In Vitro Metabolism Of Gut Microflora Of Spleen Deficiency IBS-D Cultured With Sijunzi Decoction

Objective:Irritable Bowel Syndrome（IBS）is a common digestive tract disease,which is common in IBS（IBS-D）with diarrhea due to spleen deficiency.Sijunzi Decoction is a common prescription for IBS-D of spleen deficiency type,but the direct interaction mechanism between Sijunzi Decoction and gut microflora has not been studied.In this study,gut microflora of IBS-D patients with spleen deficiency were co-cultured with Sijunzi Decoction to explain the characteristics of intestinal microecological disorders in IBS-D patients with spleen deficiency,and to dynamically reveal the target and regulation mechanism of gut microflora co-metabolism with Sijunzi Decoction in IBS-D patients.Methods:（1）Inclusion and clinical sampling of IBS-D cases with spleen deficiency.Typical cases of IBS-D with spleen deficiency were included as well as healthy control group.The feces of the volunteers were collected by a fecal collector containing 2 ml transporter.The feces of each member in the same group were mixed and separated.The feces were stored in a low-temperature refrigerator at-80 ℃ for reserve.（2）Human gut microflora was co-cultured with Sijunzi Decoction in vitro.They were divided into normal group and IBS-D group and cultured with in vitro feces incubation method.In the same group,1 g gut contents were taken from each sample and mixed with normal saline and culture medium to prepare IBS-D incubation solution with healthy human intestinal bacteria.The two groups of intestinal bacteria incubation solution were mixed with Sijunzi Decoction at 4:1 and cultured at 37（% CO₂,80% N₂）for 0,6,12,24 and 35 hours respectively.The time window samples were collected and centrifuged at 4000 g and 4 ℃ for 10 minutes.The supernatant and the bacteria were separately packed and stored in the refrigeratorat-80 ℃ for reserve.（3）16S rRNA gene sequencing of gut microflora.16 S rRNA gene sequencing platform was used to analyze the relative abundance,taxonomic tree,sample complexity and sample diversity of culture media at different time points at phylum,class,order,family and genus levels.（4）Analysis of co-metabolites of gut microflora in Sijunzi Decoction.Using LC-MS platform,the metabolites of each group at different time points were analyzed by differential metabolites analysis,correlation analysis and KEGG enrichment analysis.Spearman statistical method was used to analyze the correlation between different metabolites and relative abundance of Top 10 species at OTUs level,and to explore the co-metabolic regulation network between Sijunzi Decoction and gut microflora.Results:Using the latest diagnostic criteria of Chinese and Western medicine,5 IBS-D patients with spleen deficiency and 5 healthy volunteers of similar age were included.The fecal samples of each member were mixed to complete the co-culture of Sijunzi Decoction with gut microflora.The culture samples were obtained at 0,6,12,24 and35 hours.The results of 16 S rRNA sequencing revealed that the phylum level of the normal group and IBS-D group were dominated by sclerenchyma,proteobacteria,actinomycetes and bacteroides.The abundance of sclerenchyma in IBS-D group was lower than that in the normal group,while that of actinomycetes,Bacteroides and Proteobacteria was higher than that in the normal group.At the generic level,the two groups were mainly Lactobacillus,Streptococcus,Shigella,Bifidobacterium and Clostridium.The abundance of Streptococcus in IBS-D group was lower than that in normal group,while that of Shigella was higher than that in normal group.During co-culture,the abundance of Lactobacillus and Bifidobacterium increased,the abundance of Clostridium and Shigella decreased,and the B/E value increased gradually.Species diversity analysis showed that the core microflora of the two groupswere relatively stable during co-culture.Alpha diversity index analysis showed that there was no significant difference in species number between IBS-D group and normal group（P > 0.05）,but the species number of normal group was significantly higher than that of IBS-D group（P < 0.05）.Beta diversity analysis showed that the difference between normal group and IBS-D group was smaller than that between groups;with the prolongation of culture time,the difference between the two groups gradually narrowed.At the same time,the difference of community structure between normal group and IBS-D group was significant,and the difference between groups was smaller than that between groups（P < 0.05）.PCoA analysis and NMDS analysis showed that IBS-D group and normal group could be separated well,and the species composition was significantly different.With the prolongation of incubation time,the difference of species structure between the two groups at the same time window tended to decrease.Cluster tree analysis showed that the clustering distance between the two groups became shorter with the prolongation of culture time,indicating that Sijunzi Decoction could gradually restore the IBS-D microflora structure to normal.T-test analysis showed that there were significant differences between normal group and IBS-D group in phylum,class,order,family and genus.At the gate level,the abundance of sclerenchyma in IBS-D group was lower than that in normal group（P < 0.01）,and Proteus was higher than that in normal group（P < 0.01）.At the generic level,the abundance of Streptococcus and Paeniclostridium in IBS-D group was lower than that in normal group（P < 0.01）,while that of Enterobacter in IBS-D group was significantly higher than that in normal group（P < 0.01）.The results of LEf Se analysis showed that there were 16 biomarkers with significant difference between IBS-D group and normal group.Based on T-test and LEf Se analysis,it was found that the abundance of Proteus decreased in the normal group,especially in the IBS-D group,while that of Thickwalled Bacteria increased slightly in the normal group and slightly in the IBS-D group.At the order level,the decrease of Enterobacteriaceae abundance in IBS-D group was greater than that in normal group,while the decrease of Clostridium abundance was less than that in normal group.At generic level,the abundance of Shigella Shigella and Clostridium sporozoites decreased in the normal group,while the abundance of Lactobacillus and Bifidobacterium gradually increased in the IBS-D group.The increase of Bifidobacterium in the IBS-D group was significantly greater than that in the normal group,while the decrease of Paeniclostridium and Bacillus pararotifera was less than that in the normal group.These results suggest that Sijunzi Decoction has different effects on gut microflora structure between normal group and IBS-D group.PCA analysis of metabolites in co-culture medium showed that there was a significant difference in metabolites between normal group and IBS-D group,and the difference between groups was less than that between groups（P < 0.05）.The R2 and Q2 of PLS-DA analysis are close to 1,which indicates that the model is stable.A total of 4042 metabolites were detected in the positive ion mode,of which 458 were differentially metabolized,and 1247 in the negative ion mode,of which 166 were differentially metabolized.Enrichment analysis revealed 293 metabolites involved in KEGG function,including 10 metabolic pathways with significant difference（P < 0.05）and 14 differential metabolites,including Histamine and Histidine metabolic regulation process;Histamine,Tryptamine and Morphine participate in the process of neuroactive ligand-receptor interaction;Histamine,Histidine,Putrescine participate in protein digestion and absorption;Histamine,Arachidonic acid Participate in Fc epsilon RI signaling pathway,Arachidonic acid and Histamine participate in the regulation of tryptophan channel by inflammatory mediators.The dynamic changes of differential metabolites in co-culture of Sijunzi Decoction were analyzed.It was found that Sijunzi Decoction could improve IBS-D symptoms by up-regulating intestinal histamine and tryptamine synthesis,inhibiting TNF production and chemotaxis.The results of 16 S rRNA sequencing and metabolite correlation analysis showed that Top 20 differential metabolites were strongly correlated with relative abundance of Top 10 species at eye level（P < 0.05）,and the correlation coefficient was > 0.6.For example,metabolite Imidafenacin is negatively correlated with Streptococcus and antagonizes urination by inhibiting the contraction of bladder smooth muscle.The metabolite indeloxazine（INN）is positively correlated with Streptococcus;INN is an inhibitor of 5-hydroxytryptamine and norepinephrine uptake,which has analgesic effect and potential antidepressant properties.Escherichia coli-Shigella was negatively correlated with INN.It is speculated that Sijunzi Decoction can improve IBS-D symptoms by promoting the proliferation of Streptococcus（increasing INN synthesis）and inhibiting the growth of Escherichia coli-Shigella（inhibiting the uptake of serotonin and norepinephrine）.In addition,Sijunzi Decoction produces diuretic effect by lowering the content of Imidafenacin,which is in line with the traditional Chinese medicine thinking to treat diarrhea and unfavorable urine.Conclusion:Jiawei Sijunzi Decoction,the representative prescription of invigorating spleen and Invigorating Qi method,regulates the gut microflora structure by regulating the metabolism of neurotransmitters in gut microflora,thus regulating the immune and nervous system functions of IBS-D patients.