| Objective: This subject focused on a classical prescription Banxia Xiexin Decoction(BXD).Alcohol was applied to establish the rat model of chronic gastritis,and the mechanism of BXD in treating chronic gastritis was analyzed by observing the effects of BXD on the content of serum inflammatory factors and the expression of gastric mucosal repair factors in rats with chronic gastritis.The effect of BXD on gastrointestinal microbiota was studied using 16S rRNA high throughput sequencing technique.UHPLC-LTQ-Orbitrap-MS was utilized to analyze the metabolism of flavonoids and alkaloids in BXD by gut microbiota in rats.This article hopes to lay a foundation for the study of pharmacodynamic material basis and clinical application of BXD by studying the interaction between BXD and gastrointestinal microbiota.Methods: 1.Effect of BXD on inflammatory factors and gastric mucosa repair factors in rats with chronic gastritis Chronic gastritis was induced by oral administration of 56% ethanol solution in clean SD rats.Gastric tissues near the antrum were stained with HE method to observe the pathological changes of gastric tissues in each group and determine whether the model was successfully established.The serum levels of IL-2,IL-8 and TNF-? in normal group,model group,administration group and positive drug group were determined by double antibody sandwich ELISA,and the expression levels of EGF and Bcl-2 in gastric tissue were detected by Western blot.2.Study on the diversity of gut microbiota in rats with chronic gastritis treated with BXD 16S rRNA high throughput sequencing technique was applied to analyzed the microbial diversity of gut microbiota in rats with chronic gastritis before and after treatment,and statistical analysis of OTU was carried out at a similarity cutoff of 97%.Compared with Silva and Greengene databases,the composition of each sample community was analyzed at Phylum and Genus levels.Through species difference analysis,the differentiated species were finally searched at the level and the species function was annotated.3.Metabolism of flavonoids and alkaloids in BXD by gut microbiota Flavonoids and alkaloids in BXD group,Kujiang group,Xinkai Kujiang group and Ganbu Kujiang group were studied.Anaerobic incubation of gut microbiota in vitro was used to co-culture the drug extracts from each group with the intestinal contents of rats under anaerobic conditions at 37 ?,the metabolism of flavonoids and alkaloids in gut microbiota was analyzed by UHPLC-LTQ-Orbitrap-MS,and the contents of prototype components and their metabolites were determined.Finally,a multivariate statistical analysis method was established based on the relative content of each component to find and explain the causes of metabolic differences among different compatibilities.Results: 1.Effect of BXD on inflammatory factors and gastric mucosa repair factors in rats with chronic gastritis Compared with normal rats,lymphocytic infiltration of gastric antrum in lamina propria,submucosa,and serous layer,a few lymphocyte cells scattered in the muscular layer,and submucosal edema and congestion of lamina propria were observed in the model rats,suggesting that the experimental model of ethanol-induced chronic gastritis was successfully established.Chronic gastritis rats showed significantly increased serum IL-2,IL-8 and TNF-? levels as well as reduced expression of EGF and Bcl-2 proteins,large numbers of inflammatory factors accumulated and the integrity of gastric mucosa was damaged.After treatment,the pathological morphology of gastric tissue of rats in BXD group,mixture group of bacalin and berberine,and positive drug group improved significantly,and the levels of serum inflammatory factors and gastric mucosal repair factors gradually returned to normal.The treatment effect of high dose was better in BXD group and mixture group,indicating that the treatment of chronic gastritis was dose-dependent with BXD and the mixture of baicalin and berberine.2.Study on the diversity of gut microbiota in rats with chronic gastritis treated with BXD At the OTU level,726 taxon taxa were found,and microbial community information of 11 phyla and 145 genera were obtained by taxonomic analysis.Multivariate statistical analysis of the data showed that the differences among the sample groups are significantly greater than those within the group,and the grouping is of practical significance.Compared with the normal group,the relative abundance of Family?XIII?UCG-001?Facklamia?Rothia? norank?f?Bacteroidales?S24-7?group?norank?f?Lachnospiraceae?Unclassified?f?Family?XIII?Oscillibacter?Coprococcus?2 in the model group changed significantly.BXD group and mixture group could regulate the relative abundance of these differential bacteria and treat chronic gastritis at microbial level.3.Metabolism of flavonoids and alkaloids in BXD by gut microbiota After co-culture with gut microbiota,the prototype components of alkaloids,such as jatrorrhizine,coptis,palmatine,berberine and the prototype components of flavonoid,such as baicalin,wogonoside and liquiritin,were metabolized into 14 metabolites.The metabolites were identified by Compound Discoverer 2.1 software and their metabolic pathways were determined according to the precise molecular weight,secondary mass spectrometry fragment information and literature reports.Xcalibur 2.2.0 and SIMCA-P 14.1 software analysis showed that the metabolite of jatrorrhizine M8(VIP value 1.4084),glycyrrhizin(VIP value 1.2287),jatrorrhizine(VIP value 1.1746),the metabolite of coptisine M2(VIP value 1.0975),baicalein(VIP value 1.0891),the metabolite of glycyrrhizin M14(VIP value 1.0832),glycyrrhizin(VIP value 1.0803)and wogonin(VIP value 1.0121)contributed most to the classification results of multivariate statistical analysis,which was the main reason for the difference of metabolism of Banxia Xiexin Decoction and its different compatibilities of enterobacter.Conclusion: This subject foucs on Banxia Xiexin decoction,a classical prescription of traditional Chinese medicine.In this study,we established a rat mocel of chronic gastritis by intragastric administration with high concentration ethanol.By measuring the contents of serum inflammatory factors and the expression of gastric mucosal repair factors,we found that Banxia Xiexin decoction and the mixture of baicalin and berberine could reduce the levels of IL-2,IL-8 and TNF-?,and enhance the expression of EGF and Bcl-2 in rats with chronic gastritis.Using 16S rRNA high-throughput sequencing technology,the pathogenesis of chronic gastritis and the therapeutic effect of Banxia Xiexin decoction were explained at the microbial level.The results showed that Banxia Xiexin decoction could improve chronic gastritis by regulation the relative abundance of differentiated bacteria in normal group and model group.Using in vitro anaerobic incubation technology of gut microbiota and UHPLC-LTQ-Orbitrap-MS technology,the metabolism of gut microbiota to flavonoids and alkaloids in Banxia Xiexin decoction was analyzed,and its prototype composition and metabolites were successfully identified.Combined with multivariate statistical analysis,the causes of clustering differences after metabolism of enterobacteria in different compatibility Banxia Xiexin decoction were preliminarily clarified.It is hoped that the study on the interaction between Banxia Xiexin decoction and gastrointestinal flora will lay a foundation for the pharmacodynamic substances and clinical application of Banxia Xiexin decoction. |
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