| BackgroundFunctional dyspepsia(FD)is one of the most common Functional gastrointestinal diseases in clinical practice,and its typical clinical symptoms include upper abdominal pain,feeling of fullness,early fullness,nausea,etc.,affecting the quality of life of patients.According to the Roman IV standard,FD is divided into two subtypes:after eating uncomfortable syndrome(Postprandial discomfort syndrome,PDS)and upper abdominal pain syndrome(Epigastric pain syndrome,EPS),PDS clinical symptoms mainly for early satiety and satisfied feeling,EPS is mainly on abdominal pain or burning pain,in clinical,both can be alone,also can overlap.Visceral hypersensitivity is one of the important pathogenesis of FD.The mechanism of visceral hypersensitivity is complex,and the final result usually leads to the sensitization of visceral afferent neurons and spinal dorsal horn neurons,or the impairment of the descending excitatory and inhibitory effects of spinal nociceptive neurons,leading to the reduction of physiological stimulus threshold and a strong response to nociceptive stimuli.It is possible that these mechanisms exist independently or interact with each other,ultimately promoting visceral hypersensitivity.Visceral hypersensitivity is induced by a variety of factors,including mechanical stimulation,proximal duodenal chemical stimulation,hypothermia,psychological and spiritual factors.The induction of mechanical stimulation has always been a hotspot in the study of visceral hypersensitivity in FD.There are a variety of mechanically sensitive cells in the gastrointestinal tract,such as Enterochromaffin cells(EC),internal and external intestinal neurons,smooth muscle cells and Cajal interstitial cells,which can sense and transmit mechanical stimulation signals.In recent years,a variety of mechanically-sensitive ion channels on the surface of intestinal pheochromocyte have been studied and revealed one by one.EC cells,as important mechanically-sensitive cells in the gastrointestinal tract,play an important role in the normal physiological function of the gastrointestinal tract.At the same time,EC cells are one of the main sources of serotonin release,and abnormal serotonin release caused by mechanical stimulation can induce visceral hypersensitivity.The relationship between mechanosensitive ion channels or receptors in EC cells and serotonin release has not been fully understood,so it is of great significance to study the mechanism of visceral hypersensitivity in FD from this perspective.At present,the treatment of FD mainly focuses on symptomatic treatment.Stimulants,antacids and antidepressants are widely used,but the effect is single and prone to repeated attacks.Traditional Chinese medicine has shown its advantages in the treatment of FD on the basis of overall regulation.As one of the basic syndrome types of spleen deficiency syndrome(FD),xiangsha liujunzi decoction,as a classic recipe for treating FD spleen deficiency syndrome,has the effect of strengthening the spleen and stomach,regulating qi and reducing bloating,not only improving gastrointestinal motility,but also having a good effect in treating stomachache,stomach bloating and improving visceral hypersensitivity.The mechanism of xiangsha liuj unzi decoction in reducing visceral hypersensitivity remains unclear.In this study,the effect of xiangsha liujunzi decoction on the mechanism of viscera hypersensitivity was studied from the perspective of EC cells’ mechanical sensitivity ion channel.Part One Study on the expressio-n of PIEZO2 ion channel in the gastric fundus mucosa of patients with FDObjectiveTo clarify the expression of PIEZO2 in EC cells of gastric fundus mucosa.MethodsThe gastric fundus mucosa tissues of FD patients and normal people were collected for immunofluorescence staining and WB detection to observe the expression of EC cells and PIEZO2 protein in the gastric fundus mucosa of FD patients and normal people.ResultsBoth EC cells and PIEZO2 are mainly present in the mucosal layer.The expression levels of EC cells and PIEZO2 in the gastric fundus mucosa of FD patients were increased compared with normal peopleConclusions1 PIEZO2 is expressed in the mucosa of human gastric fundus.2 Compared with normal people,PIEZO2,EC cells and 5-HT expression levels were higher in the gastric fundus mucosa of FD patients with visceral hypersensitivity.Part Two Establishment of QGP-1 cell mechanical stimulation model and effect of inhibitor intervention on QGP-1 release of 5-HTObjectiveUsing the EC cell model QGP-1 cells,the relationship between the release of 5-HT in EC cells and PIEZ02 ion channel and EPAC1 signaling pathway in mechanical stimulation was analyzed.Methods1 QGP-1 cells were inoculated into the Flexcell stress loading system,and the cell secretion of 5-HT was observed by using stretch mechanical stress and pressure mechanical stress.The dose-effect curve of 5-HT release was established,and the stimulation time of 5-HT maximum release was calculated.2 PIEZO2 inhibitor GsMTx4 and EPAC1 antagonist ESI-09 were used to intervene cells to detect the release of 5-HT and intracellular calcium ion concentration.Results1 QGP-1 cells were subjected to cyclic tensile stress stimulation and constant pressure stress stimulation,and the supernatant collected at different time points was used to analyze the content of 5-HT.After 120s,the content of 5-HT in the culture supernatant was basically stable.The content of 5-HT in the supernatant of cultured cells tended to be stable after the stress stimulation on the cells at 150s.2 After the stimulation of tensile stress,compared with the control group,the 5-HT concentration in the positive control group and the KC1 group was significantly increased(P<0.05),while the 5-HT concentration in the two inhibitor groups was significantly decreased(P<0.05).Compared with the KCl group,the 5-HT concentration was significantly reduced in the stretch group and the two inhibitor groups(P<0.05).Compared with the stretch group,the 5-HT concentration in the two inhibitor groups was significantly reduced(P<0.05).However,there was no significant difference between the GsMTx-4 group and ESI-09 group2 after stress stimulation,compared with the control group,the concentration of 5-HT in the positive control group KCl group and the pressure group increased significantly(P<0.05),and the concentration of 5-HT in the GsMTx-4 group and ESI-09 group decreased significantly(P<0.05).Compared with the KCl group,the concentration of 5-HT in the pressure group and the inhibitor group was decreased,and the difference was statistically significant(P<0.05).The concentration of 5-HT in GsMTx-4 group and ESI-09 group was significantly lower than that in the pressure group(P<0.05).There was no significant difference between inhibitor groups.3 After stress stimulation,compared with the control group,the concentration of 5-HT in the positive control group KCl group and the pressure group increased significantly(P<0.05),and the concentration of 5-HT in the GsMTx-4 group and ESI-09 group decreased significantly(P<0.05).Compared with the KCl group,the concentration of 5-HT in the pressure group and each inhibitor group was significantly reduced(P<0.05).Compared with the pressure group,the 5-HT concentrations in the GsMTx-4 group and ESI-09 group were significantly reduced(P<0.05).There was no significant difference between the two groups.4 After mechanical stress stimulation,calcium ion concentration in each group increased significantly compared with the control group(P<0.05).Compared with the KCl group,the intracellular calcium concentration in each group was significantly reduced(P<0.05).There was no difference between the tensile group,pressure group,GsMTx-4 group and ESI-09 group.Calcium ion concentrations in GsMTx-4 group and ESI-09 group were significantly lower than those in the tensile group(P<0.05).Calcium ion concentrations in GsMTx-4 group and ESI-09 group were significantly lower than those in the pressure group(P<0.05).Conclusions1 Qgp-1 cells can be used as the model of EC cells.2 EPAC1 participates in PIEZ02-mediated mechanical signal transduction,and synergizes to increase intracellular calcium ion level,which eventually leads to the release of 5-HT.Part Three The establishment of rat model of FD disease and syndrome and the intervention mechanism of xiangsha liujunzi decoctionObjectiveBased on the FD rat model of spleen deficiency,the intervention of xiangsha liujunzi decoction on EPAC1-PIEZO2 axis was observed,and the mechanism of xiangsha liujunzi decoction in reducing visceral hypersensitivity was studiedMethods1 The viscera hypersensitive model related to spleen deficiency syndrome of FD was established by the legislation of xiaoping station superimposed with iodoacetamide.2 The model rats were evaluated from the general situation,body weight,drinking water and food intake,VFH fiber experiment,grasping power,sugar water preference experiment,serum lactic acid,d-xylose,amylase content determination experiment.3 The treatment of xiangsha liujunzi decoction on model rats was observed from body weight,water intake and intake,VFH fiber experiment,grasping power,serum lactic acid,d-xylose,am ylase content determination experiment and pathological tissue section staining.4 WB method was used to determine the contents of EPAC1 protein and PIEZO2 protein in the gastric fundus mucosa tissues of rats in each group,and Elisa method was used to determine the contents of 5-HT,cAMP and calcium ion in the gastric fundus mucosa tissues.Results1 Compared with the control group,the body weight,amount of food and water,pain threshold,grasping power,serum d-xylose content and amylase content of rats in the model group with disease and syndrome were significantly reduced(P<0.05).Serum lactic acid concentration increased,and the difference was statistically significant(P<0.05).2 After TCM intervention,compared with the control group,the rats in the model group combined with disease and syndrome and the high,medium and low dose groups of TCM all had significantly lower body weight(P<0.05),but there was no significant difference in body weight between the high and medium dose groups of TCM compared with the positive drug fluoxetine group.Compared with the control group,the food intake of the model group combined with disease and syndrome and the low-dose group of traditional Chinese medicine was still significantly reduced(P<0.05).Compared with the control group,the water intake of the model group with disease and syndrome combination,the fluoxetine group,and the high-dose,medium-dose and low-dose groups of traditional Chinese medicine was still significantly reduced(P<0.05).Compared with the control group,the pain threshold of the model group combined with disease and syndrome,the medium and low dose group of traditional Chinese medicine was still significantly lower(P<0.05).Compared with the control group,the grasping power of rats in the disease-and-syndrome combination model group was still significantly reduced(P<0.05),and there was no significant difference between the other groups and the control group.After administration,the contents of d-xylose and amylase in serum of the rats in each group were significantly increased(P<0.05),and the contents of lactic acid were significantly decreased(P<0.05).There was no significant difference between the two groups compared with the control group.HE staining showed that the gastric fundus mucosa of each group was intact and normal in shape,without obvious hyperemia,edema,erosion or ulcer.3 The levels of 5-HT and cAMP in rats in the symptomatic combination model group were significantly higher than those in the control group(P<0.05),and all indexes tended to return to the normal level after the intervention of traditional Chinese medicine.Compared with the model group,EPAC1 protein and PIEZ02 protein expression in gastric mucosa of rats in the high-dose and medium-dose groups were significantly decreased(P<0.05).Compared with the disease model group,the content of 5-HT in the gastric mucosa of rats in the medium dose group was significantly reduced(P<0.05),which was similar to that in the fluoxetine group.Conclusions1 Intragastric administration of iodoacetamide is a classical method for the establishment of visceral hypersensitive rat model.Standing on a small platform with iodide acetamide superposition is a good method for the establishment of FD spleen deficiency rat model.2 EPAC1-PIEZO2 axis can be used to interfere with the release of EC cells 5-HT,so as to reduce the degree of viscerally hypersensitivity. |
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