Research About The Mechanism Of IBS-C Treated With Electroacupuncture Based On 5-HT Signaling System

Background:Irritable bowel syndrome is the most common functional gastrointestinal disease i,and is one of the most diagnostic rates of gastrointestinal tract diseases.The etiology and pathogenesis of IBS are complicated,and the increase of visceral sensitivity and abnormal intestinal motility caused by abnormal brain intestinal interaction is considered to constitute the pathological physiological basis of IBS.IBS is very difficult to cure and consumes a lot of social medical resources.Patients and families suffer from illnesses,psychological and economic triple pressures.Acupuncture can alleviate the symptoms of abdominal pain and defecation dysfunction in IBS patients,and improve the quality of life of patients.But the mechanism for acupuncture treatment of IBS has not been fully elucidated.Objective:5-HT is an important chemical signal and neurotransmitter in intestinal motility and sensory regulation.5-HT₃-Type and 5-HT₄-type receptors and 5-HT transporters are hot spots in research of bowel diseases in recent years.In view of the important regulating effect of 5-HT signaling system on intestinal function and the close correlation with IBS pathogenesis,this paper is based on the scientific hypothesis that "Acupuncture treats IBS-C by regulating the 5-HT signaling system".The purpose is to expound the important mechanism of electroacupuncture in treating IBS-C.Methods:In this study,the Htr4 gene partially knocked out mice?Htr4+/-?and wild type?Wildtype,WT?C57BL6 mice were as the research object.The C57BL6 mice were divided into WT blank group,WT model group and WT model electroacupuncture treated group.IBS-C model was constructed by using ice water gavage method and evaluated.Using Crispr/Cas9 method to construct Htr4+/-mice model and evaluate.After the successful construction,it was divided into Htr4+/-model group and Htr4+/-model electroacupuncture treated group.The effect of electroacupuncture on the gastric transmission function of IBS-C model mice was evaluated by detecting the time of excretion of the first black stool,the number of fecal particles and the water content,colonic electromyography;The mood change of IBS-C model mice was evaluated by sugar consumption;The effects of electro acupuncture on serum 5-HT concentration and 5-HT expression,5-HT₃ receptor,5-HT₄ receptor,SERT protein content and mRNA expression of the local colon tissues in mice were observed by enzyme-linked immunosorbent assay,immunohistochemical method,Western Blot method and RT-PCR respectively.To evaluate the regulative effect of electroacupuncture on 5-HT signaling system.Results:?1?Model evaluation:Compared with WT blank group,the first black stool in WT model group was prolonged,the amount of stool particles decreased,the moisture content decreased,sugar consumption decreased.the colonic electromyography increased,the amplitude increased,the coefficient of variation increased,and the difference was remarkable?P<0.05?.The first black stool in Htr4+/-model group was prolonged,the amount of stool particles decreased,the moisture content decreased remarkably,sugar consumption decreased.the colonic electromyography increased,the amplitude increased,the coefficient of variation increased,and the difference was remarkable?P<0.05?.?2?Effect of Electroacupuncture on gastrointestinal transmission function:Compared with the WT model group,the initial black stool of the mice in WT model electroacupuncture treated group was shortened,the amount of feces particles increased and the moisture content increased.The frequency of myoelectric slow discharge decreased,the coefficient of discharge frequency decreased,and the difference was significant?P<0.05?.Compared with the Htr4+/-model group,the first black stool expulsion time of the mice in Htr4+/-model was shortened?P<0.05?,the amount of fecal particles increased?P>0.05?,and the moisture content was elevated?P>0.05?.The frequency of myoelectric slow discharge decreased,the coefficient of discharge frequency decreased,and the difference was significant?P<0.05?.The amplitude and amplitude variation coefficients tended to decrease without significant difference.?3?The effect of electroacupuncture on 5-HT:Compared with WT blank group,the serum 5-HT content and 5-HT expression of colon tissue were significantly increased?P05?in the WT model group and Htr4+/-model group.Compared with the WT model group,the serum and colonic 5-HT level of mice in WT model was lowered,and the difference was significant?P<0.05?.Compared with Htr4+/-model group,the serum and colon 5-HT level of mice in Htr4+/-model were lowered,and there was no significant difference?P>0.05?.?4?The effect of Electroacupuncture on 5-HT₄R and 5-HT₃R in the colon:Compared with WT blank group,the content of colonic 5-HT₄R protein and mRNA expression decreased markedly in WT model group and Htr4+/-model group,and the difference was significant.?P<0.05?After electroacupuncture intervention,the content of colonic 5-HT₄R protein and its mRNA expression in WT model electroacupuncture treated group were increased?P<0.05?,and the difference was significant.The content of 5-HT₄R protein was increased in Htr4+/-model electroacupuncture treated group,but there was no significant difference?P>0.05?.There was a significant difference in the expression of 5-HT₄R mRNA?P<0.05?.Compared with the WT blank group,the 5-HT₃R protein content and mRNA expression in the colon of the WT model group were significantly decreased,the difference was significant?P<0.05?.The content of colonic 5-HT₄R protein was increased in Htr4+/-model electroacupuncture group,but was with no significant difference?P<0.05?.After electroacupuncture intervention,the content of colonic 5-HT₃R protein and mRNA expression in the WT electroacupuncture treated model were increased?P<0.05?,the difference was significant?P<0.05?.The content of 5-HT₃R protein and 5-HT₄R mRNA expression in colon in Htr4+/-model group and Htr4+/-model electroacupuncture treated group were significantly reduced,and the difference was significant?P<0.05?.?5?The effect of electroacupuncture on colonic SERT:There was no significant difference?P>0.05?of the content of the SERT protein and its mRNA expression in the colon in the WT model group,the Htr4+/-model group,the Htr4+/-model electroacupuncture treated group.Conclusion:?1?Ice-water irrigation method can be used to replicate constipation-type irritable bowel syndrome animal model.The time of the first black stool of the model mice was prolonged,the amount of fecal particles decreased and the moisture content decreased.Chronic ice-water stimulation promotes excessive release of 5-HT in EC,inducing 5-HT signaling disorders,resulting in intestinal motility and sensory abnormalities;The Htr4+/-model group showed that the expression of congenital 5-HT₄R receptor was lowered,and the 5-HT signaling system was disturbed.IBS-C symptoms such as decreasing the number of fecal particles and decreasing moisture content,confirmed that 5-HT₄ receptor was the key receptor of 5-HT signaling system in gastrointestinal tract.The successful establishment of two kinds of IBS-C models shows that the abnormal signal of HT signaling system was the important pathogenesis of IBS-C.It also showed that in the 5-HT signaling system,if any balance was broken,would be secondary to the entire metabolic process dysfunction,resulting in disease.?2?Electroacupuncture can well reduce serum 5-HT content and 5-HT expression in colon tissues in ice water irrigation IBS-C model and Htr4+/-model mice that 5-HT abnormally elevated;Increase 5-HT₄R protein content and mRNA expression of ice-water irrigation and gastric IBS-C model and Htr4+/-model mice colon.The 5-HT₃R expression in colonic tissues of mice with ice-water irrigation and gastric IBS-C model was raised,and the 5-HT₃R expression abnormal higher in Htr4+/-model electroacupuncture treated group was lowered,5-HT,5-HT₄R,5-HT₃R were promoted to be returned to normal level.It was suggested that electroacupuncture can be used to regulate the function of 5-HT signaling system in different targets.It embodied the advantages of acupuncture multi-target treatment and bidirectional benign regulation.?3?Electroacupuncture can significantly improve the gastrointestinal motility disorder caused by the ice water irrigation and Htr4+/-part gene knockout,enhance gastrointestinal motility,increase the number of stool particles,moisture content,restore the normal rhythm of colonic myoelectric activity,and alleviate the IBS-C model mice defecation difficult symptoms.The mechanism?Conclusion 2?was by acting on 5-HT,5-HT₄R,5-HT₃R multiple targets,regulating the 5-HT signaling system,improving intestinal motility and intestinal secretion,achieving the therapeutic effect of IBS-C.