FMT and TCM to treat diarrhoeal irritable bowel syndrome with induced spleen deficiency syndrome- microbiomic and metabolomic insights

Abstract

Background: Diarrheal irritable bowel syndrome (IBS-D) is a functional bowel disease with diarrhea, and can be associated with common spleen deficiency syndrome of the prevelent traditional Chinese medicine (TCM) syndrome. Fecal microbiota transplantation (FMT) could help treating IBS-D, but may provide variable effects. Our study evaluated the efficacy of TCM- shenling Baizhu decoction and FMT in treating IBS-D with spleen deficiency syndrome, with significant implications on gut microbiome and serum metabolites.

Methods: The new borne rats were procured from SPF facility and separated as healthy (1 group) and IBS-D model ( 3 groups) rats were prepared articially using mother's separation and senna leaf treatment. 2 groups of IBS-D models were further treated with TCM- shenling Baizhu decoction and FMT. The efficacy was evaluated by defecation frequency, bristol stool score, and intestinal tight junction proteins (occludin-1 and claudin-1) expression. Microbiomic analysis was conducted using 16 S rRNA sequencing and bioinformatics tools. Metabolomics were detected in sera of rats by LC-MS and annotated by using KEGG database.

Results: Significant increment in occludin-1 and claudin-1 protein expression alleviated the diarrheal severity in IBS-D rats (P < 0.05) after treatment with FMT and TCM. FMT and TCM altered the gut microbiota and regulated the tryptophan metabolism, steroid hormone biosynthesis and glycerophospholipid metabolism of IBS-D rats with spleen deficiency syndrome.The microbial abundance were changed in each case e.g., Monoglobus, Dubosiella, and Akkermansia and othe metabolic profiles.

Conclusion: FMT and TCM treatment improved the intestinal barrier function by regulating gut microbiota and improved metabolic pathways in IBS-D with spleen deficiency syndrome.

Keywords: Fecal microbiota transplantation; Gut microbiota; Irritable bowel syndrome; Metabolomics; Spleen deficiency syndrome.

Fig. 1

Experimental design. Besides the healthy group, newborn rats were separated from their mothers for 3 weeks, subjected to restraint stress for another 3 weeks, and given senna leaf intragastric administration for 2 weeks. After the modeling, the rats in each group were treated for 2 weeks, and tight junction protein, intestinal flora and metabolome were detected

Fig. 2

Defecation frequency and stool form of IBS-D rats with spleen deficiency syndrome. (A) Defecation frequency; (B) Bristol Stool Scale. Note: *P < 0.05, ***P < 0.001

Fig. 3

Analysis of diarrhea severity and intestinal barrier function. (A) Defecation frequency; (B) Bristol stool scale; (C) Occludin-1 protein expression levels; (D) claudin-1 protein expression levels (E) The formal WB results of Western blot. Note: Compared to healthy group, *P < 0.05, **P < 0.01, ***P < 0.001; Compared to IBS-D model group, #P < 0.05, ##P < 0.01, ###P < 0.001; Compared to FMT group, ^^P < 0.01

Fig. 4

Analysis of gut microbiota of rats in each group. (A) Principal coordinates analysis of Bray Curtis showing the beta diversity distances among the four groups to respresent the compositional dissimilarities; (B) Microbial community bar plot on the phylum level showing that Fermicutes were in greater strength than other major phyla; (C) Microbial community bar plot on the genes level; (D) LEfSe analysis of gut microbiota in healthy and IBS-D model groups showing differences in the abundance of bacterial communities such as p__Verrucomicrobiota, p__Firmicutes, and p__Spirochaetota between the two groups; (E) LEfSe analysis of gut microbiota in IBS-D model and FMT groups showing differences in the abundance of bacterial communities such as p__Verrucomicrobiota and o__Monoglobales between the two groups; (F) LEfSe analysis of gut microbiota in IBS-D and TCM-shenling baizhu decoction groups showing differences in the abundance of bacterial communities such as p__Campilobacterota, and p__Cyanobacteria between the two groups. Note: LEfSe analysis, different color nodes represent the microbial groups that are significantly enriched in the corresponding group and have a significant effect on the difference between the groups; while the light yellow nodes represent microbial groups that have no significant differences between groups. ZC, Healthy group; MX, IBS-D model group; FMT, FMT group; ZY, TCM group

Fig. 5

Volcano plot of fecal and serum metabolites of rats in each group. (A) Fecal metabolites: IBS-D vs. healthy group; (B) Fecal metabolites: FMT vs. IBS-D group; (C) Fecal metabolites: TCM vs. IBS-D group; (D) Serum metabolites: IBS-D vs. healthy group; (E) Serum metabolites: FMT vs. IBS-D group; (F) Serum metabolites: TCM vs. IBS-D group. Note: The volcano plot showed all the differentially expressed metabolites between the two groups. Each dot represents a specific metabolite, and the size of the dot indicates the Vip value. Red dots represent significantly upregulated metabolites, blue dots represent significantly downregulated metabolites, and gray dots represent metabolites with no significant difference

Fig. 6

KEGG pathway analysis of metabolites in IBS-D group and healthy group. (A) VIP value analysis of fecal differential metabolites; (B) KEGG enrichment analysis of fecal differential metabolites; (C) KEGG topological analysis of fecal differential metabolites; (D) VIP value analysis of serum differential metabolites; (E) KEGG enrichment analysis of serum differential metabolites; (F) KEGG topological analysis of serum differential metabolites

Fig. 7

KEGG pathway analysis of differential metabolites between the FMT and untreated IBS-D model groups. (A) VIP value analysis of fecal differential metabolites; (B) KEGG enrichment analysis of fecal differential metabolites; (C) Analysis of fecal serotonin levels; (D) Analysis of fecal kynurenine levels; (E) KEGG topological analysis of fecal differential metabolites; (F) VIP value analysis of serum differential metabolites; (G) KEGG enrichment analysis of serum differential metabolites; (H) KEGG topological analysis of serum differential metabolites

Fig. 8

KEGG pathway analysis of differential metabolites between TCM-shenling baizhu decoction group and IBS-D model group. (A) VIP value analysis of fecal differential metabolites; (B) KEGG enrichment analysis of fecal differential metabolites; (C) KEGG topological analysis of fecal differential metabolites; (D) VIP value analysis of serum differential metabolites; (E) KEGG enrichment analysis of serum differential metabolites; (F) KEGG topological analysis of serum differential metabolites

Fig. 9

Procrustes analysis of gut microbiota and metabolites. (A) Correlation analysis between gut microbiota and fecal metabolites in the FMT and IBS-D groups; (B) Correlation analysis between gut microbiota and fecal metabolites in the TCM and IBS-D groups;. Note: MX, IBS-D model group; FMT, FMT group; ZY, TCM-shenling baizhu decoction group

Fig. 10

Correlation heatmap between differential metabolites and gut microbiota in the FMT and IBS-D groups. (A) Correlation analysis between gut microbiota and differential fecal metabolites; (B) Correlation analysis between gut microbiota and differential serum metabolites

Fig. 11

Correlation heatmap between differential metabolites and gut microbiota in the healthy and IBS-D groups. (A) Correlation analysis between gut microbiota and differential fecal metabolites; (B) Correlation analysis between gut microbiota and differential serum metabolites

Fig. 12

Correlation heatmap between differential metabolites and gut microbiota in the TCM and IBS-D groups. (A) Correlation analysis between gut microbiota and differential fecal metabolites; (B) Correlation analysis between gut microbiota and differential serum metabolites