Investigation On The Mechanism Of Yangyin Runchang Decoction Treating Slow Transmit Constipation In Mice

The global incidence of chronic constipation is about 14%,of which slow transit constipation(STC)accounts for the first of four subtypes of chronic idiopathic constipation.Its main features are slow colonic transit,prolonged passage of stool,with or without abdominal distention,dry stool.Studies have shown that reduction of ICC cell number and abnormal expression of aquaporins in colon tissue play an important role in the pathology of STC.As a novel 5-HT4 receptor agonist,prucalopride is beneficial to the relief of chronic simple constipation,but the evidence for the treatment of STC is still insufficient.Non-drug therapy,including transcutaneous electrical stimulation,sacral nerve stimulation and fecal microbiota transplantation,which are difficult to carry out in clinical.The recurrence rate of surgical treatment of STC is higher.Chinese medicine has a long history of treating constipation.Yangyin Runchang Decoction is an anorectal specialist Zhu Bingyi’s experience in the treatment of STC,which is effective in clinical.This study aims at elucidating the mechanism of Yangyin Runchang Decoction on ICC and aquaporin 3/9 and at providing a scientific basis for better inheriting and application of the experienced prescription from famous experts.Objective:1.In animal,to observe the effects of Yangyin Runchang Decoction on the expression of SCF and c-kit in the colon tissue of mice with STC,the ultrastructural changes of ICC,and the expression of aquaporins 3 and 9(AQP3/9).2.At the cellular level,observe the effect of Yangyin Runchang Decoction on the proliferation of ICC in vitro,and the intracellular Ca2+ concentration of ICC.Methods:1.The STC mouse model was established by compound diphenoxylate tablets.The differences in SCF and c-kit expression in the colon tissue were observed by ELISA,immunohistochemistry,qPCR and Western Blot.The ultrastructure of ICC in colon tissue was observed by transmission electron microscope.2.Immunohistochemistry,qPCR,and Western Blot were used to observe the differences in the expression of AQP3 and AQP9 in mice colon tissues;3.ICC was obtained by enzymatic hydrolysis,and indentified by immunofluorescence.Observation the effect of Yangyin Runchang Decoction on ICC proliferation;4.The purified ICC was obtained by flow cytometric sorting.ICC was identified by immunofluorescence staining.Concentration of intracellular Ca2+ in the colonic ICC of mice was observed by laser confocal microscopy.Results:1.STC model mice were successfully established by compound diphenoxylate tablets.After intervention of Yangyin Runchang Decoction,the total feces and fecal water content of STC mice increased(P<0.05),and the colonic transmission rate increased(P<0.05);2.Compared with mice in the model group,serum SCF increased after intervention with Yangyin Runchang Decoction(P<0.05),the expression of SCF and c-kit in colon tissue were increased.(P<0.05),and the damaged ICC ultrastructure was partially repaired;3.Compared with mice in the model group,the expression of AQP3 in the colon tissue o was significantly decreased after intervention with Yangyin Runchang Decoction(P<0.05),and the expression of AQP9 was increased.4.In vitro proliferation of ICC was not observed with ICC obtained by enzymatic hydrolysis,no matter Yangyin Runchang Decoction was administrated by serum or directly.5.Purified ICC was obtained by flow cytometry and identified by immunofluorescence staining.After intervention of Yangying Runchang Decoction,the intracellular Ca2+concentration of ICC was increased(P<0.01).Conclusion:1.Yangyin Runchang Decoction can relieve the symptoms of constipation in STC mice,increase the total amount of feces and fecal water content,and promote the recovery of intestinal motility;2.No direct effect of Yangyin Runchang Decoction on cells was observed by In vitro culture of ICC.Therefore,it is inferred that the mechanism of Yangyin Runchang Decoction in restoring colonic ICC cell number in vivo may be achieved by restoring the SCF/c-kit pathway instead of directly affecting ICC;3.Yangyin Runchang Decoction can increase concentration of intracellular Ca2+ of ICC,to a certain extent,suggesting that Yangyin Runchang Decoction may affect the pacing function of ICC from a functional perspective;4.Yangyin Runchang Decoction can reduce AQP3 expression while increase AQP9 expression in colonic tissues of STC mice,which can increase fecal moisture content.