拉克替醇和婴儿双歧杆菌对便秘大鼠AQP3及ICC的影响

Effects of lactitol and Bifidobacterium infantis on AQP3 and ICC in a rat model of constipation

目的探讨拉克替醇和婴儿双歧杆菌改善大鼠便秘的作用机制及其对结肠水通道蛋白3（aquaporin 3，AQP3）、Cajal间质细胞（interstitial cells of Cajal，ICC）的影响。方法SD大鼠30只，随机选取6只为对照组，其余大鼠连续5d给予洛哌丁胺灌胃建立便秘模型后随机分为模型组、拉克替醇组、婴儿双歧杆菌组、联合组。连续治疗7d，测定大鼠摄食量、摄水量、体重、粪便含水量及小肠推进率。ELISA法测定各组大鼠血清中P物质（substance P，SP）、血管活性肠肽（vasoacfive intestinal peptide，VIP）含量的变化。Real—time PCR检测结肠蛋白激酶A（protein kinase A，PKA）、速激肽受体1（neurokinin-1 receptor，NK-1）mRNA的表达情况；Western blot、real-time PCR检测结肠AQP3、ICC蛋白和mRNA表达量的变化。结果与模型组相比，各治疗组的粪便含水率、小肠推进率、血清中VIP、SP含量均增加，结肠中PKA、NK-1 mRNA的表达量提高，AQP3、ICC蛋白及mRNA的表达量也明显提高，且差异有统计学意义（P〈0．05）。其中联合组效果最为明显，拉克替醇组次之，婴儿双歧杆菌组效果欠佳。结论拉克替醇和婴儿双歧杆菌能够提高大鼠血清中SP和VIP的含量，SP可以与ICC表面的NK-1受体相结合，促进胃肠道平滑肌收缩，加快肠道蠕动，利于粪便排出。VIP亦可通过cAMP—PKA途径增强AQP3 mRNA和蛋白的表达，促进肠道内水分吸收，软化粪便，改善便秘。

Objective To analyze the roles and mechanisms of lactitol and Bifidobacterium infantis in the treatment of rat constipation and to investigate their effects on aquaporin3 （AQP3） and interstitial cells of Cajal （ICC） in colon tissues. Methods Thirty SD male rats were recruited in this study, 6 of which were randomly selected as the control and the rest were given 4 mg/kg, d of loperamide for 5 consecutive days to construct the 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. General indexes including food intake, water intake, body weight, fecal water content and intestinal transit rate of each rat were measured after receiving corresponding treatments for 7 consecutive days. The levels of substance P （SP） and vasoactive intestinal peptide （VIP） in serums samples were detected by ELISA. The expression of protein kinase A （PKA） and neurokinin-1 receptor （NK-1） at mRNA level in colon tissues were detected by real-time polymerase chain reaction （ real-time PCR）. Western blot assay and real-time PCR analysis were used to detect the expression of AQP3 and c-kit at protein and mRNA levels, respectively. Results Compared with the rats in model group, the levels of fecal water content and intestinal transit rate, the concentrations of SP and VIP in serums samples, the expression of PKA and NK-1 at mRNA level and the expression of AQP3 and c-kit at mRNA and protein levels were significantly increased in rats from the three treatment groups （P〈0.05）. The most effective treatment was lactitol in combination with Bifidobacterium infantis, followed by the lactitol treatment and then the Bifidobacterium infantis treatment. Conclusion The combination therapy with lactitol and Bifidobacterium infantis increased the serum levels of SP and VIP in rats with constipation. 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. VIP could promote the absorption of water in intestinal tracts, soften stools and alleviate constipation by upregulating the expression of AQP3 at both protein and mRNA levels via the cyclic adenosine monophosphate-PKA （cAMP-PKA） signaling pathway.