Study On The Interaction Between DBT And Gut Bacterials Based On Metabolome And Microbiome

Objective:To study the interaction between Danggui Buxue Tang（DBT）and gut bacterial,including the effect of gut bacterial on the absorption of DBT active ingredients,and the effect of DBT on gut bacterial and gut function.Methods:1)The effect of gut bacterial on the absorption of DBT active ingredients:We optimized the detection method of reticulocytes and detected the change of DBT efficacy after destroying the balance of gut bacterial by antibiotics.16S rRNA microbial sequencing was used to analyze the changes of gut bacterial.UPLC-MS/MS was used to detect the changes of serum metabolites and find the potential components of DBT.We established the correlation between the potential components and the efficacy of DBT to determine the DBT active ingredients.The network pharmacology was used to find the signal pathway of the efficacy of DBT.We analyzed the correlation between the gut bacterial and the DBT potential components by Spearman.The functional anotation of the above gut bacterial were finished by PICRUSt 2,so as to determine whether the gut bacterial can affect metabolism and absorption of the DBT active ingredients.2)The effect of DBT on the gut bacterial and gut function:The model of antibiotic was made in mice and Danggui Buxue Tang were infused into stomach.UPLC-MS/MS was used to detect the changes of metabolic components and pathways in serum.The changes of gut bacterial in faeces were detected by 16S rRNA microbial sequencing.The correlation between gut bacterial and metabolic components was analyzed by Spearman.We analyzed obvious correlation between gut bacterial and the index of gut function.PICRUSt 2 was used to predict the metabolic pathway and function of gut bacterial.HE staining of ileum and colon was used to observe the effect of antibiotics and DBT.We explored the changes of gut villi and goblet cells,so as to judge whether DBT can alleviate gut disorder and restore gut function.Result:1)Optimization of reticulocyte detection method:After optimization of dyeing time and concentration,MTG/TMRE/Hoechst method can detect the proportion of reticulocytes in peripheral blood,which was about（5.11±0.53）%.At the same time,it can be divided into three development stages,including R1(MTG ^neg/TMRE ^high),R2(MTG ^high/TMRE ^high)and R3(MTG ^high/TMRE ^neg).Compared with the traditional method（new methylene blue manual method）,the results were similar.R=0.817（P<0.01）and the CV value was9.75,which was smaller than the traditional method.2)The effect of gut bacterial on the absorption of DBT:Compared with the Control group,the efficacy indexes of DBT group were significantly increased,including the proportion of peripheral blood reticulocytes（P=0.031）,BFU（P=0.011）,CFU（P=0.009）and the number of bone marrow cells（P=0.018）.After antibiotic treatment,the Ret%of the ABXDBT group decreased significantly（P=0.036）.The results of UPLC-MS/MS showed that there were 194 metabolic components and 18 DBT potential compounds contents changed in ABXDBT group,compared with DBT group.11 potential compounds such as caffeic acid and anthocyanin were significantly decreased.The correlation analysis showed that 6 DBT potential components such as caffeic acid and formononetin were related to the decrease of DBT efficacy.Network pharmacology analysis showed that the above active ingredients involved in the regulation of DBT hematopoiesis through PI3K-Akt and HIF-1 signaling pathways.The results of correlation analysis showed that Bacteroides and Rickenellaceae RC9 gut group were related to the absorption of DBT.The function of the above gut bacteria was predicted by PICRUST 2,and it was found that the drug metabolizing pathway of gut bacteria was significantly reduced after the action of antibiotics（P=0.033）.It was predicted that DBT efficacy was affected by Bacteroides and Rickenellaceae＿RC9＿gut＿group through affecting the absorption of caffeic and formononetin.3)The effect of DBT on gut bacterial and gut function:The results of 16S rRNA microbial diversity analysis showed that compared with the Control group,82 genera of gut bacterial in mice were significantly changed after antibiotic treatment;12 genera of gut bacterial had a recovery trend after DBT gavage,such as Bacteroides.The serum metabolites were detected by UPLC-MS/MS and VIP≥1,or fold change≥2 and fold change≤0.5 were used as screening conditions.Compared with the Control group,26 metabolites decreased significantly in ABX group including bile acid such as deoxycholic acid and 23 metabolites increased significantly such as erucic acid.Compared with ABX group,20 metabolites decreased significantly in ABX group such as coumaric acid and 16 metabolites increased significantly such as taurocholate.According to the analysis of KEGG,the metabolic pathways that had great influence after the action of antibiotics include bile acid biosynthesis and phenylalanine metabolism.By Spearman’s analysis,Bacteroides,Lachnospiraceae＿NK4A136＿group and Rikenellaceae＿RC9＿gut＿group were related to the potential differential metabolites such as taurodeoxycholic acid,deoxycholic acid and chenodeoxycholic acid.The results of PICRUSt 2 on functional prediction showed that the biosynthesis of primary bile acid and secondary bile acid had significant effects.It is speculated that DBT can affect the metabolism of bile acids by restoring the abundance of Bacteroides.4)Effect of DBT on gut function:Compared with the Control group,inflammation happened in the ileum and colon of mice in the ABX group.The number of goblet cells was significantly reduced（P<0.001）,and the length of lleum villi was significantly shorter（P<0.01）.Compared with ABX group,the inflammation in ileum in ABXDBT group was reduced,the number of goblet cells was increased and the villi became longer.By MaAslin analysis,we found that Bacteroides and Alloprevotella was significantly correlated with goblet cells and villi length.Through Spearman’s analysis,we can conclude that the metabolites of deoxycholic acid and chenodeoxycholic acid have a significant positive association with the number of goblet cells in ileum and colon.Conclusion:1)Based on the change of mitochondrial membrane potential,MTG/TMRE/Hoechst method can detect reticulocytes in peripheral blood at low cost and accurately.Meanwhile,reticulocytes can be divided into three stages:R1(MTG ^neg/TMRE ^high),R2(MTG ^high/TMRE ^high)and R3(MTG ^high/TMRE ^neg).2)Gut bacterial affects the absorption and metabolism of DBT.Bacterial such as Bacteroides and Rikenellaceae＿RC9＿gut＿group affect the efficacy of DBT by affecting the absorption of effective components such as caffeic acid and formononetin.3)DBT restored the effect of antibiotics on gut bacterial disorder,metabolic pathway and gut inflammation.DBT can restore the abundance of Bacteroides and other bacteria,thus affecting the metabolism of bile acid in vivo.Bacteroides is positively correlated with goblet cells and villi length in ileum,while metabolites such as chenodeoxycholic acid is positively correlated with goblet cells in ileum and colon.