Effects of electroacupuncture on urinary metabolome and microbiota in presenilin1/2 conditional double knockout mice

Abstract

Aim: The treatment of Alzheimer's disease (AD) is still a worldwide problem due to the unclear pathogenesis and lack of effective therapeutic targets. In recent years, metabolomic and gut microbiome changes in patients with AD have received increasing attention, and the microbiome-gut-brain (MGB) axis has been proposed as a new hypothesis for its etiology. Considering that electroacupuncture (EA) efficiently moderates cognitive deficits in AD and its mechanisms remain poorly understood, especially regarding its effects on the gut microbiota, we performed urinary metabolomic and microbial community profiling on EA-treated AD model mice, presenilin 1/2 conditional double knockout (PS cDKO) mice, to observe the effect of EA treatment on the gut microbiota in AD and find the connection between affected gut microbiota and metabolites.

Materials and methods: After 30 days of EA treatment, the recognition memory ability of PS cDKO mice was evaluated by the Y maze and the novel object recognition task. Urinary metabolomic profiling was conducted with the untargeted GC-MS method, and 16S rRNA sequence analysis was applied to analyze the microbial community. In addition, the association between differential urinary metabolites and gut microbiota was clarified by Spearman's correlation coefficient analysis.

Key findings: In addition to reversed cognitive deficits, the urinary metabolome and gut microbiota of PS cDKO mice were altered as a result of EA treatment. Notably, the increased level of isovalerylglycine and the decreased levels of glycine and threonic acid in the urine of PS cDKO mice were reversed by EA treatment, which is involved in glyoxylate and dicarboxylate metabolism, as well as glycine, serine, and threonine metabolism. In addition to significantly enhancing the diversity and richness of the microbial community, EA treatment significantly increased the abundance of the genus Mucispirillum, while displaying no remarkable effect on the other major altered gut microbiota in PS cDKO mice, norank_f_Muribaculaceae, Lactobacillus, and Lachnospiraceae_NK4A136 group. There was a significant correlation between differential urinary metabolites and differential gut microbiota.

Significance: Electroacupuncture alleviates cognitive deficits in AD by modulating gut microbiota and metabolites. Mucispirillum might play an important role in the underlying mechanism of EA treatment. Our study provides a reference for future treatment of AD from the MGB axis.

Keywords: Alzheimer’s disease; PS cDKO mice; electroacupuncture; gut microbiota; metabolomics.

FIGURE 1

Electroacupuncture ameliorates cognitive deficits in PS cDKO mice. The duration (A) and frequency (B) of entries in the novel arm of the Y maze. The symbol “°” means an individual. (C,D) The preference index in the novel object recognition task. One-way ANOVA, *P < 0.05, ^**P < 0.01, N = 6.

FIGURE 2

Scores plots of multivariate statistical analysis on urinary metabolites. PCA scores plot (A) and PLS-DA scores plot (B) of the WT, cDKO, and cDKO + EA groups. N = 6.

FIGURE 3

Heat map of the differential metabolites in the cDKO group and the WT group (A), and the cDKO group and the cDKO + EA group (B), N = 6.

FIGURE 4

Pathway analysis of urinary metabolites using metaboanalyst (impact factor ≥0.1). (A) Disturbed metabolic pathways in PS cDKO mice. (B) Influenced metabolic pathways of PS cDKO mice after electroacupuncture. (1) Glyoxylate and dicarboxylate metabolism, (2) citrate cycle (TCA cycle), (3) alanine, aspartate, and glutamate metabolism, (4) glutathione metabolism, (5) arginine biosynthesis, (6) pentose and glucuronate interconversions, (7) glycine, serine, and threonine metabolism, and (8) d-glutamine and d-glutamate metabolism. N = 6.

FIGURE 5

Influence of electroacupuncture on gut taxa in PS cDKO mice. The α-diversity indexes of Shannon (A) and Chao1 (B) of gut microbiota. *P < 0.05. PCoA (C) and PLS-DA (D) of the gut microbiome composition of WT, cDKO, and cDKO + EA groups on the OUT level. (E) Relative abundance of the gut microbiome in the three groups, colored at and genus level, N = 6.

FIGURE 6

The taxa of gut microbiota affected by electroacupuncture. LEfSe analysis from the phylum to genus level in the WT and cDKO groups (A), the cDKO and cDKO + EA groups (B). Taxa enriched in three groups are indicated by LDA scores (green for the WT group, blue for the cDKO group, and red for the cDKO + EA group). The LDA score threshold is ≥3. (C,D) The cladogram of enriched taxa from the phylum to genus level. Differential abundance analysis of taxa on the genus level in the WT group and the cDKO group (E), the cDKO group, and the cDKO + EA group (F). Student’s t-test, *P < 0.05, ^**P < 0.01, N = 6.

FIGURE 7

The relevance between the gut microbiota of genus level and the differential urinary metabolites. (A–C) Spearman’s correlation heat map: red indicates a positive correlation, while green indicates a negative correlation. The deeper color means a greater correlation (*P < 0.05, ^**P < 0.01). (B–D) The gut microbiota of the genus level, predicted by metabolic variation (|r| > 0.4), is labeled with a similar value. Lines connecting with metabolites show the direction of the relevance to each genus of microbe with the red (positive) or blue (negative) lines, N = 6.

FIGURE 8

Schematic diagram representing the effect of electroacupuncture on urinary metabolome and microbiota in PS cDKO mice and correlation of gut microbiota and urinary metabolome.