Constipation, characterized by infrequent stools and/or difficult stool passage, is one of the most common gastrointestinal complaints, especially in children (1,2). Constipation can adversely affect an individual's health, with disorders of host immunity and oxidative stress (3–5). It is well known that constipation can cause changes in intestinal permeability. Besides the systemic immune response, constipation affects mainly the local gut immunity (3,4). Furthermore, it has been proved that chronic constipation can cause potential oxidative stress and free radical damage (5). In oxidative stress, the key antioxidase-superoxide dismutase (SOD) decreases and the representative oxidative product, malondialdehyde (MDA), accumulates (6). Oxidative damage is widely accepted to be the underlying mechanism of many pathological processes, including aging, diabetes, and tumorigenesis (7–9); however, effective prevention and curative methods of constipation and its harmful effects are deficient.
It is increasingly accepted that some gut microbiota may play a beneficial role in constipation (10). Species such as the bifidobacteria and the lactobacilli have various health-promoting functions, including immunostimulation and inhibition of harmful bacteria (11,12). Although there has been increased interest in manipulating the gut microbial community in constipation, prebiotics may be an alternative strategy with even more benefits (13).
As lower-molecular-weight nondigestible carbohydrates, prebiotics, engineered to be metabolized by specific desirable bacteria, can selectively stimulate 1 or a limited number of bacteria in the bowel and thus improve host health (14,15). The prebiotics of proven efficacy for selective stimulation of the indigenous bifidobacteria and lactobacilli include galactooligosaccharides (GOS), xylooligosaccharides (XOS), fructooligosaccharides, inulin, oligofructose (OF), and lactulose. Oligosaccharides, an ideal model of prebiotics in milk, are the third largest solid component of human milk (16). GOS can stimulate intestinal bifidobacteria and lactobacilli, decrease fecal pH, and improve stool frequency (17). XOS may increase lactobacilli and bifidobacteria and decrease cecal pH, with hypolipidemic activity (18,19). Fructooligosaccharide supplementation significantly modifies the composition of the gut microbiota and is helpful for creating increasing softer stools and the mean number of stools per day (18,20). It was proved that inulin could support larger total bacterial counts stimulating bowel movements and positively influencing lipids in hyperlipidemic individuals (21). Inulin and OF are effective prebiotics that stimulate lactobacilli and bifidobacteria, and they may enhance calcium absorption (22,23). In addition, prebiotics also may increase cation absorption, have certain effects associated with dietary fiber, and have a low energy value (24). In an effort to prevent constipation, many patients try to consume prebiotics (25), but the effectiveness of this strategy is not clear.
Although it is proved that a single prebiotic has a limited beneficial effect, the effect of combined prebiotics on constipation and optimal dosage have not been clarified. Furthermore, because the relief of constipation was associated with immune response and oxidative stress, the effects of prebiotics on the content of intestinal secretory immunoglobulin A (sIgA; the main local immunity of gut) and of SOD and MDA in constipation need elucidation. Therefore, the present study was designed to elucidate these issues.
Diphenoxylate, a derivative of pethidine, is used to induce constipation (26). The role of diphenoxylate on the intestinal tract is a direct effect on the smooth muscle, by inhibiting its intestinal receptor. It can eliminate the peristaltic reflex and thus weaken peristalsis, promoting the intestinal absorption of water.
In the present study, constipation was induced in female Sprague-Dawley rats by the administration of diphenoxylate intragastrically, as a proof of the concept. The combined prebiotics, dissolved in milk for drinking, were used as an intervention. The indicators of the intestinal peristalsis, the intestinal local immunity-sIgA, and the contents of SOD and MDA were measured to evaluate the effects of prebiotics in constipation.
MATERIALS AND METHODS
Experimental Design and Data Collection
The experimental protocol followed the guidelines established by the Ethics Review Committee for Animal Experimentation of Hebei Medical University. Ninety female Sprague-Dawley rats with body weight of 179.40 ± 11.90 g were supplied by the Department of Experimental Animals, with animal certificate no. 0018142. All of the animals were fed a standard laboratory diet (containing crude fiber <5%) and housed at (23 ± 1)°C, with 12 hours of light. Depending on the weight, rats were divided randomly into 6 groups: control (Con), control plus prebiotics (Con + Preb), constipation (Cons), constipation plus high-dose prebiotics (Cons + Preb[H]), constipation plus middle-dose prebiotics (Cons + Preb[M]), and constipation plus low-dose prebiotics (Cons + Preb[L]). After being divided into 6 groups, all rats were given a standard diet. In the Con + Preb group, rats drank middle-dose prebiotics. In the latter 4 groups, constipation was induced in rats by an intragastric administration of diphenoxylate (MinSheng, Henan, China). The dosage of diphenoxylate (5.0 mg/kg body weight per day) to induce constipation was determined by the reports and our preexperiment. In the last 3 groups, rats drank different dosages of the prebiotics, including high-dose prebiotics (2.51 g/kg body weight per day), middle-dose prebiotics (0.84 g/kg body weight per day), and low-dose prebiotics (0.42 g/kg body weight per day), which were according to the 15-, 5-, and 2.5-fold of the recommended dosage of human. The dosage of middle-dose prebiotics used for rats was equivalent to the dosage recommended for humans. The prebiotics used in the study were a combination of GOS, XOS, OF, and inulin (Jiangmen Quantum Hi-Tech, Guangdong, and HengYe ZhongYuan, Beijing, China); the ratio of GOS:XOS:OF:inulin is 3.6:1:0.4:5. Similar to diphenoxylate, the composition and dosage of prebiotics were evaluated and determined by the recommendation for humans and our preexperiment. The prebiotics in different groups were dissolved in milk (Nestle, Vevey, Switzerland), with the same concentration of milk (6.25 g/kg body weight per day). The rats first drank the prebiotics containing milk, and only after that were they given water to ensure the dosage. After 5 days, the rats were treated with diphenoxylate. The treatment was preceded by a 16-hour fast during which the prebiotic intervention continued. Black ink was administered by gavage 30 minutes after the diphenoxylate treatment, after which the rats were housed individually and observed every 30 minutes for passage of black fecal pellets initially, the number of which was recorded at 24 hours. During follow-up, the rats received the standard diet and the prebiotic treatment continued. After 7 days, the rats were treated with diphenoxylate. The treatment was preceded by a 16-hour fast during which the prebiotic intervention continued. Thirty minutes later, rats were given intragastric administration of ponceau and housed in cages individually. After 15 minutes, rats were killed by femoral artery bleeding. The procedures above were illustrated in a flowchart (Fig. 1). Blood samples were collected, incubated at 37°C, and centrifuged for 15 minutes at 2500 rpm to allow serum collection. The intestine from pylorus to ileocecus was separated and placed gently in a straight line. The total length of the intestine and the length from the pylorus to the forward position of ponceau were measured and their ratio was calculated to show the advance rate of ponceau. Then the intestinal tissue at the ileocecus was removed quickly and rinsed with 0.9% sodium chloride. A piece of intestinal tissue was preserved in 4% paraformaldehyde for morphological detection and the rest was preserved at −20°C for future molecular biological detection.
sIgA in Intestinal Tissue
A section of intestinal tissue was homogenized in 1 mL lysis buffer (2.9 g/L NaCl, 3.6 g/L Na2HPO4·12H2O, 0.2 g/L KH2PO4, 0.1 g/L sodium dodecyl sulfate) and centrifuged at 10,000 rpm at 4°C for 15 minutes. The supernatant was collected and the total protein content was determined by the Lowry method. The content of sIgA in the intestinal tissue was analyzed by enzyme-linked immunosorbent assay (ELISA) (Jiancheng, Nanjing, China). Standards and samples were added to the wells of the plate and incubated for 1 hour. After the wells were washed with the ELISA wash buffer, the conjugated antibody was added and incubated for 1 hour. Then the wells were again washed with the ELISA wash buffer. The substrate was added in the wells and incubated for 15 minutes. Then the stop solution was added and absorption was measured with an ELISA reader at 450 nm. The tests were carried out in duplicate.
SOD in Serum and Intestinal Tissue
The content of SOD in serum and intestinal tissue was measured by the xanthine oxidase method. A section of intestinal tissue was homogenized in 1 mL lysis buffer (2.9 g/L NaCl, 3.6 g/L Na2HPO4·12H2O, 0.2 g/L KH2PO4, 0.1 g/L sodium dodecyl sulfate) and centrifuged at 10,000 rpm at 4°C for 15 minutes. The supernatant was collected and the total protein content was determined by the Lowry method. The contents of SOD in serum and intestinal tissue were measured by its detection kit. The procedures followed the kit (Jiancheng).
MDA in Serum and Intestinal Tissue
The content of MDA in serum and intestinal tissue was measured using the thiobarbituric acid method. A section of intestinal tissue was homogenized and centrifuged as the method in the analysis of SOD. The supernatant was collected and the total protein content was determined by the Lowry method. The contents of MDA in serum and intestinal tissue were measured by its detection kit. The procedures followed the kit (Jiancheng).
The data were presented as mean (± SD), and the SPSS 13.0 statistical package (SPSS Inc, Chicago, IL) was used to analyze the data. One-way analysis of variance was used to analyze the differences among the groups, and the differences between 2 groups were evaluated by the Student-Newman-Keuls test. P < 0.05 was considered statistically significant.
Changes in Intestinal Peristalsis
The time of passing black stool initially, the grains of black stool in 24 hours, and the advance rate of ponceau are detected to reflect the changes in intestinal peristalsis (Table 1). Compared with the Con group, the time of passing black stool initially increased, the grains of black stool in 24 hours decreased, and the advance rate of ponceau decreased in the Cons group (P < 0.01 or P < 0.05), suggesting that the model of constipation has been built successfully. Compared with the Cons group, the above alterations attenuate significantly in the Cons + Preb(L) group (P < 0.05), showing that prebiotics could promote intestinal peristalsis, with beneficial effects on constipation; however, the effects of prebiotics are not dose dependent, and prebiotics at low dosages in milk have greater beneficial effects.
Content of Intestinal sIgA
Compared with the Con group, the content of sIgA decreased in the Cons group (P < 0.05), which increased significantly in the Cons + Preb(M) group and the Cons + Preb(L) group (P < 0.05) (Fig. 2). The results suggest that constipation results in decreased content of sIgA, weakening intestinal immunity; however, the decreased content of sIgA can be elevated by prebiotics to the levels in the Con group, improving the intestinal immunity of constipated rats.
Content of SOD in Serum and Intestinal Tissue
Compared with the Con group, the content of SOD in serum reduces in the Cons group (P < 0.05); however, the content of SOD elevates significantly in the Cons + Preb(L) group and Cons + Preb(M) (P < 0.05) (Fig. 3). Compared with the Con group, the content of SOD in intestinal tissue reduces in the Cons group (P < 0.05). The content of SOD elevates significantly in Cons + Preb(L) and the Cons + Preb(M) group (P < 0.05) (Fig. 4). These results show that constipation decreases the content of SOD, weakening the body's antioxidative ability, which can be attenuated by prebiotics. The dosage seems to determine the effects of prebiotics, and the low dosage has a positive effect on the SOD in serum and the middle dosage has a positive effect on the SOD in intestinal tissue, but prebiotics at a high dosage in milk may have adverse effects.
Content of MDA in Serum and Intestinal Tissue
Compared with the Con group, the content of MDA in serum elevates in the Cons group (P < 0.05). Compared with the Cons group, the content of MDA reduces significantly in the Cons + Preb(L) group (P < 0.05) (Fig. 5). Compared with the Con group, the content of MDA in intestinal tissue elevates in the Cons group (P < 0.01). Compared with the Cons group, the content of MDA reduces significantly in the Cons + Preb(M) group (P < 0.05) (Fig. 6). The results show that constipation can cause the accumulation of the oxidative product-MDA, which can be attenuated by prebiotics. Similarly, the dosage seems to determine the effects of the prebiotics; the low dosage has a positive effect on the MDA in serum and the middle dosage has a positive effect on the MDA in intestinal tissue. The intestinal MDA in the Cons + Preb(L) group shows a tendency toward increasing, which needs further explanation.
Prebiotics can affect host health with many beneficial effects (14,15,27); however, the effectiveness of prebiotics on constipation is not fully clarified. In the present study, the prebiotics could attenuate the decreased intestinal immunity-sIgA, the decreased antioxidative defense-SOD, and the increased oxidative product-MDA in the constipated rats, in addition to reduced intestinal peristalsis.
In the study, constipation was successfully induced by diphenoxylate with obviously weakened intestinal peristalsis, including the prolonged time of passing black stool initially, the decreased grains of black stool in 24 hours, and the reduced advance rate of ponceau. Furthermore, the morphology also confirmed the pathological changes of the intestinal wall in constipation (not shown).
The sIgA is the main local immunity of gut (28,29). In the study, the intestinal sIgA of the constipated rats decreased significantly, leading to weakened intestinal immunity. In constipation-caused oxidative stress, the SOD and MDA represent the antioxidative defense and the oxidative product, respectively (6,30). In the study, decreased SOD and increased MDA in both serum and intestinal tissue were detected in the constipated rats, indicating that constipation could cause weakening of antioxidative defense and accumulation of oxidative product.
As lower-molecular-weight nondigestible carbohydrates, prebiotics have various beneficial effects on the body (24). The main effects are the improved resistance to pathogenic bacteria colonization and the enhanced host systemic and mucosa immunity (31–34). In addition, prebiotics could stimulate intestinal peristalsis by increasing daily stool frequency and fecal bulk and moisture (35). Whether prebiotics have more beneficial effects on constipation is not clarified.
In the study, it was confirmed that prebiotics at low dosage improved the intestinal peristalsis of the constipated rats, including the shortened time of passing black stool initially, the increased grains of black stool in 24 hours, and the enhanced advance rate of ponceau. Also, the low-dose prebiotics had an obviously protective effect on the pathological changes in the intestinal wall (not shown). The results indicated that the effect was not dose dependent and the low dosage of prebiotics in milk was more suitable for constipation. In addition, low-dose prebiotics (169.00 ± 11.40) had a tendency to reduce the body weight of the constipated rats (175.89 ± 6.85), and this may be because of the intrinsic character of the prebiotics. Also, middle-dose prebiotics (187.50 ± 8.52) could reduce the body weight of the control rats (208.80 ± 20.50), indicating the beneficial role of prebiotics in maintaining weight.
In the experiment, middle- and low-dose prebiotics increased the content of intestinal sIgA to the levels found in the control rats, which improved the intestinal immunity of the constipated rats. Furthermore, low- or middle-dose prebiotics increased the content of SOD in serum or intestinal tissue, suggesting the improving effects of prebiotics on the decreased antioxidative defense induced by constipation. Also, low- or middle-dose prebiotics decreased the content of MDA in serum or intestinal tissue, suggesting antagonistic effects of the prebiotics on the accumulated oxidative product induced by constipation.
The dosage seemed to determine the effects of the prebiotics, and low dosage of prebiotics in milk may have beneficial effects on constipation. In addition, both middle- and low-dose prebiotics increased intestinal sIgA. Also, the low dosage had a positive effect on the SOD and MDA in serum and the middle dosage had a positive effect on the SOD and MDA in intestinal tissue. The prebiotics at high dosage in milk may have adverse effects. The intestinal MDA in the low-dosage group showed a tendency toward increasing, which needed further explanation.
In conclusion, prebiotics could attenuate the decreased intestinal immunity and enhanced oxidative stress in the constipated rats, in addition to the reduced intestinal peristalsis, having beneficial effects on constipation.
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Keywords:Copyright 2011 by ESPGHAN and NASPGHAN
constipation; malondialdehyde; prebiotics; secretory immunoglobulin A; superoxide dismutase