| Background and AimsConstipation is a common clinical disease experienced by most persons at some point during their lifetimes. It is characterized by the high incidence and intricate pathogenesis. Various risk factors are associated with constipation, ranging from changes in diet, physical activity, or lifestyle to primary motor dysfunctions due to colonic myopathy. Constipation may cause gastrointestinal nerve functional disturbance, induce cardia-cerebrovascular diseases, hepatic encephalopathy, etc. It is a common gastrointestinal symptom with a tendency to cause discomfort and affect patient quality of life. Therefore, it is very important to elaborate the pathogenesis and the therapeutic mechanisms of constipation thoroughly.Some studies have shown that the water transport and the intestinal motility play a significant role in the pathogenesis of constipation. Aquaporins(AQPs) are a kind of proteins located on the cell membrane, which are identified as water transporters in the cell. What’s more, AQP3 plays a critical role in regulation of the colon water metabolism. Interstitial cell of Cajal(ICC) is neither a kind of neuronal cells nor a kind of smooth muscle cell. It is known as a pacemaker of the gastrointestinal activities, it can initiate rhythmic electrical activity and generate slow wave activity which determines the contractile activity of the gastrointestinal. However, the mechanism of how AQP3 regulate the water transport in the gastrointestinal has not been fully elucidated, and the role of ICC in control of colonic motor patterns is far from understood.As for the treatment of constipation, clinical mangements include expansibility agents, osmotic and stimulant laxatives, and so on. Although these methods have achieved very good effects, they might have some side effects such as diarrhea, drug dependence and dysbacteriosis. Lactitol is a kind of prebiotic, it can increase the quantity of the probiotics in the colon, produce organic acids, increase the osmotic pressure of the intestinal, thus relieves constipation. Bifidobacterium infantis can produce lactic acid, acetic acid and other organic acids when they colonized in the intestine, promote intestinal peristalsis, prevent constipation. Though lactitol and Bifidobacterium infantis can improve constipation, their specific molecular mechanisms remains unclear.The objectives of this study are to establish a standard constipation model of SD rats which are induced by loperamide,then treat the constipation rats with lactitol and Bifidobacterium infantis to investigate their therapeutic effects, and to explore their effects on AQP3 and ICC in colon tissues. This paper will provide experimental data for treating constipation in clinic. MethodsThirty SD male rats were recruited in this study, 6 of which were randomly selected as the control and the rest were given 4mg/kg.d of loperamide for 5 consecutive days to construct a rat model of constipation. The rats with constipation were randomly divided into four groups including model group, lactitol treatment group, Bifidobacterium infantis treatment group and lactitol in combination with Bifidobacterium infantis treatment group. Rats in lactitol group, Bifidobacterium infantis group and combination group respectively received daily intragastric administration of lactitol(10g/kg) 1ml, Bifidobacterium infantis(1×1010cfu/ml) 1ml, lactitol and Bifidobacterium infantis(10g/kg +1×1010cfu/ml)1ml. While rats in control group and model group were treated with equal volume of saline. Interventions continued for 7 days. During and after the experiment, general index including food intake, water intake, body weight, fecal water content and intestinal transit rate were measured. The levels of SP、VIP in serums samples were detected by ELISA. The expression of PKA、NK-1 at m RNA level in colon were detected by real time polymerase chain reaction(real time-PCR).Western-blot assay and real time-PCR were used to detect the expression of AQP3 and c-kit at protein and m RNA levels, respectively.Results 1. Before the constipation model establishment, there was no statistical difference among the five groups in weight(p>0.05). On the fifth day after modeling, body weight of rats in the control group increased significantly than those in loperamide groups(p<0.05). Except the control group, the stool in the loperamide groups showed the traits of granule and fecal water content showed a significant reduction(p<0.05), this indicated that the constipation model was successful. On the seventh day after treatment, compared with the model group, the levels of fecal water content were increased significantly in the three treatment groups(p<0.05). However, no difference was seen between the control group and the three treatment groups.(P>0.05). The intestinal transit rates were observed after receiving corresponding treatment for 7 consecutive days, results showed that compared with the control group, intestinal transit rate were increased significantly in the model group(P<0.05); intestinal transit rate in the three treatment groups were increased significantly compared with the model group(P<0.05),however, no difference was seen between the control group and the three treatment groups(P>0.05). 2. Compared with the control group, the level of SP, VIP in serum were decreased in model group(P<0.05). Compared with the model group, the levels of SP, VIP in serum were increased with the treatment of Bifidobacterium infantis(P<0.05); and were remarkably increased with the management of lactitol and combination(P<0.01), however, no difference was seen between the control group and the three treatment groups(P>0.05). 3. Compared with the control group, the expression of AQP3, ICC, PKA, NK-1 at m RNA level were decreased in the model group(P<0.05), but they were increased in rats from the three treatment groups as compared to the model group(P<0.05). 4. The expression of AQP3,ICC protein were decreased in model group as(P<0.01) compared to the control group, and they were increased in the three treatment groups as compared to the model group. The most effective treatment was lactitol in combination with Bifidobacterium infantis, followed by the lactitol treatment and then the Bifidobacterium infantis treatment Conclusions 1. Loperamide can successfully induce constipation model. Both lactitol and Bifidobacterium infantis can alleviate the rats’ constipation in some degree. What’s more, the most effective treatment is lactitol in combination with Bifidobacterium infantis. 2. Both lactitol and Bifidobacterium infantis can increase the level of VIP in rats’ serum, VIP could bind to the receptors which localized on epithelial cells lining the intestines then activate the adenylyl cyclase on the cell membrance, thus increase the concentration of c AMP in the cells and activate PKA, which can promote the transcription factors phosphorylation. Finally they upregulate the expression of AQP3 at m RNA level and protein level, promote the absorption of water in intestinal tracts, soften stools and alleviate constipation. 3. Both lactitol and Bifidobacterium infantis can increase the concentration of SP in rats’ serum. SP could enhance the contraction of gastrointestinal smooth muscles and improve the intestinal motility by binding to the NK-1 receptor on the membrane of ICC. ICC can generate spontaneous inward pecemaker currents that depolarize membrance. This membrance depolarization leads to the smooth muscles contraction via the generating of action potential. |
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