Polygalae Radix Oligosaccharide Esters May Relieve Depressive-like Behavior in Rats with Chronic Unpredictable Mild Stress via Modulation of Gut Microbiota

Abstract

Polygalae radix (PR) is a well-known traditional Chinese medicine that is used to treat depression, and polygalae radix oligosaccharide esters (PROEs) are the main active ingredient. Although gut microbiota are now believed to play key role in depression, the effects of PROEs on depression via modulation of gut microbiota remain unknown. In this article, we investigate the effect of PROEs on the gut microbiota of a depression rat and the possible mechanism responsible. The depression rat model was induced by solitary rearing combined with chronic unpredictable mild stress (CUMS). The depression-like behavior, the influence on the hypothalamic-pituitary-adrenal (HPA) axis, the contents of monoamine neurotransmitter in the hippocampus, and the quantity of short-chain fatty acids (SCFAs) in the feces were each assessed, and the serum levels of lipopolysaccharide (LPS) and interleukin-6 (IL-6) were measured by ELISA. Additionally, ultrastructural changes of the duodenal and colonic epithelium were observed under transmission electron microscope, and the gut microbiota were profiled by using 16S rRNA sequencing. The results show that PROEs alleviated the depression-like behavior of the depression model rats, increased the level of monoamine neurotransmitters in the brain, and reduced the hyperfunction of the HPA axis. Furthermore, PROEs regulated the imbalance of the gut microbiota in the rats, relieving intestinal mucosal damage by increasing the relative abundance of gut microbiota with intestinal barrier protective functions, and adjusting the level of SCFAs in the feces, as well as the serum levels of LPS and IL-6. Thus, we find that PROEs had an antidepressant effect through the restructuring of gut microbiota that restored the function of the intestinal barrier, reduced the release of intestinal endotoxin, and constrained the inflammatory response.

Keywords: chronic unpredictable mild stress; depression; gut microbiota; intestinal barrier; polygalae radix oligosaccharide esters.

Figure 1

The effects of PROEs on CUMS-induced depression-like behaviors in rats. (A) The body weights of rats; (B) the sucrose preference rate; (C) the immobility time in the FST; (D–F) the total movement distance, the number of rearings, and the time spent in the central area of the OFT; data represent mean ± SEM (n = 8); ^## p < 0.01, and ^### p < 0.001 compared to the NC group; ** p < 0.01 and *** p < 0.001 compared to the CUMS group.

Figure 2

The effects of PROEs on HPA axis hormone, amino acid, and pro-inflammatory factors. (A–C) The levels of CORT, ACTH, and CRF in Serum; (D–F) the levels of TRP and KYN in plasma and the ratio of TRP/KYN; (G,H) the levels of IL-6 and LPS in serum; data represent mean ± SEM (n = 6); ^## p < 0.01 and ^### p < 0.001 compared to the NC group; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the CUMS group.

Figure 3

Changes in gut microbiota diversity. (A,B) Shannon index and Chao 1 index in alpha diversity analysis (n = 8); (C) principal co−ordinate analysis (PCoA); (D) unweighted pair group method with arithmetic mean (UPGMA); ^## p < 0.01 and ^### p < 0.001 compared to the NC group; ** p < 0.01 and *** p < 0.001 compared to the CUMS group.

Figure 4

The effects of PROEs on the relative abundance of gut microbiota (A) and significantly changed gut microbiota (B) at the phylum level in depression-model rats (n = 8); ^# p < 0.05, ^## p < 0.01 compared to the NC group; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the CUMS group.

Figure 5

The effects of PROEs on the redundancy analysis (RDA) of gut microbiota (A) and significantly changed gut microbiota (B) at the family level in depression-model rats (n = 8); ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to the NC group; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the CUMS group.

Figure 6

Heat map analysis of the effects of PROEs on gut microbiota (A) and significantly changed gut microbiota (B) at the genus level in depression-model rats (n = 8); ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to the NC group; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the CUMS group.

Figure 7

(A) Linear discriminant analysis of effect size (Lefse) with p < 0.01 and LDA score (log 10) > 4.0; (B) correlation analysis among brain neurotransmitters, HPA axis hormone, and relative abundances of gut microbiota. Red and green squares indicate negative and positive correlations, respectively, and the intensities of the colors are proportional to the degree of correlation. * p < 0.05, ** p < 0.01.

Figure 8

Predicted functions for the altered metagenome of gut microbiota in each group shown with KEGG pathways. (A–C) KEGG pathways at level 2; (D) KEGG pathways at level 3; the extended error bar plot of significantly differential KEGG pathways predicted using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt). Only p < 0.05 are shown.

Figure 9

H&E staining of duodenum and colon tissue (n = 3). (A,E) The effects of PROEs on duodenum and colon histopathological changes; (B,F) the length of duodenal and colonic villi; (C,G) the depth of duodenal and colonic crypts; (D,H) the ratio of V/C in the duodenum and colon; data represent mean ± SEM (n = 10); ^## p < 0.01 and ^### p < 0.001 compared to the NC group; *** p < 0.001 compared to the CUMS group.

Figure 10

The effects of PROEs on duodenum (A) and colon (B) epithelium ultrastructures (n = 3) (scale bar = 50, 20 µm; original magnification ×1.5 k, ×5.0 k).

Figure 11

The effects of PROEs on the protein expression of occludin in the colon; data represent mean ± SEM (n = 3); ^# p < 0.05 compared to the NC group; * p < 0.05, ** p < 0.01 compared to the CUMS group.

Figure 12

The effects of PROEs on the expression of 5-HT_1A, 5-HT_2A, IDO1, and TNF-α mRNA, and the concentrations of SCFAs. (A,B) The expression of 5-HT_1A and 5-HT_2A mRNA in the cerebral cortex; (C,D) the expression of IDO1 mRNA in the cerebral cortex and duodenum; (E,F) the expression of TNF-α mRNA in the cerebral cortex and duodenum; (G–I) the concentrations of acetic acid, propionic acid, and butyric acid in feces; data represent mean ± SEM (n = 6); ^## p < 0.01 and ^### p < 0.001 compared to the NC group; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to the CUMS group.