| Background With the development of society,the pace of human life is getting faster and faster,coupled with changes in the diet structure and population aging,the effects of gastrointestinal motility disorders are becoming more and more significant on human health.The number of patients with slow transit constipation has increased year by year,and the incidence rate has increased year by year.The slow transit constipation has seriously affected the quality of people’s life.A large number of clinical studies have suggested that the etiology and pathogenesis of slow transit constipation are related to the abnormal function of intestinal nervous system,colon smooth muscle and interstitial cells of Cajal in the gastrointestinal tract,and are also related to mental and psychological factors.At present,patients with milder conditions are treated with lifestyle changes,through dietary and behavioral therapies improve defecation habits.If the treatments above are invalid,drug therapy,such as mosapride,lactulose and polyethylene glycol,would be recommended instead.However,drug therapy may has little effect on some patients with severe slow transit constipation.As a cathartic agent,the traditional Chinese medicine senna has been used in China for more than a thousand years,and its clinical application is still extensive.A large number of clinical practices have found that senna can promote gastrointestinal motility and have a good therapeutic effect on many gastrointestinal motility disorders.At present,it is found that sennoside A,B,C,and D in the senna leaf components are the main cathartic components,in which sennoside A and B are isomers,and sennoside A are more abundant in senna leaves and more relatively easy to be extracted.Our previous preliminary experiments found that sennoside A and sennoside B have a significant effect on gastrointestinal motility,and that sennoside A and sennoside B are not only found in senna,but are also found in rhubarb.Therefore,the development of this kind of ingredients has a potential market utilization value.Therefore,it is a great significance for the development of drugs to study the mechanism of sennoside A and sennoside B to promote gastrointestinal motility.Our preliminary pre-experimental results showed that the gastrointestinal motility effect of sennoside A is significantly better than that of sennoside B.Therefore,the focus of our research is sennoside A.Numerous studies have shown that the interstitial cells of Cajal are the pacemaker and disseminator of gastrointestinal slow wave rhythm,and also participate in the transmission of gastrointestinal neurotransmitter signaling,playing a vital role in the formation and regulation of gastrointestinal motility.Cajal interstitial cells are also involved in the process of gastrointestinal sensory formation.Recent studies have shown that hyperpolarization-activated cyclic nucleotide-gated channels on the surface of Cajal interstitial cells are the originating ion channels of their rhythmic electrical activity.The roles of ICC and HCN1 are not known in the formation of slow transit constipation.In the previous study,we found that the crude senna extract can significantly promote intestinal peristalsis in slow transit constipation model animals.And sennoside A is the main active ingredient of senna to play a laxative effect,but the pharmacological mechanism is unclear.Objective In view of the above problems,this study is based on the successful production of STC animal models,using immunochemistry,pharmacological,electrophysiological methods to study the roles of ICC and HCN1 in the formation of slow transit constipation.At the same time,the pharmacological effects and pharmacological mechanisms of sennoside A in slow transit constipation were studied to clarify the pathogenesis of slow transit constipation,and to lay the theoretical foundation for the senna to treat slow transit constipation.Methods 1.The method of model construction and the pharmacological method:(1)The rat of slow transit constipation is constructed by ice water gavage;(2)The specific blockers:(a)We selectively inhibit the proliferation of Cajal interstitial cells in the gastrointestinal tract by intraperitoneal injection of STI-571,(b)We selectively blocked HCN1 by tail vein injection of ZD7288;(3)The interventional drug: sennoside A,mosapride and saline were given by intragastric administration.2.The test of gastrointestinal motility: Intestinal carbon foam advancement test,colonic bead expulsion test were used to detect the changes of intestinal propulsion rate in vivo;the changes of intestinal contractile tension movement was detected by using muscle strip tension test in vitro.3.The morphological change of gastrointestinal ICC is detected by immunofluorescence staining.4.The electrophysiological activity of gastrointestinal ICC is detected by patch clamp.5.The stimulation of mechanical expansion were given by colonic balloon dilatation,and the gastrointestinal sensory sensitivity was assessed by detecting the rectus abdominis electrical activity.6.The levels of neurotransmitter secretion in the gastrointestinal tract is detected by ELISA.Results In the first part of the experiment,our main findings are as follows:(1)The morphological experiment showed that the number of ICC in the gastrointestinal tract of STC rats was significantly reduced,and the expression of HCN1 on the ICC membrane was decreased.(2)The experiment of vivo showed that the rate of small intestine and colon propulsion in STC rats was lower than that in normal rats,and sennoside A could significantly increase the rate of small intestine and colon propulsion in STC rats,and the effect was better than that of mosapride.The effect of sennoside A can be blocked by HCN1-specific inhibitors.(3)The experiment of muscle strip showed that the amplitude and frequency of contraction of muscle strips in STC rats were lower than those in normal rats,and sennoside A could significantly increase the amplitude and frequency of contraction of muscle strips in STC rats.The effect of sennoside A can be blocked by HCN1-specific inhibitors.(4)The experiment of patch clamp showed that the frequency and amplitude of spontaneous depolarization of gastrointestinal ICC in STC rats were lower than those in normal rats,and sennoside A could significantly increase the frequency and amplitude of spontaneous depolarization of gastrointestinal ICC in STC rats.The effect of sennoside A can be blocked by HCN1-specific inhibitors.In the second part of the experiment,our main findings are as follows:(1)Inhibition of ICC proliferation or blocking of HCN1 may lead to a disorder of colonic mechanical sensation,suggesting that ICC is involved in the formation of gastrointestinal mechanical sensation,and HCN1 is an important functional protein in this process.(2)The stimulation of mechanical dilatation can promote the release of excitatory neurotransmitters SP and MOT,and inhibit the release of inhibitory neurotransmitters VIP and CGRP,suggesting that mechanical stimulation can regulate the release of gastrointestinal neurotransmitter.Inhibition of ICC proliferation or blockade of HCN1 significantly attenuates the regulation of colonic stimulation on the release of intestinal neurotransmitter,suggesting that ICC can be involved in the regulation of intestinal neurotransmitter release by mediating the formation of gastrointestinal mechanical sensation,whereas HCN1 plays an important role in this process.(3)The STC rat is less sensitive to the stimulation of mechanical dilatation than that of normal rats.At the same time,the levels of excitatory neurotransmitters SP and MOT in STC rats were lower than those in normal rats,while the levels of inhibitory neurotransmitters VIP and CGRP were higher than normal rats,which suggests that the abnormal level of different neurotransmitters is an important cause of STC.(4)Sennoside A can improve the sensitivity of intestinal mechanical sensation in STC rats.It can also increase the levels of excitatory neurotransmitters SP and MOT in the intestinal tract of STC rats,meanwhile reducing the release of inhibitory neurotransmitters VIP and CGRP.However,The sensitization of mechanical sensory and the neurotransmitter regulation can be blocked by HCN1 blockers.Conclusion(1)The decrease in the number of Cajal interstitial cells and the decrease in the expression of HCN1 can result in the decrease in the frequency and amplitude of spontaneous depolarization of ICC.As a result,the frequency and amplitude of the slow wave are decreased,thereby directly causing the dysfunction of gastrointestinal muscular layer and finally resulting in chronic constipation.(2)The decrease in the number of ICC and the abnormal function of ICC can also reduce the sensitivity of intestinal mechanical sensory,resulting in abnormal regulation of intestinal neurotransmitter secretion,thereby indirectly causing contraction disorder of gastrointestinal muscular layer.This is another important factor leading to chronic constipation.(3)On one hand,sennoside A can increase the frequency and amplitude of slow wave by activating the HCN1.On the other hand,it improves the sensitivity of intestinal mechanical sensory and regulates the release of neurotransmitters in the gastrointestinal tract.Finally,the regulation of gastrointestinal motility is achieved by sennoside A... |
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