Topic basis: Xiao Jian Zhong Tang(XJZ),from the Treatise on Cold Damage,consists of six herbs: Cinnamomum cassia(L.)J.Presl,Paeonia lactiflora Pall.,Ziziphus jujuba Mill.,Zingiber officinale Roscoe,Glycyrrhiza uralensis Fisch.and Saccharum granorum.It is mainly used to treat abdominal pain caused by weakness in the middle part of the body and loss of harmony between the liver and spleen.In modern clinical practice,it is mostly used in the treatment of gastrointestinal diseases such as Chronic atrophic gastritis(CAG)and gastric ulcer,and has definite clinical effects.However,there is still a lack of research on the mechanism of action of XJZ in the treatment of CAG,and most studies have only briefly summarized the efficacy of it.Metabolomics is a technical tool to study the changes of metabolites and their relationship with biological functions in biological systems after external interventions during the life course.Metabolomics technology can be used to study the changes of metabolites in body with disease states and after drug treatment,and thus provide data to support the interpretation of drug action mechanism.Meanwhile,as a digestive system disease,CAG is inextricably related to microbiota,so it is also important to study the changes of gastrointestinal microbiota in CAG rats after treatment with XJZ.The stomach is not a sterile environment,and the microbiota of the stomach shows a rich diversity in the disease state.The gut microbiota is considered to be a new major organ of human body,and the colon is the intestinal segment with the most abundant microbiota.Therefore,the study of microbial communities in gastric juice and in colon contents can reflect the microbial differences in CAG rats under disease state and after XJZ treatment more comprehensively.The combination of metabolomics and microbiomics can study the relationship between gastrointestinal microbiota and organism metabolism more deeply,which opens a new perspective to elucidate the efficacy of XJZ.Objective: This study investigated the potential mechanism of XJZ in the treatment of CAG by combining metabolomics and microbiomics.CAG rats were used as the animal pathological model and XJZ as the therapeutic drug.Methods:(1)Firstly,the CAG rat model was constructed,and on this basis,the XJZ low-dose group,XJZ high-dose group,positive drug group,model-antibiotic group,XJZ low-dose-antibiotic group and XJZ high-dose-antibiotic group were set up.During the modeling period,we observed and recorded the changes of rat appearance,body weight,pH and pepsin activity of gastric juice,serum TNF-α,IL-6 and total bile acids.Pathological sections of gastric antrum tissues were performed.At the same time,the inhibitory effect of antibiotics on gastrointestinal microbiota was detected by micro-turbidimetry.Subsequently,the optimal dose was selected based on traditional pharmacodynamic indicators.And LCMS metabolomic analysis of gastric juice and colon contents was performed to obtain differential metabolites for comprehensive evaluation of the efficacy of XJZ.According to the changes in the metabolite of the antibiotic group,it was speculated whether the change in the metabolite content came from the direct regulation of XJZ or the indirect effect of Xiaojianzhong on the regulation of the gut microbiota.(2)The 16 S rRNA high-throughput sequencing technology was used to analyze the changes of gastric juice and colon content microbiota of rats in each group.Then,the effect of XJZ on the bacterial structure of CAG rats was studied,and the microbiotas that were significantly regulated were finally located.(3)The differential metabolites obtained from metabolomics and the differential microbiotas obtained from 16 S rRNA high-throughput sequencing were subjected to spearman correlation analysis,and differential metabolite-difference microbiota pairs with strong correlation were screened out and analyzed.The possible interactions between correlated pairs were finally verified by molecular docking.Results:(1)The apparent pharmacodynamics of rats in each group was tested,and it was found that the rats in the model group had withered and yellow fur,white tails,low mental state and weak response to external stimuli.In addition,their pH value of gastric juice increased,the activity of pepsin decreased,the gastric mucosa atrophied and inflammation occurred,and the contents of serum TNF-α,IL-6 and total bile acid all increased significantly,indicating that the CAG model was successfully replicated.After treatment with XJZ,all indicators can be significantly corrected,but the callback effect of the high-dose group is better than that of the low-dose group.After antibiotics intervened in the administration group,the callback effect decreased,indicating that the microbiota played an important role in the efficacy of XJZ.Micro-turbidimetry assay results showed that after antibiotic intervention,the fecal microbiota content of rats was significantly reduced.And its proliferation rate was significantly reduced.Metabolomics results showed that compared with control group,metabolites in the model group changed significantly.There were 14 differential metabolites in gastric juice,and 7 differential metabolites could be recalled after XJZ treatment.It is speculated that XJZ can directly regulate 2 of the metabolites,and affect the remaining 5 metabolites by regulating the microbiota.There are 19 differential metabolites in the colon contents,11 of which can be significantly reversed after XJZ treatment.It is speculated that 4 of the metabolites are directly regulated by XJZ,1 is jointly regulated by XJZ and the microbiota,and the changes of the remaining 6 metabolites come from the indirect regulation of XJZ on the microbiota.(2)The 16 S rRNA sequencing results showed that the gastrointestinal microbiota of CAG rats was significantly changed.In the gastric juice,3 phylum,4 families,6 genera and 6 species were significantly altered,and the abundance of 1 phylum,3 families,4 genera and 5 species were significantly regressed after drug administration.In the colon,3 phyla,8 families,9 genera and 9 species were significantly altered,and the abundance of 1 phylum,3 families,4 genera and 5 species could be significantly adjusted after administration.It indicates that the pathogenesis of CAG is closely related to the disorder of gastrointestinal microbiota,and XJZ treatment can improve this disorder of microbiota.(3)Spearman’s correlation analysis was performed to find the correlation between metabolome and microbiome,in which there were 9 pairs of strong correlations with |r|greater than 0.6 in gastric juice.There were 16 strongly correlated pairs with |r| greater than0.6 in the colon contents.Subsequently,the active compounds of each single herb of XJZ were collected,and the molecular docking verification was performed for the pairs of "XJZmetabolite" and "XJZ-microbiota" that might have mutual regulation.It is speculated that the active compounds of XJZ can regulate the synthesis and metabolism of L-Tyrosine,LPhenylalanine,PE(17:0/0:0),Cervonoyl ethanolamide,Glycocholic acid,Urocanic acid and Tyramine,while the active compounds of XJZ can inhibit the proliferation of Escherichia coli and Streptococcus miti in gastric juice,inhibit the proliferation of Bifidobacterium pseudolongum,Lachnospiraceae bacterium G11 and Phascolarctobacterium faecium in colon,and promote the proliferation of Lactobacillus intestinalis.Conclusion: This study was conducted from the perspective of "XJZ-CAGGastrointestinal microbiota".The efficacy of XJZ was evaluated using traditional pharmacodynamic indicators,and the optimal dose was selected.Metabolomics was used to screen for disease-differentiating metabolites in CAG rats.Microbiomics identified species closely associated with CAG.Subsequent metabolomics and microbiomics correlation analysis showed that the gastrointestinal microbiota played an important role in the efficacy of XJZ,especially the inhibition of some harmful bacteria and the promotion of beneficial bacteria may be the potential mechanism for the efficacy of XJZ. |