| Objective:To investigate the effect of cAMP-PKA signal pathway in the regulation of gastrointestinal motility and water metabolism in the role of the VIP-AQP3, and Xiao Fu Tong Jie Fang on the intervention effect of this pathway, which supposes Xiao Fu Tong Jie Fang the scientific basis in the treatment of functional constipation, open up new research method for the traditional Chinese medicine in the treatment of functional constipation.Methods:① Effect of VIP on functional constipation rats in defecation and AQP3, cAMP-PKA:60 SD rats were randomly divided into 4 groups:blank group, model group (compound diphenoxylate gavage), VIP group (VIP injection), model+VIP group (compound diphenoxylate gavage+VIP injection),15 rats in each group, and administrated with 2 weeks, performed with the following detection. (2)The regulation role of cAMP-PKA in VIP-AQP3 signaling pathway:Isolated and cultured the colonic epithelial cells of SD rats and the cells were divided into 4 groups: blank control group (culture medium), VIP group (VIP agonist), cAMP-PKA signal pathway inhibitor group (VIP+cAMP-PKA inhibitor), cAMP-PKA signaling pathway agonist group (VIP+cAMP-PKA agonist),6 repeated holes in each group, incubated for 3h,6h,12h and the following test were conducted at each time.③The intervention effect of Xiao Fu Tong Jie Fang on functional constipation rat of intestinal motility and VIP-cAMP-PKA-AQP3 pathway:150 SD rats were randomly divided into 5 groups:blank control group, functional constipation model group, Xiao Fu Tong Jie Fang high, medium and low dose group,30 rats in each group. Model group and other treatment groups were given compound diphenoxylate 15mg/kg/day gavage for 4 weeks, and then treated with Xiao Fu Tong Jie Fang. Each group were treated for 1,2,3 weeks, and 10 rats in each group were performed with the detection below.Detecting indexes:The detection of first black defecating feces excretion time and fecal water contention were performed; Power Lab recording the colonic electromyography; Q-PCR and Western bolt were used to detect the expression of AQP3, cAMP, PKA mRNA and protein in colon tissue or colonic epithelial cells.Results:Comparing with the blank group, the time of first black defecation was extended and fecal water contention reduced in the model group; comparing with the model group, the model+VIP group and Xiao Fu Tong Jie Fang each dose group the time of first black defecation was shorted and fecal water contention increased in different degree. Colonic electromyography test results:comparing with the blank group, the slow wave frequency decreased and amplitude increased and waveform was irregular in model group, the frequency of VIP group increased but amplitude did not change significantly; comparing with the model group, the frequency increased and amplitude reduced in the model+VIP group and Xiao Fu Tong Jie Fang high, medium dose groups. Q-PCRand Western bolt test results:comparing with the control group, levels of AQP3, cAMP, PKA mRNA and protein in colon tissue decreased in model group, and VIP group increased; comparing with the model group, the upper index expression level in model+VIP group increased. In the colonic epithelial cells, comparing with the control group, levels of AQP3, cAMP, PKA mRNA and protein increased at each time in VIP group; comparing with the VIP group, the cAMP-PKA signaling pathway inhibitor group at each time point the upper index expression level decreased, while cAMP-PKA signaling pathway agonist group were increased, and the changes of 6h is obviously. Xiao Fu Tong Jie Fang ach dose group the levels of AQP3, cAMP, PKA mRNA and protein increased, the effect in the high dose group and 3 weeks are most obviously.Conclusion.①VIP can promote intestinal peristalsis, regulate the intestinal fluid metabolism, and influence the expression level of the AQP3, cAMP、PKA in rat colon tissue.②AQP3 may be regulated by VIP with the cAMP-PKA signaling pathway. ③Xiao Fu Tong Jie Fang can regulate the intestinal peristalsis and intestinal fluid metabolism and cure the function constipation, and has time and dose dependence; Xiao Fu Tong Jie decoction improve gastrointestinal motility and improve intestinal fluid metabolism in rats may be achieved by the intervention of VIP-cAMP-PKA-AQP3 signaling pathway. |
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