| BackgroundFunctional dyspepsia(FD)is characterized by one or more of the following symptoms:postprandial fullness,early satiation,epigastric pain,and epigastric burning,in the absence of any organic,systemic or metabolic disease that readily explains the complaints.FD is one of the most common internal medicine diseases which has a severe effect on the life quality of patients.Jian-pi-li-qi prescription(JPLQ)presented good curative effect on FD by spleennourishing and qi-regulating,which aimed at the core pathogenesis of FD.The pathogenesis of FD is complicated.Enterochromaffin(EC)cells is proved to be relative to visceral hypersensitivity and abnormal motility in functional gastrointestinal disorders.But whether EC cells participate in the gastrointestinal mucosal barrier injury is still unclear.Therefore,this study measured the therapeutic effect of JPLQ and investigate the relationship between EC cells and mucosal barrier tight junctions(TJs)injury in order to reveal the underlying mechanism of FD and provide evidences for the clinical application of JPLQ.Part One:Clinical ResearchSpleen-nourishing and Qi-regulating Principle for FD:Meta-Analysis of Randomized Controlled TrialsObjectives:To assess the efficacy and safety of Chinese herbal medicine of spleennourishing and qi-regulating principle for treating FD.Methods:Literature searches were carried out on PubMed,Embase,Cochrane Library,CNKI database,Wanfang database,and VIP database up to December 31,2019.Study selection,data extraction,quality assessment,and data analyses were performed according to the Cochrane standards.Results:Thirty-one publications in total were suitable for inclusion.There was evidence that Chinese herbal medicine of spleen-nourishing and qi-regulating principle increased symptom improvement(OR 4.15,95%CI 3.40 to 5.07)compared with PPI prokinetic drugs or placebo.Conclusion and significance:Chinese herbal medicine of spleen-nourishing and qi-regulating principle might be more effective compared with PPI prokinetic drugs or placebo in the treatment of FD,and caused less side effects in the included trials.However,the potential benefit needs to be confirmed in rigorously designed,multi-center,and large-scale trials.Part Two:Experimental ResearchObjectiveTo observe the effect of JPLQ on the gastric morphology,gastric compliance and gastric sensitivity of FD rats,so as to verify the FD model of iodoacetamide combined with tail clamping method,and to evaluate the effect of JPLQ on FD model rats.And to detect the effect of JPLQ on the expression of 5-HT and TJs expression in FD rats’ duodenum,and to observe the TJs changes before and after treatment of JPLQ serum,5-HT and blank serum,so as to verify the effects of JPLQ and 5-HT on TJs.And to investigate the changes in the expression and 5-HT secretion function of ECs in FD rats,as well as the effect of JPLQ,so as to reveal the pathogenesis of ECs related to FD and the therapeutic mechanism of JPLQ treatment.MethodsExperiment One:Effect of JPLQ on Morphology and Function of FD Rats’ Stomach36 healthy male Sprague-Dawley(SD)rats were randomly divided into normal group,model group,Chinese medicine intervention group,12 in each group.In addition to the normal group,model group and intervention group used iodoacetamide combined with tail clamping method to establish FD model.Afterwards,intervention group received JPLQ decoction by gavage continuous for 7 days.The model group received saline with equal volume.During this administration,the intervention group and model group consistently received tail clamping.At the end of the intervention,6 rats were randomly selected from each group for anesthesia,gastric tissues were collected for HE staining.And the other 6 rats each group were measured for gastric compliance and sensitivity.Experiment Two:Effect of JPLQ on 5-HT and TJs in Duodenum of FD Rats18 healthy male SD rats were randomly divided into normal group,model group,Chinese medicine intervention group,6 in each group.In addition to the normal group,model group and intervention group used iodoacetamide combined with tail clamping method to establish FD model.Afterwards,intervention group received JPLQ decoction by gavage continuous for 7 days.The model group received saline with equal volume.During this administration,the intervention group and model group consistently received tail clamping.After intervention,18 rats were anesthetized for sampling.Duodenal tissues were detected by Elisa and qPCR methods.And frozen sections were prepared for immunofluorescence(IF)staining.HUTU-80 cells cultured in 12-well plates were randomly divided into 4 groups,normal control group,5HT treatment group,5-HT+Chinese medicine serum treatment group,and 5-HT+blank serum control group.The expression of TJs was detected after the treatment.Experiment Three:Effect of JPLQ on Expression and Function of ECs18 healthy male SD rats were randomly divided into normal group,model group,Chinese medicine intervention group,6 in each group.In addition to the normal group,model group and intervention group used iodoacetamide combined with tail clamping method to establish FD model.Afterwards,intervention group received JPLQ decoction by gavage continuous for 7 days.The model group received saline with equal volume.During this administration,the intervention group and model group consistently received tail clamping.After intervention,18 rats were anesthetized for sampling.The duodenal tissues were detected with qPCR,immunohistochemistry(IHC)and IF methods.ResultsExperiment One:Effect of JPLQ on Morphology and Function of FD Rats’ StomachHE staining showed that the morphology and structure of gastric tissue in each group was intact.The gastric compliance test showed that the gastric compliance of the model group was significantly lower than that of the normal group(P<0.05),while after JPLQ treatment,the gastric compliance of the rats in intervention group was higher than that in the model group(P<0.05).The gastric sensitivity of FD rats were predictably higher than normal rats(P<0.05),while treatment of MLJZ considerably reduced gastric sensitivity of FD rats(P<0.05).Experiment Two:Effect of JPLQ on 5-HT and TJs in Duodenum of FD RatsELISA showed that the expression of 5-HT in the duodenum of model group was higher than that of normal group(P<0.01).And in intervention group it was significantly decreased compared with model group(P<0.05).The results of qPCR showed that the mRNA expression of TJs in the duodenum of model rats was significantly lower than that of normal rats(P<0.01).After the treatment of JPLQ,TJs in intervention group partially recovered(P<0.01).IF staining showed that the fluorescence intensity of TJs was weakened and intermittent after 5HT treatment,and it was enhanced after JPLQ treatment,while the fluorescence remains intermittent after blank serum treatment.IF staining was also used to detect TJs of HUTU-80 cells.The fluorescence of each protein component in normal control group was clearly and coherently distributed along the surface of the cell membrane,which of the 5-HT treatment group was weakened and discontinuous.After Chinese medicine serum treatment,the fluorescence signal partially recovered,while it of blank serum control group was still deficient.Experiment Three:Effect of JPLQ on Expression and Function of ECsPCR showed that the mRNA expressions of PAX4 and TPH1 increased significantly in model group compared with the normal group(P<0.01),while they decreased significantly after JPLQ treatment(P<0.01).And the expressions of TRPA1 and TRPC4 were basically the same in each group(P>0.05).IHC staining showed that the number of 5-HT positive staining cells,PAX4,TPH1 expression,integrated optical density(IOD)in model group were increased(P<0.01)compared with normal group,while after Chinese medicine intervention the number of 5-HT positive staining cells decreased(P<0.05),so as the IOD of 5-HT,PAX4 and TPH1(P<0.01).IF double-staining of 5-HT and TPH1 in duodenum showed that the fluorescence intensity of model rats was enhanced compared with the normal group,while the fluorescence intensity of the intervention group was decreased compared with model group.Conclusion and significanceIodoacetamide combined with tail clamping methods can simulate the pathological state of FD with visceral hypersensitivity and decreased gastric compliance.And JPLQ improved the gastric compliance and sensitivity of FD rats.The abnormal increase of 5-HT expression and the defect of TJs were found in the duodenum of FD rats,and 5-HT could cause the defect of TJs in HUTU-80 cells.JPLQ improved duodenal TJs,and down-regulated the level of 5-HT,and recovered intercellular TJs injury caused by redundant 5-HT.The expression of EC cells,PAX4 and TPH1 in the duodenum of FD rats increased abnormally which may induced overactivation of 5-HT synthesis.By reducing the levels of PAX4 and TPH1,JPLQ reduced the number of EC cells and their secretion of 5-HT. |
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