Combining Intestine Microbiota And Metabolomics To Explore The Integrated Regulatory Mechanism Of Ji-Chuan Decoction For Slow Transit Constipation (STC)

Aim: Slow transit constipation(STC)is a common functional digestive system disease with a high incidence and many patients.It is a disease related to various factors such as enteric nervous system(enteric neurons,enteric glial cells,enteric neurotransmitters),intestinal flora,metabolic changes,smooth muscle dysfunction,abnormal levels of gastrointestinal hormones,pelvic floor muscle dysfunction and abnormal interstitial cells of Cajal,etc.Ineffective treatment of modern medicine.Ji-chuan Decoction(JCD)is a classic prescription for the treatment of constipation.It has been widely used for the treatment of STC for a long time with good curative effect,but its specific pharmacological mechanism is still unclear.This experiment will reveal the pharmacological mechanism of JCD in the treatment of STC by combining microbiome,metabolomics and network pharmacology with molecular experiments.Materials and Methods: 1.Experimental grouping,STC model-made and JCD treatment.30 C57BL/6J mice were randomly divided into 6 groups with 5 mice in each group,namely: healthy control group(HC),STC model(STC),positive drug treatment(mosapride,MSP),JCD low dose group(JCDL),JCD middle dose group(JCDM)and JCD high dose group(JCDH).The mice in the HC group were intragastrically administered with normal saline(0.1 ml/10 g.d)as a negative control,and the remaining mice were induced to become the STC model by intragastric administration of compound diphenoxylate tablets(10mg/kg.d)for 14 days.After model identification,the mice in the MSP group were given mosapride(2.5 mg/kg);JCDL,JCDM and JCDH were given 3.04 g/kg,6.08 g/kg and 12.16 g/kg JCD,respectively;the HC group and STC group were given Normal saline(0.1 ml/10 g.d)was administered by gavage.Each mouse was dosed once a day for 14 days.The food consumption,water consumption and body weight changes of the mice were detected weekly during modeling and treatment.After the treatment,the feces and colon tissues of the mice in each group were collected,and the number of defecation grains,wet weight,dry weight and water content within 6 hours,and the propulsion rate of activated carbon were detected.The content of colonic acetylcholine(ACH)was detected by Elisa,and the pathological changes of colon were observed by HE staining.2.Intestinal microbiota analysis.Detect the fecal microbiota of mice in each group by 16 S r RNA sequencing technology.Rarefaction Curve,Rank Abundance,and species accumulation boxplot were used to judge whether the sequencing depth and sample size were appropriate.The top ten species in abundance at the phylum and genus levels were observed,and differences in abundance between species were analyzed by T-test.The similarity between groups was observed by UPGMA clustering tree.The difference species between groups and their influence on the difference were found by LEf Se analysis.3.Metabolomic analysis of the intestinal contents.Differentially expressed metabolites and metabolic pathways were detected by LC-MS.Total sample PCA analysis observed system stability and data quality.Between-group PCA was used to analyze between-group separation trends and the presence or absence of abnormal samples.PLS-DA identified differences between the two groups,as well as the interpretation and prediction rates of the model.Visualize differential metabolites with volcano plots.Cluster analysis of total differential metabolites showed the overall difference size between groups.Metabolite pathway analysis identified metabolic pathways that were significantly altered by JCD treatment.4.Network pharmacology combined with metabolomics analysis and molecular experimental validation.JCD targets were collected by TCMSP and BAT-MAN,and STC targets were collected by OMIM and Gene Cards.Collect common targets of drugs and diseases through Omicshare.By sorting out the relationship between compounds and common targets,it is presented with Cytoscape software,and important compounds and targets are determined by the degree of connectivity.The interaction relationship between common targets was established by STRING11.0,and the top 30 nodes in the betweenness centrality algorithm were used as Hub nodes by the cyto Hubba plug-in of Cytoscape software.The expression of AKT in Hub nodes was detected.Combined analysis of common targets and differential metabolites,combined with literature reading,put forward the hypothesis that JCD affects STC by regulating enteric glial apoptosis.Using immunofluorescence double staining to confirm our suspicions,and then we detected the expression of GDNF and the expression of excitatory enteric nerve marker Ch AT and inhibitory enteric nerve marker n NOS.Results: 1.JCD significantly improved the defecation indexes and pathological changes of STC mice.After modeling,there were significant differences between the model group and the healthy control group in terms of body weight,6-hour defecation grain count,stool water content,activated carbon propulsion rate,colonic acetylcholine content and colonic inflammatory infiltration,and the modeling was successful.The treatment of each dose group of JCD can correct the changes in body weight,6-hour bowel movement,stool water content,activated carbon propulsion rate,colonic acetylcholine content and changes in colonic inflammation caused by modeling,and the treatment is effective.2.JCD repairs the structure and function of intestinal flora at multiple levels.Among the top ten species in abundance at the phylum and genus levels,Verrucomicrobia and Akkermansia were decreased during modeling and increased after JCDH treatment,which are considered to be the key bacteria affecting constipation.The UPGMA clustering tree showed that the fecal microbiota of the HC group and the JCDH group had a high similarity,but had a greater difference with the STC group.The LEf Se branch evolution map shows that the HC group mainly includes Verrucomicrobiae,Verrucomicrobiales and Akkermansiaceae as the dominant flora,the JCDH group is dominated by Gammaproteobacteria Burkholderiales and Oxalobacteraceae,and the STC group is Bacterodia,Bacteroidales and Muribaculaceae.The family is the dominant flora.The LDA value distribution histogram shows that the HC group mainly includes Verrucomicrobiota and Akkermansia as the dominant flora,the JCDH group has the Proteobacteria and Herbaspirillum as the dominant flora,and the STC group has the Bacteroidota and Muribaculaceae and Enterococcaceae as the dominant flora. 3.JCD regulates metabolic pathways such as Taurine and hypotaurine metabolism.The PCA analysis of the total sample shows that the system is stable and the data quality is high.Compared with the STC group,the distance between the HC group and the JCDH group was closer,indicating that the mice in the HC group and the JCDH group had similar metabolic patterns.Principal component analysis(PCA)showed that the samples of the STC group and the HC group had a certain degree of discrimination,and there was also a certain degree of discrimination between the STC group and the JCDH group.The STC model and the treatment of JCDH all made the mice’s susceptibility.Metabolic patterns have changed somewhat.PLS-DA(Partial Least SquaresDiscriminant Analysis)shows that the model is robust with good interpretation and prediction rates.Differential metabolite screening results showed that 42 metabolite concentrations(negative ion mode)changed during modeling,86 metabolites changed during JCDH treatment,and 18 of these metabolites were altered during compound diphenoxylate modeling,and then reversed after JCDH treatment.Clustering analysis of total differential metabolites showed that the metabolic patterns of HC and JCDH were similar,with a greater difference from the STC group.Metabolic pathway analysis showed that Taurine and hypotaurine metabolism and arachidonic acid metabolism changed significantly during JCDH treatment.4.JCD regulates intestinal motility by regulating the expression of AKT,the apoptosis of enteric glial cells and the number of enteric nerves.280 drug targets,1372 disease targets and 132 common targets were screened.The compound target network identified five important active components: quercetin,kaempferol,wogonin,nobiletin and naringenin;and five important targets: SCN5 A,PTGS2,AR,ESR1 and PTGS1.The top 30 nodes with the largest Betweenness value in the PPI network were used as Hub targets,and the changes of AKT were confirmed by RT-PCR and immunohistochemistry.The combined network pharmacology and metabolomics analysis of common targets and differential metabolites showed that JCD may affect constipation through signaling pathways such as apoptosis and PI3K/Akt.Experiments confirmed that the apoptosis of enteric glial cells was increased during STC modeling and decreased during JCD treatment.GDNF decreased during STC modeling and increased during JCD treatment.Excitatory enteric nerve Ch AT decreased during STC modeling and increased during JCD treatment.Inhibitory enteric nerve n NOS increased during STC modeling and decreased during JCD treatment.Conclusion: 1.JCD can restore the intestinal flora of disordered STC mice,and Akkermansia muciniphila plays a key role.2.JCD can restore the metabolic disorders of STC mice,especially the metabolism of Taurine and hypotaurine.3.JCD treats STC by regulating the apoptosis of intestinal glial cells and the number of enteric nerves.