Studies On The Effects And Underlying Mechanisms Of Chang-Run-Fang In Treating Functional Constipation In Rats

Objective:Functional constipation(FC)is a refractory chronic disease,which brings serious trouble and economic burden to patients.Clinical application of Chang-Run-Fang in the treatment of functional constipation has a better effect and effectively relieves the symptoms of constipation,but the mechanism of its effect needs to be further clarified.The purpose of this study was to explore the therapeutic effect and related molecular mechanism of Chang-Run-Fang in rats with functional constipation.Methods:Diphenoxylate compound was used to establish a functional constipation rat model,and the rats were randomly divided into a normal control group(Control),a constipation model group(Model),a Maren Pill positive drug group(Maren Pills),and Chang-Run-Fang High dose(22 g/kg/day),Medium dose(11 g/kg/day),Low dose(5.5 g/kg/day)group.After the model replicating and Chang-Run-Fang treating in each groups,the indexes of the time of the first black stool,6h defecation count and their total weight were detected respectively;Western blot and RT-qPCR were used to detect the mRNA and protein expression levels from the interstitial cells of Cajal(ICCs)marker proteins----c-KIT,SCF,and hyperpolarization-activated cyclic nucleotide-gated cation channel(HCN)-1,HCN2;Immunohistochemistry was used to detect c-KIT and SCF Expression and distribution;Immunofluorescence was used to observe the expression and co-localization of HCN1 and c-KIT,substance P(Substance P,SP),and vasoactive intestinal polypeptide(VIP);Polyphysiology instrument was used to detect the effect of Chang-Run-Fang extract on rat colon tension,shrinkage wave amplitude.Results:The time of the first black stool in the constipation model group was significantly longer than that in the normal control group,the number of defecation particles was less within 6 hours,and the total weight of defecation was lighter,when the time of the first black stool in Chang-Run-Fang of different dosages of and Maren Pills groups were shortened,the number of defecation particles and total weight for 6h were increased.RT-qPCR and WB test results showed that compared with the normal group,the mRNA and protein expression levels of c-KIT,SCF,HCN1,and HCN2 in the model group were significantly reduced;Compared with the model group,The mRNA levels and protein levels of c-KIT,SCF,HCN1,and HCN2 were up-regulated in different doses group of Chang-Run-Fang and Maren Pills group.The results of immunohistochemistry showed that compared with the normal group,the ICCs of the model group were smaller and sparsely arranged,and the expressions of c-KIT and SCF were significantly reduced,while the above indexes of Chang-Run-Fang and Maren Pill treatment groups were improved to varying degrees.Immunofluorescence results showed that HCN1 can co-localize with c-KIT,SP,and VIP,and the fluorescence intensity of HCN1,c-KIT,and SP in the model group was significantly reduced compared to the normal control group,while VIP fluorescence intensity was significantly enhanced.The results of the intestinal motility experiment in vitro showed that different concentrations of water extracts of Chang-Run-Fang can improve the decrease of rat colonic tension induced by diphenoxylate and increase the amplitude of contraction waves.The therapeutic effects of Chang-Run-Fang on constipation and the expression level of related molecules showed a certain dose dependence,and the high-dose group had the most significant effect.Conclusion:Chang-Run-Fang can excite the isolated colon smooth muscle in rats,enhance colonic motor function,and improve gastrointestinal motility transmission disorder and corresponding constipation symptoms in functional constipation rats.The specific mechanism may be related to up-regulating the expression levels of c-KIT,SCF and HCN channels,maintaining the normal development of colonic ICCs and gastrointestinal slow-wave pacing and conduction functions,and promoting the expression of gastrointestinal regulatory peptide SP and down-regulating VIP levels to enhance colonic smooth muscle contraction and to promote colonic movement.