notably, dietary supplementation with NAC substantially increased the levels of claudin-1 in the colon mucosa, indicating that NAC may improve the colonic epithelial barrier function and alleviate the AA-induced mucosal damage in young pigs.
Ulcerative colitis is a chronic recurrent inflammatory bowel disease in which oxidative stress and cellular injury have been implicated [11, 57]. This is consistent with elevated levels of TNF-α in the colonic mucosa of AA-treated piglets (Table 7). NAC appears to act primarily by increasing thiol antioxidant activity , thereby minimizing oxidative stress and the downstream negative effects of the stress . MPO is an enzyme found predominantly in neutrophils and has been used as an effective quantitative index of inflammation due to a positive correlation between MPO activities and neutrophil infiltration in the colon [11, 60]. MDA is an important indicator to reflect the extent of ROS accumulation in the body in response to oxidative damage . Toxic colitic injury has been shown to increase MDA levels in rats [61, 62]. Consistent with this report, we found that NAC supplementation decreased MPO in the plasma as well as MDA and TNF-α concentrations in the colon. These findings suggest that NAC could alleviate AA-induced oxidative injury in the colonic mucosa of piglets and may have positive effects on reducing the severity of colonic inflammation.
Oxidative stress and resultant tissue damage are the hallmark of cell death. Of particular note, NAC attenuated the production of active caspase-3 in the colon of AA-induced pigs (Figure 1). Apoptosis is typically accompanied by the activation of a class of "death" proteases (caspases) . Caspase-3 stands out among the known caspases, because it is commonly activated by numerous "death" signals and cleaves a variety of important cellular proteins . Thus, this protein is either partially or totally responsible for the proteolytic cleavage of many proteins. Our results demonstrated that NAC could effectively inhibit AA-induced apoptosis and promote cell growth and survival, indicating a protective effect of NAC against AA-induced colonocyte death through inhibiting the activation of caspase-3. These findings support the notion that NAC is effective in preventing intestinal oxidative injury and inflammatory disease in neonates .
Another novel and important observation of this study is an increase in EGF concentration in the plasma of NAC-supplemented pigs (Table 6). EGF can promote proliferation, repair, and migration of epithelial cells in the small intestine during the process of regeneration after its damage [30, 65]. EGF can accelerate gastric ulcer healing by reducing bacterial colonization of the ulcer . Epithelial mRNA levels for EGFR appears to be reduced or unchanged in patients with IBD . In our porcine model of colitis, EGFR expression in the colon mucosa was not affected. Moreover, AR (a heparin-regulated growth factor) is a bifunctional growth modulator: it interacts with the EGF/TGF-α receptor to promote the growth of normal epithelial cells and inhibits the growth of certain aggressive carcinoma cell lines . The AR's mRNA level is markedly elevated in the colon of NAC-supplemented piglets. AR could facilitate colonic injury recovery via its growth-regulatory effect.
A piglet model of ulcerative colitis was successfully developed by intrarectal administration of 10 mL of 10% AA. This disorder was characterized by a deregulation of the colonic mucosal immune system along with the presence of architectural distortion and infiltration of neutrophils and macrophages. Dietary supplementation with 500 mg/kg NAC alleviated ulcerative colitic injury in AA-induced piglets. The beneficial effects of NAC were associated with the following changes: 1) alleviated colonic injury (indicated by a reduction in the AA-induced damage of the colonic structure), 2) reduced oxidative stress (indicated by decreased activities of MPO in the plasma, elevated levels of MDA in the plasma and colon), 3) reduced cell apoptosis (indicated by decreased expression of the caspase-3 protein in the colonic mucosa of AA-induced piglets), 4) enhanced recovery of the injured colon (increases in plasma EGF concentrations and colonic mucosal AR mRNA levels), and 5) enhanced formation of the tight junction (indicated by increased expression of claudin-1 proteins in the colonic mucosa of AA-induced piglets). Because AA produces colonic inflammation in rodents that resembles many histological characteristics of human ulcerative colitis , and because intestinal physiology and physiopathology are very similar between pigs and humans , our study helps to identify a beneficial role for dietary NAC supplementation as an adjuvant therapy for ulcerative colitis. Thus, findings from the porcine model may have important implications for the treatment of human intestinal disease (Crohn’s and ulcerative colitis).
Epidermal growth factor
Epidermal growth factor receptor
Ribosomal protein L4
Real-time polymerase-chain reaction
Toll-like receptor 4
Tumor necrosis factor-alpha
Transforming growth factor-α.
This research was jointly supported by National Basic Research Program of China (No. 2012CB126305), Hubei Provincial Research and Development Program (No. 2010BB023), Natural Science Foundation of Hubei Province (No. 2011CDA131), the Thousand-People Talent program at China Agricultural University, Chinese Universities Scientific Fund (2012RC024), and National Research Initiative Competitive Grants from the Animal Growth & Nutrient Utilization Program (2008-35206-18764) of the USDA National Institute of Food and Agriculture, and Texas AgriLife Research (H-82000). All these funding agencies had no role in the design, analysis or writing of this article.
- Wu G, Fang YZ, Yang S, Lupton JR, Turner ND: Glutathione metabolism and its implications for health. J Nutr. 2004, 134: 489-492.PubMedGoogle Scholar
- Hou Y, Wang L, Zhang W, Yang Z, Ding B, Zhu H, Liu Y, Qiu Y, Yin Y, Wu G: Protective effects of N-acetylcysteine on intestinal functions of piglets challenged with lipopolysaccharide. Amino Acids. 2012, 43: 1233-1242. 10.1007/s00726-011-1191-9.View ArticlePubMedGoogle Scholar
- Wu G: Amino acids: metabolism, functions, and nutrition. Amino Acids. 2009, 37: 1-17.View ArticlePubMedGoogle Scholar
- Sridharan S, Nalini R, Shyamala DCS: In vitro evaluation of the anticancer effect of N-acetylcysteine on oral carcinoma cell line. Indian J Pharmacol. 2001, 33: 343-349.Google Scholar
- Sadowska AM, Manuel-y-Keenoy B, De Backer WA: Antioxidant and anti-inflammatory efficacy of NAC in the treatment of COPD: Discordant in vitro and in vivo dose-effects. Pulm Pharmacol Ther. 2007, 20: 9-22. 10.1016/j.pupt.2005.12.007.View ArticlePubMedGoogle Scholar
- Arakawa M, Ito Y: N-acetylcysteine and neurodegenerative diseases. Basic and clinical pharmacology. Cerebellum. 2007, 6: 308-314. 10.1080/14734220601142878.View ArticlePubMedGoogle Scholar
- Dekhuijzen PNR: Antioxidant properties of N -acetylcysteine: their relevance in relation to chronic obstructive pulmonary disease. Eur Respir J. 2004, 23: 629-636. 10.1183/09031936.04.00016804.View ArticlePubMedGoogle Scholar
- Cuzzocrea S, Mazzon E, Dugo L, Serraino I, Ciccolo A, Centorrino T, De Sarro A, Caputi AP: Protective effects of n-acetylcysteine on lung injury and red blood cell modification induced by carrageenan in the rat. FASEB J. 2001, 15: 1187-1200. 10.1096/fj.00-0526hyp.View ArticlePubMedGoogle Scholar
- Stadnicki A, Colman RW: Experimental models of inflammatory bowel disease. Arch Immunol Ther Exp (Warsz). 2003, 51: 149-155.Google Scholar
- Kovvali G, Das KM: Molecular mimicry may contribute to pathogenesis of ulcerative colitis. FEBS Lett. 2005, 579: 2261-2266. 10.1016/j.febslet.2005.02.073.View ArticlePubMedGoogle Scholar
- Cetinkaya A, Bulbuloglu E, Kurutas EB, Ciralik H, Kantarceken B, Buyukbese MA: Beneficial effects of N-acetylcysteine on acetic acid-induced colitis in rats. Tohoku J Exp Med. 2005, 206: 131-139. 10.1620/tjem.206.131.View ArticlePubMedGoogle Scholar
- Jewell DP, Patel C: Immunology of inflammatory bowel disease. Scand J Gastroenterol. 1985, 20: 119-126. 10.3109/00365528509093772.View ArticleGoogle Scholar
- Keshavarzian A, Morgan G, Sedghi S, Gordon JH, Doria M: Role of reactive oxygen metabolites in experimental colitis. Gut. 1990, 31: 786-790. 10.1136/gut.31.7.786.View ArticlePubMedPubMed CentralGoogle Scholar
- Millar AD, Rampton DS, Chander CL, Claxson AW, Blades S, Coumbe A, Panetta J, Morris CJ, Blake DR: Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis. Gut. 1996, 39: 407-415. 10.1136/gut.39.3.407.View ArticlePubMedPubMed CentralGoogle Scholar
- Jurjus AR, Khoury NN, Reimund JM: Animal models of inflammatory bowel disease. J Pharmacol Toxicolo Methods. 2004, 50: 81-92. 10.1016/j.vascn.2003.12.002.View ArticleGoogle Scholar
- MacPherson BR, Pfeiffer CJ: Experimental production of diffuse colitis in rats. Digestion. 1978, 17: 135-150. 10.1159/000198104.View ArticlePubMedGoogle Scholar
- Pawlik WW, Hottensten OD, Palen TE, Pawlik T, Jacobson ED: Adenosine modulates reactive hyperemia in rat gut. J Physiol Pharmacol. 1993, 44: 119-137.PubMedGoogle Scholar
- Menguy R, Desbaillets L, Masters YF: Mechanism of stress ulcer: Influence of hypovolemic shock on energy metabolism in the gastric mucosa. Gastroenterology. 1974, 66: 46-55.PubMedGoogle Scholar
- Wang L, Walia B, Evans J, Gewirtz AT, Merlin D, Sitaraman SV: IL-6 induces NF-κB activation in the intestinal epithelia. J Immunol. 2003, 171: 3194-3201.View ArticlePubMedGoogle Scholar
- Feldmann M, Brennan FM, Maini RN: Role of cytokines in rheumatoid arthritis. Annu Rev Immunol. 1996, 14: 397-440. 10.1146/annurev.immunol.14.1.397.View ArticlePubMedGoogle Scholar
- Polk WH, Dempsey PJ, Russell WE, Brown PI, Beauchamp RD, Barnard JA, Coffey RJ: Increase production of transforming growth factor alpha following acute gastric injury. Gastroenterol. 1992, 102: 1467-1474.View ArticleGoogle Scholar
- Konturek PC, Ernst H, Brzozowski T, Ihlm A, Hahn EG, Konturek SJ: Expression of epidermal growth factor and transforming growth factor alpha after exposure of rat gastric mucosa to stress. Scand J Gastroenterol. 1996, 31: 209-216. 10.3109/00365529609004868.View ArticlePubMedGoogle Scholar
- Blikslager AT, Moeser AJ, Gookin JL, Jones SL, Odle J: Restoration of barrier function in injured intestinal mucosa. Physiol Rev. 2007, 87: 545-564. 10.1152/physrev.00012.2006.View ArticlePubMedGoogle Scholar
- Bergen WG, Wu G: Intestinal nitrogen recycling and utilization in health and disease. J Nutr. 2009, 139: 821-825. 10.3945/jn.109.104497.View ArticlePubMedGoogle Scholar
- Liu Y, Huang J, Hou Y, Zhu H, Zhao S, Ding B, Yin Y, Yi G, Shi J, Fan W: Dietary arginine supplementation alleviates intestinal mucosal disruption induced by Escherichia coli lipopolysaccharide in weaned pigs. Br J Nutr. 2008, 100: 552-560. 10.1017/S0007114508911612.View ArticlePubMedGoogle Scholar
- Hou Y, Wang L, Ding B, Liu Y, Zhu H, Liu J, Li Y, Wu X, Yin Y, Wu G: Dietary alpha-ketoglutarate supplementation ameliorates intestinal injury in lipopolysaccharide-challenged piglets. Amino Acids. 2010, 39: 555-564. 10.1007/s00726-010-0473-y.View ArticlePubMedGoogle Scholar
- Ademoglu E, Erbil Y, Tam B, Barbaros U, Ilhan E, Olgac V, Mutlu-Turkoglu U: Do vitamin E and selenium have beneficial effects on trinitrobenzenesulfonic acid-induced experimental colitis. Dig Dis Sci. 2004, 49: 102-108.View ArticlePubMedGoogle Scholar
- Yoshida N, Yoshikawa T, Yamaguchi T, Naito Y, Tanigawa T, Murase H, Kondo M: A novel water-soluble vitamin E derivative protects against experimental colitis in rat. Antioxid Redox Signal. 1999, 1: 555-562. 10.1089/ars.1999.1.4-555.View ArticlePubMedGoogle Scholar
- Kuralay F, Yildiz C, Ozutemiz O, Islekel H, Caliskan S, Bingol B, Ozkal S: Effects of trimetazidine on acetic acid-induced colitis in female swiss rats. J Toxicol Environ Health A. 2003, 66: 169-179. 10.1080/15287390306402.View ArticlePubMedGoogle Scholar
- Hou Y, Wang L, Yi D, Ding B, Yang Z, Li J, Chen X, Qiu Y, Wu G: N-acetylcysteine reduces inflammation in the small intestine by regulating redox, EGF and TLR4 signaling. Amino Acids. 2013, 45: 513-522. 10.1007/s00726-012-1295-x.View ArticlePubMedGoogle Scholar
- Hou Y, Yao K, Wang L, Ding B, Fu D, Liu Y, Zhu H, Liu J, Li Y, Kang P, Yin Y, Wu G: Effects of α-ketoglutarate on energy status in the intestinal mucosa of weaned piglets chronically challenged with lipopolysaccharide. Br J Nutr. 2011, 106: 357-363. 10.1017/S0007114511000249.View ArticlePubMedGoogle Scholar
- Hou Y, Wang L, Ding BY, Liu Y, Zhu H, Liu J, Li Y, Kang P, Yin Y, Wu G: α-ketoglutarate and intestinal function. Front Biosci. 2011, 16: 1186-1196. 10.2741/3783.View ArticleGoogle Scholar
- Tan B, Yin Y, Liu Z, Li X, Xu H, Kong X, Huang R, Tang W, Shinzato I, Smith SB, Wu G: Dietary L-arginine supplementation increases muscle gain and reduces body fat mass in growing-finishing pigs. Amino Acids. 2009, 37: 169-175. 10.1007/s00726-008-0148-0.View ArticlePubMedGoogle Scholar
- Nofrarías M, Manzanilla EG, Pujols J, Gibert X, Majó N, Segalés J, Gasa J: Effects of spray-dried porcine plasma and plant extracts on intestinal morphology and on leukocyte cell subsets of weaned pigs. J Anim Sci. 2006, 84: 2735-2742. 10.2527/jas.2005-414.View ArticlePubMedGoogle Scholar
- Tan B, Li XG, Kong X, Huang R, Ruan Z, Yao K, Deng Z, Xie M, Shinzato I, Yin Y, Wu G: Dietary L-arginine supplementation enhances the immune status in early-weaned piglets. Amino Acids. 2009, 37: 323-331. 10.1007/s00726-008-0155-1.View ArticlePubMedGoogle Scholar
- Li P, Kim SW, Li X, Datta S, Pond WG, Wu G: Dietary supplementation with cholesterol and docosahexaenoic acid affects concentrations of amino acids in tissues of young pigs. Amino Acids. 2009, 37: 709-716. 10.1007/s00726-008-0196-5.View ArticlePubMedGoogle Scholar
- Wang J, Chen L, Li D, Yin Y, Wang X, Li P, Dangott LJ, Hu W, Wu G: Intrauterine growth restriction affects the proteomes of the small intestine, liver and skeletal muscle in newborn pigs. J Nutr. 2008, 138: 60-66.View ArticlePubMedGoogle Scholar
- Wang X, Ou D, Yin J, Wu G, Wang J: Proteomic analysis reveals altered expression of proteins related to glutathione metabolism and apoptosis in the small intestine of zinc oxide-supplemented piglets. Amino Acids. 2009, 37: 209-218. 10.1007/s00726-009-0242-y.View ArticlePubMedGoogle Scholar
- Sartor RB, Bond TM, Schwab JH: Systemic uptake and intestinal inflammatory effects of luminal bacterial cell wall polymers in rats with acute colonic injury. Infect Immun. 1988, 8: 2101-2108.Google Scholar
- Heller F, Fuss IJ, Nieuwenhuis EE, Blumberg RS, Strober W: Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity. 2002, 17: 629-638. 10.1016/S1074-7613(02)00453-3.View ArticlePubMedGoogle Scholar
- Medina C, Videla S, Radomski A, Radomski M, Antolín M, Guarner F, Vilaseca J, Salas A, Malagelada JR: Therapeutic effect of phenantroline in two rat models of inflammatory bowel disease. Scand J Gastroenterol. 2001, 36: 1314-1319. 10.1080/003655201317097182.View ArticlePubMedGoogle Scholar
- Prasad AS, DeMouchelle E, Koniuchi D: A simple fluorimetric method for the determination of RNA and DNA in tissue. J Lab Clin Med. 1972, 80: 598-601.PubMedGoogle Scholar
- Munro HN, Fleck A: Analysis of tissues and body fluids for nitrogenous constituents. Mammalian protein metabolism. Edited by: Munro HN. 1969, New York: Academic press, 465-483.Google Scholar
- Lowry OH, Rosebrough NJ, Farr AL: Protein measurement with the folin phenol reagent. J Biol Chem. 1951, 193: 265-275.PubMedGoogle Scholar
- Fu WJ, Stromberg AJ, Viele K, Carroll RJ, Wu G: Statistics and bioinformatics in nutritional sciences: analysis of complex data in the era of systems biology. J Nutr Biochem. 2010, 21: 561-572. 10.1016/j.jnutbio.2009.11.007.View ArticlePubMedPubMed CentralGoogle Scholar
- Wei JW, Carroll RJ, Harden KK, Wu G: Comparisons of treatment means when factors do not interact in two-factorial studies. Amino Acids. 2012, 42: 2031-2035. 10.1007/s00726-011-0924-0.View ArticlePubMedGoogle Scholar
- Fuss IJ, Boirivant M, Lacy B, Strober W: The interrelated roles of TGF- and IL-10 in the regulation of experimental colitis. J Immunol. 2002, 168: 900-908.View ArticlePubMedGoogle Scholar
- Jensen M, Puiman P, Stoll B, Dorst K, Renes I, Sangild P: Van GoudoeverJ: Improved gut barrier function via increased threonine utilization may explain enhanced resistance to necrotizing enterocolitis in preterm pigs fed colostrum. Acta Paediatr. 2009, 98: 44-Google Scholar
- Choudhary S, Keshavarzian A, Yong S, Wade M, Bocckino S, Day BJ, Banan A: Novel antioxidants zolimid and aeol11202 ameliorate colitis in rats. Dig Dis Sci. 2001, 46: 2222-2230. 10.1023/A:1011975218006.View ArticlePubMedGoogle Scholar
- Laroui H, Ingersoll SA, Liu HC, Baker MT, Ayyadurai S, Charania MA, Laroui F, Yan Y, Sitaraman S, Merlin D: Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon. PLoS ONE. 2012, 7: e32084-10.1371/journal.pone.0032084.View ArticlePubMedPubMed CentralGoogle Scholar
- Alex P, Zachos NC, Nguyen T, Gonzales L, Chen TC, Conklin LS, Centola M, Li XH: Distinct cytokine parttens identified from multiplex profiles of murine DSS and TNBS-induced colitis. Inflmm Bowel Dis. 2009, 15: 341-352. 10.1002/ibd.20753.View ArticleGoogle Scholar
- Fasina YO, Moran ET, Ashwell CM, Conner DE, Leslie M, Mckee SR: Effect of dietary gelatin supplementation on the expression of selected enterocyte genes, intestinal development and early chick performance. Int J Poultry Sci. 2007, 6: 944-951. 10.3923/ijps.2007.944.951.View ArticleGoogle Scholar
- Iji PA, Saki A, Tivey DR: Intestinal development and body growth of broiler chicks on diets supplemented with non-starch polysaccharides. Anim Feed Sci Technol. 2001, 89: 175-188. 10.1016/S0377-8401(00)00223-6.View ArticleGoogle Scholar
- Jeurissen SH, Lewis F, van der Klis JD, Mroz Z, Rebel JM, ter Huurne AA: Parameters and techniques to determine intestinal health of poultry as constituted by immunity, integrity and functionality. Curr Issues Intest Microbiol. 2002, 3: 1-14.PubMedGoogle Scholar
- Tsukita S, Furuse M: The structure and function of claudins, cell adhesion molecules at tight junctions. Ann NY Acad Sci. 2000, 915: 129-135.View ArticlePubMedGoogle Scholar
- Howe KL, Reardon C, Wang A, Nazli A, McKay DM: Transforming growth factor-1 regulation of epithelial tight junction proteins enhances barrier function and blocks enterohemorrhagic Escherichia coli O157:H7-induced increased permeability. Am J Pathol. 2005, 167: 1587-1597. 10.1016/S0002-9440(10)61243-6.View ArticlePubMedPubMed CentralGoogle Scholar
- Seril DN, Liao J, Yang GY, Yang CS: Oxidative stress and ulcerative colitis-associated carcinogenesis: studies in humans and animal models. Carcinogenesis. 2003, 24: 353-362. 10.1093/carcin/24.3.353.View ArticlePubMedGoogle Scholar
- Gürer H, Ozgünes H, Neal R, Spitz DR, Erçal N: Antioxidant effects of N-acetylcysteine and succimer in red blood cells from lead-exposed rat. Toxicology. 1998, 128: 181-189. 10.1016/S0300-483X(98)00074-2.View ArticlePubMedGoogle Scholar
- Dodd S, Dean O, Copolov DL, Malhi GS, Berk M: N-acetylcysteine for antioxidant therapy: pharmacology and clinical utility. Expert Opin Biol Ther. 2008, 8: 1955-1962. 10.1517/14728220802517901.View ArticlePubMedGoogle Scholar
- Krawisz JE, Sharon P, Stenson WF: Quantitative assay for acute intestinal inflammation based on myeloper-oxidase activity. Gastroenterology. 1984, 87: 1344-1350.PubMedGoogle Scholar
- Liu SP, Dong WG, Wu DF, Luo HS, Yu JP: Protective effect of angelica sinensis polysaccharide on experimental immunological colon injury in rats. World J Gastroenterol. 2003, 9: 1790-2786.Google Scholar
- Mahgoub AA, El-Medany AA, Hager HH, Mustafa AA, El-Sabah DM: Evaluating the prophylactic potential of zafirlukast against the toxic effects of acetic acid on the rat colon. Toxicol Lett. 2003, 145: 79-87. 10.1016/S0378-4274(03)00269-8.View ArticlePubMedGoogle Scholar
- Nicholson DW, Thornberry NA: Caspases: killer proteases. Trends Biochem Sci. 1997, 22: 299-306. 10.1016/S0968-0004(97)01085-2.View ArticlePubMedGoogle Scholar
- Jänicke RU, Sprengart ML, Wati MR, Porter AG: Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem. 1998, 273: 9357-9360. 10.1074/jbc.273.16.9357.View ArticlePubMedGoogle Scholar
- Nair RR, Warner BB, Warner BW: Role of epidermal growth factor and other growth factors in the prevention of necrotizing enterocolitis. Semin Perinatol. 2008, 32: 107-113. 10.1053/j.semperi.2008.01.007.View ArticlePubMedGoogle Scholar
- Elliott SN, Wallace JL, McKnight W, Gall DG, Hardin JA, Olson M, Buret A: Bacterial colonization and healing of gastric ulcers: the effects of epidermal growth factor. Am J Physiol Gastrointest Liver Physiol. 2000, 278: G105-G112.PubMedGoogle Scholar
- Chowdhury A, Fukuda R, Fukumoto S: Growth factor mRNA expression in normal colorectal mucosa and in uninvolved mucosa from ulcerative colitis patients. J Gastroenterol. 1996, 31: 353-360. 10.1007/BF02355024.View ArticlePubMedGoogle Scholar
- Plowman GD, Green JM, McDonald VL, Neubauer MG, Disteche CM, Todaro GJ, Shoyab M: The amphiregulin gene encodes a novel epidermal growth factor-related protein with tumor-inhibitory activity. Mol Cell Biol. 1990, 10: 1969-1981.View ArticlePubMedPubMed CentralGoogle Scholar
- Sodhi C, Richardson W, Gribar S, Hackam DJ: The development of animal models for the study of necrotizing enterocolitis. Dis Model Mech. 2008, 1: 94-98. 10.1242/dmm.000315.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/13/133/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.