Study On The Mechanism Of Visceral Hypersensitivity In FD With Liver Depression And Qi Stagnation Syndrome And Intervention Study Of Qi Stagnation And Stomach Pain Prescription Based On Mtmr2/Piezo2/5HT₃R Axi

BackgroundFunctional dyspepsia(FD)refers to a group of clinical syndromes without organic diseases,such as postprandial discomfort syndrome(PDS),epigastric pain syndrome(EPS)and overlapping(PDS+EPS).FD is a common clinical functional gastrointestinal disease(FGIDs).In addition to the above typical symptoms,FD can also have reflux,heartburn,abdominal pain,abnormal defecation and other overlapping symptoms of FGIDs.At the same time,the disease has the characteristics of repeated attack or chronic delay.The course of disease can reach several years to decades,and can be accompanied by a variety of mental symptoms,which has a serious impact on the quality of life of patients.The pathogenesis of FD involves many aspects.Visceral hypersensitivity is an important link in the occurrence of FD symptoms.Enterochromaffin cells(EC)in gastrointestinal mucosa can promote the synthesis and release of 5-hydroxytryptamine(5-HT)when they are stimulated by mechanical and chemical factors,and then act on 5-HT receptor(5-HTR)in nerve endings to induce nerve signals to transmit to the center,resulting in gastrointestinal sensation such as fullness discomfort and pain.Recent studies have shown that Neuropod cells in the gastrointestinal tract,as a special type of EC cells,play an important role in the rapid transmission of gastrointestinal mucosal sensory signals.The abnormal transmission of 5-HT rapid mechanical stimulation based on Neuropod structure may be an important reason for the visceral hypersensitivity of FD.FD can be classified as "fullness" and "epigastric pain" in traditional Chinese medicine.Disharmony of liver and spleen,block of Qi mechanism and abnormal rise and fall are the basic pathogenesis of this disease.Liver depression syndrome,spleen deficiency syndrome and damp heat syndrome are the common clinical syndrome types of FD.Clarifying the differences of symptoms and syndrome characteristics among the three syndrome types and their correlation with visceral hypersensitivity may provide an important reference for the accurate differentiation and treatment of FD and the exploration of its mechanism.Traditional Chinese medicine has the advantages of multi-target and individualization in the treatment of FD.Qizhi Weitong(QZWT)decoction.has the effects of regulating qi and relieving pain,soothing the liver and stomach.Many studies have shown that QZWT can effectively improve the clinical manifestations,emotional state and life quality of FD patients,but its specific mechanism and whether it is related to the improvement of visceral hypersensitivity need to be further clarified.Part Ⅰ clinical observation and researchObjectiveObserve the differences of FD liver qi stagnation syndrome,spleen deficiency qi stagnation syndrome and spleen stomach damp heat syndrome in clinical symptoms,TCM syndrome characteristics,psychological function and quality of life;To observe the differences of gastrointestinal hormones and visceral sensitivity among the above three FD syndrome types.MethodUsing the clinical observational research method,with the help of FD main symptom integral scale,TCM Syndrome Scale,SF-36 quality of life scale and functional gastrointestinal disease Pro scale,the differences of symptoms,TCM syndrome characteristics,serum gastrointestinal hormone content and visceral sensitivity of FD patients with liver qi stagnation syndrome,spleen deficiency qi stagnation syndrome and spleen stomach damp heat syndrome were compared from multiple dimensions.The serum and gastric mucosa of healthy volunteers and enrolled patients were collected.The serum levels of 5-HT,secretain(SEC),cholecystokinin(CCK),motilin(MTL),gastrin(gas),vasoactive intestinal peptide(VIP),somatostatin(SS)and The contents of substance P(SP)and nitric oxide(NO)and other gastrointestinal hormones;He staining was used to observe the morphology of gastric fundus mucosa in each group;Tryptophan hydroxylase 1(Tphl)protein was labeled by immunofluorescence staining to observe the distribution of EC cells;Tphl,5HT3R,postsynaptic density-95(PSD95)and other Neuropod structural proteins were simultaneously labeled by immunofluorescence staining to observe the distribution of Neuropod like structures;Western blot(WB)was used to detect the expression levels of high sensitivity related proteins Tphl and Piezo2 in gastric fundus viscera.Results1.The common syndrome types of FD are mainly overlapping subtypes(PDS+EPS),accounting for more than 60%of each group.The symptoms of epigastric pain and upper abdominal burning sensation in patients with liver qi stagnation syndrome were significantly higher than those in patients with spleen qi deficiency syndrome and spleen stomach damp heat syndrome(P<0.05),TCM syndrome score showed that the scores of fullness of epigastric fullness,epigastric pain and pharyngeal obstruction in patients with liver qi stagnation syndrome were significantly higher than those in the other two types(P<0.05,P<0.01);The scores of physical function,physiological function,emotional function and mental health of SF-36 quality of life scale in patients with liver depression and qi stagnation syndrome were significantly lower than those of the other two groups(P<0.05,P<0.01),while the scores of physical pain were significantly higher(P<0.05,P<0.01);The results of Pro scale showed that the scores of dyspepsia symptoms,reflux,psychological function and social function of patients with liver qi stagnation syndrome were higher than those of the other two types(P<0.05,P<0.01).2 The contents of serum 5-HT,CCK,SP and other visceral hypersensitivity related gastrointestinal hormones in patients with liver qi stagnation syndrome were significantly increased(P<0.05,P<0.01).The distribution of EC cells in gastric fundus of patients with liver qi stagnation syndrome was higher than that of other syndrome types(P<0.05),At the same time,there may be a large number of Neuropod like structures,and the expression of 5-HT synthase Tph1 and EC mechanical sensitive protein Piezo2 increased(P<0.05,P<0.01).Conclusions1 Compared with spleen deficiency qi stagnation syndrome and spleen stomach damp heat syndrome,FD patients with liver stagnation qi stagnation syndrome have more serious dyspepsia symptoms such as pharyngeal obstruction,fullness of epigastric fullness,epigastric pain,and lower quality of life,and are accompanied by more serious overlapping symptoms such as anxiety,depression and reflux.2 There are differences in the content of gastrointestinal hormones in serum and visceral hypersensitivity related indicators in gastric mucosa in FD patients with common syndrome types.Gastrointestinal hormones such as 5-HT,CCK and SP are significantly increased in patients with liver depression and qi stagnation syndrome,and the distribution of EC cells in gastric fundus tissue is increased.At the same time,there may be a large number of Neuropod like structures,and the expression of 5-HT synthase Tphl and mechanical sensitive protein Piezo2 in EC cells are significantly increased.Part Ⅱ animal experimental researchObjectiveTo observe the intervention effect of QZWT on FD model rats with liver qi stagnation syndrome;The mechanism of QZWT on visceral hypersensitivity of FD rats with liver qi stagnation syndrome was studied by taking the EC cell of gastrointestinal mucosal receptor and mechanical sensitive channel protein Piezo2 as the breakthrough point and the conduction process of Mtmr2/Piezo2/5HT3R signal axis between gastric mucosa and nerve endings as the main line.MethodThe FD syndrome combined rat model was established by intragastric administration of iodoacetamide(IA)combined with tail clamping stimulation.The QZWT was used for 14 days.The reliability of the model and the curative effect of traditional Chinese medicine were evaluated by taking the general situation,body weight,water intake,sucrose water preference test,balloon expansion test and pathological manifestations of he as observation indexes.ELISA method was used to detect the content of 5-HT in serum and gastric fundus of rats in each group;Tphl in rat gastric fundus mucosa was fluorescently labeled by immunofluorescence staining,and the distribution of EC cells was observed;The Neuropod structural proteins Tphl,5HT3R and PSD95 in rat gastric fundus mucosa were labeled by immunofluorescence staining to observe the distribution of Neuropod like structure;Piezo2,5HT3R and PSD95 proteins in gastric fundus mucosa of rats in each group were labeled by immunofluorescence method,and the co expression of piezo2 and Neuropod like structure was observed;The fluorescence expression of myotubulin related protein-2(Mtmr2)and PSD95 protein in the gastric fundus of rats in each group were observed by immunofluorescence;Real time PCR was used to detect the expression of Mtmr2 mRNA and Piezo2 mRNA in the gastric fundus of rats in each group;WB method was used to detect the expression levels of Mtmr2,Piezo2,Tphl,5HT3R and other proteins in the gastric fundus of rats in each group,and to observe the regulatory effect of QZWT on Mtmr2/Piezo2/5HT3R signal axis.Results1 During the experiment,the rats in the disease syndrome model group showed agitation in varying degrees,intense reaction,irritability,rough and dirty fur,soft or loose stool,sour smell,lower body weight than the normal group(P<0.01),slower increase in food intake and drinking water,and lower preference rate of sugar water(P<0.01,P<0.05).Balloon dilation test showed that the changes of EMG in disease model group and disease syndrome model group increased(P<0.01).After 14 days of administration,the overall state,agitation,hair color recovery,stool formation and body weight of rats in each treatment group were significantly higher than those in the disease syndrome model group(P<0.01,P<0.05).The amount of food and drinking water increased in varying degrees,the preference rate of sucrose water increased,and the change value of electromyography decreased(P<0.01,P<0.05).2 The results of ELISA showed that compared with the disease model group and syndrome model group,the content of 5-HT in serum and gastric fundus of rats in each treatment group decreased in varying degrees(P<0.01,P<0.05);Immunofluorescence results showed that compared with the disease syndrome model group,the expression level of Tphl in gastric fundus of rats in each dose group of TCM and fluoxetine group decreased in varying degrees(P<0.01,P<0.05).The number of Neuropod like structure in gastric fundus mucosa of rats in each dose group of TCM was lower than that in disease model group,disease syndrome model group,mosapride group and fluoxetine group,The co expression of Piezo2 and Neuropod like structure in gastric fundus of rats in each dose group of TCM was lower than that in disease model group and syndrome model group at the same time.The Mtmr2 fluorescence expression in gastric fundus of rats in each dose group of TCM and Mosapride group increased in varying degrees(P<0.01,P<0.05),and the expression of PSD95 in gastric fundus of rats in each dose group was lower than that in disease syndrome model group(P<0.01);PCR results showed that the relative expression of Mtmr2 mRNA in each dose group was higher than that in disease model group and disease syndrome model group(P<0.01,P<0.05),the relative expression of Piezo2 mRNA in each dose group was significantly lower(P<0.01),the relative expression of Piezo2 mRNA in medium and high dose groups was lower than that in low dose group(P<0.01,P<0.05),and the relative expression of Piezo2 mRNA in medium dose group was significantly lower than that in mosapride group(P<0.01);The results of Western blot showed that the expression of Mtmr2 in gastric fundus increased,and the contents of Piezo2,Tphl and 5HT3R decreased in different degrees in each treatment group(P<0.01,P<0.05).Conclusions1 The rat model of disease syndrome combination established by IA gavage plus tail clamping stimulation is highly reliable.QZWT can improve the disease syndrome performance and visceral high sensitivity of the model,and the effect is remarkable.2 QZWT can significantly reduce the content of 5-HT in serum and gastric fundus of FD liver stagnation syndrome model rats,regulate the expression of Mtmr2/Piezo2/5HT3R pathway related proteins by reducing the distribution of enterochromaffin cells and Neuropod like structure in gastric fundus mucosa,and improve the visceral hypersensitivity of FD liver stagnation syndrome model rats.