| Research BackgroundGastric electrical rhythm disorders are very common,they are closely related to the occurrence of gastric functional disorders.And it is an important cause of gastric motility dysfunction.Gastric dyskinesia can lead to delayed gastric emptying,the main clinical manifestations are epigastric pain,post-prandial abdominal distension,early satiety,belching,nausea or vomiting,and other symptoms of dyspepsia.Modern medicine calls it Gastric Dysrhythmia Syndrome(GDS).Chinese medicine in the name of symptoms of these diseases,including the fullness syndrome,belching,vomiting,hiccups,nausea and so on.In clinical,30%-40%of patients with digestive tract diseases are eventually diagnosed with gastrointestinal motility disorders,which have increasingly affected people's health and quality of life.Banxia Xiexin Tang is an famous prescription recognized by ancient physicians as"eliminating distention and fullness and dissolving knots"(regulating gastrointestinal motility disorders),commonly used to treat the epigastric fullness of cold-heat complex and Qi lift imbalance.It is mainly applied to acute and chronic gastritis,functional dyspepsia(FD),bile reflux gastritis,reflux esophagitis(RE),and peptic ulcer(PU),irritable bowel syndrome(IBS),helicobacter pylori(Hp)infection,diabetic gastroparesis(DGP)and other diseases in modern clinical practice.Pharmacological studies have found that Banxia Xiexin Tang has many functions,such as up-regulating the expression of gastric mucosal protective factors,reducing inflammatory injury of gastric mucosa epithelial cells,promoting repair of injured gastric mucosa,increasing ghrelin levels in gastric antrum,reducing the main virulence factors of Helicobacter pylori Expression,regulating immunization,regulating interstitial cells of Cajal,and regulating neuroendocrine systems.Therefore,it is of great clinical value to explore the mechanism of regulating gastrointestinal motility by Banxia Xiexin Tang from the angle of microRNA molecular pathogenesis.MicroRNA(miRNA)is a class of non-coding,single-stranded small molecule RNA consisting of 18?22 nucleotides that is complementary to the 3'nd non-coding region of the mRNA,leading to mRNA degradation or translation inhibition,has become an important way of gene regulation after transcription.The human genome contains>1,800 miRNA-coding genes,and about>60%of human protein-coding genes are predicted to bemicroRNA targets.Functionally,miRNAs can target mRNA molecules involved in various biological processes,such as development,differentiation,proliferation,apoptosis and tumorigenesis.In recent years,it has been found that miRNAs are involved in the regulation of genes such as IBS and Hp infection.MiRNA as an important post-transcriptional gene regulatory element,its expression directly affects the function of the relevant coding protein gene and participates in a number of systemic pathophysiological processes including the digestive system.At present,the regulation of miRNA in digestive diseases and the specific mechanism,mainly in its relationship with the tumor,immune,and the relationship between gastrointestinal motility disorders has little reported.Therefore,it is necessary to carry out further research.ObjectiveRat model of gastric dysrhythmia was established,to study the changes of gastric tissue miRNA expression profile in rats with gastric dysrhythmia,and to explore the intervention effect of Banxia Xiexin Tang on specific miRNAs of gastric tissue with rat model of gastric electrical rhythm disorders.As a carrier,to seek the relationship between miRNA and gastric motility disorder,to reveal the molecular pathogenesis gastric motility disorders,to further grope for the mechanism of Banxia Xiexin Tang on gastrointestinal motility disorders.Methods30 healthy male SD rats were divided into normal control group and model group.The model group used a single day diet and a double fasting method(drinking diluted hydrochloric acid water during the period)to replicate the gastric dysrhythmia model.And evaluate model by detecting the rat gastric electric rhythms,calculating slow wave frequency and slow wave frequency coefficient of variation.Rats with successful modeling were randomly divided into model control group and Banxia Xiexin Tang group.The normal control group and model control group were given deionized water to gavage,and Banxia Xiexin Tang group was given Banxia Xiexin Tang liquid gavage.The microarray technology was used to analyze the microRNA expression profiles of gastric tissue in rat model of gastric dysrhythmia.Hierarchical clustering was performed to show the distinguishable miRNAs expression pattern among samples.The Targetscan databases and microRNAorg databases were used to perform target gene prediction on the differential miRNAs,and then GO analysis and KEGG analysis were applied to determine the roles of these target genes.RT-PCR was used to verify the results of the selected miRNA chip.The effect of Banxia Xiexin Tang on the differential expression of microRNA was detected by real-time quantitative PCR.Results(1)The frequency of slow wave in normal control group rats was 3.59±0.28 times/min.Compared with the normal control group,the slow wave frequency coefficient of variation increased in the model group(P<0.05).(2)MicroRNA expression profile and bioinformatics analysis of gastric tissue in gastric dysrhythmia model ratsThere were 10 differentially expressed miRNAs,of which only one was up-regulated(rno-miR-3102)and nine were down-regulated(rno-miR-7b,rno-miR-221-3p,rno-miR-451-5p,mo-miR-148a-3p,rno-miR-33-5p,rno-miR-3068-3p,rno-miR-582-5p,rno-miR-370-3p,rno-miR-770-3p).GO analysis:A total of 889 potential target genes were predicted,the biological processes associated with it have brain development,positive regulation of transcription from RNA polymerase ? promoter,transcription,DNA-templated,vesicle-mediated transport,protein dephosphorylation,MAPK cascade,neuro projection development,nervous system development,intracellular protein transport,etc.;cellular components include Golgi membrane,extracellular exosome,cytoplasm,membrane,dendrite,neuronal cell body,cell junction,cell surface,endoplasmic reticulum,etc.;has molecular functions such as protein binding,calmodulin binding,voltage-gated potassium channel activity,protein kinase binding,ligase activity,GTPase binding,myosin light chain binding,and magnesium ion binding and so on.Pathway Analysis:MAPK signaling pathway,cell cycle,cAMP signaling pathway,TNF signaling pathway,cGMP-PKG signaling pathway,gastric acid secretion and other pathways may be related to the regulation of miRNA expression in gastric electric rhythm disorders.(3)A total of 10 distinct differentially expressed miRNAs screened by microarray hybridization were verified by RT-PCR.In addition to the melting curves of the genes mo-miR-33-5p and rno-miR-370-3p which were double-peaked and contain non-specific amplification,the melting curves of the remaining genes were all single peaks,suggesting that the product was unique,and the specificity of PCR amplification was better.2-??ct analysis showed that the results of rno-miR-3102,rno-miR-7b,rno-miR-451-5p,rno-miR-148a-3p,rno-miR-3068-3p,rno-miR-582-5p,rno-miR-370-3p,rno-miR-770-3p were all consistent with the results of miRNA gene chip detection.(4)In the 8 specific miRNAs detected by RT-PCR,the expression of rno-miR-3102 was increased compared with the normal control group,rmo-miR-7b,no-miR-451-5p,4no-miR-148a-3p,rno-miR-3068-3p,rno-miR-582-5p,rno-miR-370-3p,rno-miR-770-3p expression decreased;compared with the model control group,Banxia Xiexin soup can increase expression of rno-miR-7b,rno-miR-451-5p,rno-miR-148a-3p,decreased the expression of rno-miR-3102,rno-miR-3068-3p,rno-miR-582-5p,rno-miR-370-3p,rno-miR-770-3p.ConclusionMicroRNA was significantly differently expressed in gastric tissue of gastric electric rhythm disorders model rats.Bioinformatics analysis showed that multiple microRNAs were involved in gastric electrical rhythm disturbance.These differential expressions may be related to the formation of gastric motility disorder,which can provide a basis for further study of the molecular mechanism of gastric motility.Combined with miRNA microarray results,Banxia Xiexin Tang can down-regulate the expression of rno-miR-3102 and up-regulate the expression of rno-miR-7b,rno-miR-451-5p and rno-miR-148a-3p,so as to regulate the dysregulation of gastric electrical rhythm.It can provide certain conditions for exploring the mechanism of Banxia Xiexin Tang in the gastrointestinal motility disorders. |
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