Experimental Study On Qingfu Tongchang Granules In The Treatment Of Slow Transit Constipation By Inhibiting Autophagy Of Colonic Cajal Interstitial Cells

Objective:The aim of this study was to investigate the mechanism of autophagy of colonic Cajal interstitial cells in a rat with slow transit constipation(STC)by Qingfu Tongchang Granules.Methods: 1.100 SD rats were randomly divided into 2 groups: the blank group and the model group.The model group was given with Compound Diphenoxylate by continuous gavage for 14 days.The rats were observed for general conditions,changes in body mass,24 h fecal water content,and discharge time to first white stool;Rats were injected with Iohexol Injection into their stomachs,and then X-ray films were taken to observe the degree of intestinal patency and the accessible location of Iohexol Injection;Activated carbon was poured into the stomach of rats with semi-solid paste,and the intestinal tube length and the distance of charcoal were measured from the pylorus to the ileocecal part,then the intestinal propulsion rate of rats was calculated;Western Blot was used to detect the expression levels of ICC and autophagy related proteins C-Kit and LC3Ⅰ/LC3Ⅱ in colic muscle layer,so as to comprehensively judge the success of the modeling.2.At the rat model,the surviving rats in the modeling group were randomly divided into model group,Moxapride group,Qingfu Tongchang Granules low-dose group,medium-dose group and high-dose group,which were administered for 14 days.The general condition,body mass growth,fecal moisture content in 24 h and the first white stool discharge time of rats were observed;Rats were injected with Iohexol Injection into their stomachs,and then X-ray films were taken to observe the degree of intestinal patency and the accessible location of Iohexol Injection;The electromyographic conduction of colon was detected in vivo;HE staining was used to observe the histopathological changes of colon in rats;The morphology of ICC autophagy was observed by transmission electron microscopy.ICC and autophagy related proteins C-Kit,Beclin1 and LC3 were detected by immunofluorescence.The expression levels of C-Kit and Beclin1 in colonic muscle layer were observed by Western Blot.Results: 1.Modelling experiment: The general condition of the rats in the modelling group became worse and their body weight increased slowly after the administration of the drug(P<0.05).The water content of faeces was significantly decreased in the modelling group,and the first white stool discharge time was significantly prolonged(P<0.01).The results of Iohexol Injection radiography showed that the distance of Iohexol Injection in 1min,30 min,90min and 6h was closer than that in the blank group.The length of the charcoal end segment of the intestine was significantly shorter and the charcoal end advancement rate was significantly lower in the 14 d staging group(P<0.01);the expression of C-Kit was significantly lower in the staging group when ICC and autophagy-related proteins were detected by Western-Blot(P<0.01);the grey ratio of LC3Ⅰ/LC3Ⅱ was significantly increased(P<0.01);2.Experiment on the therapeutic mechanism of Qingfu Tongchang Granules: In the 14 d of drug intervention,2 rats died in the Qingfu Tongchang Granules high-dose group and 1 rat died in the Moxaburi group,while no rats died in the remaining groups and their vital signs were stable.The general condition of the rats after drug administration was better than that of the model group,and their weight increased faster than that of the model group(P<0.05).The results of Iohexol Injection radiographs showed that the distance reached by the contrast agent was significantly prolonged in the 1min,30 min,90min and 6h groups than in the model group;the results of colonic electromyography showed that the frequency and amplitude of the drug intervention group were significantly increased compared with the model group.HE staining and pathological observation showed that the model group could see intramucosal lymphocyte infiltration,interstitial edema,mucosal edema and serosal layer edema,and each drug administration group was significantly improved compared with the model group.ICC microscopic observation under transmission electron microscope showed that the necrosis degree of ICC cells and autophagy were serious in the model group,while the STC rats treated with Qingfu Tongchang Granules reduced the damage of ICC and inhibited the occurrence of autophagy.Immunofluorescence staining showed that the expression of C-Kit was inhibited and Beclin1 and LC3 were enhanced in the model group.Qingfutongchang granule inhibited the expression of Beclin1 and LC3 in colon tissue,and restored the expression of C-Kit in colon of STC rats.Western Blot method was used to observe the expression levels of C-Kit and Beclin1 in the colonic muscle layer,and it was found that the gray ratio of C-Kit decreased and the gray ratio of Beclin1 increased in the model group.However,the intervention of Qingfu Tongchang Granules inhibited the expression of Beclin1 in the colonic tissue and increased the expression of C-Kit.Conclusion: Qingfu Tongchang Granules can improve the symptoms of STC rats,enhance the colon conduction function,promote the expression of C-Kit,inhibit the expression of autophagy related proteins LC3 and Beclin1,and thus play a therapeutic role in STC rats.