Sleep deprivation-induced anxiety-like behaviors are associated with alterations in the gut microbiota and metabolites

Abstract

The present study aimed to characterize the gut microbiota and serum metabolome changes associated with sleep deprivation (SD) as well as to explore the potential benefits of multi-probiotic supplementation in alleviating SD-related mental health disorders. Rats were subjected to 7 days of SD, followed by 14 days of multi-probiotics or saline administration. Open-field tests were conducted at baseline, end of SD (day 7), and after 14 days of saline or multi-probiotic gavage (day 21). Metagenomic sequencing was conducted on fecal samples, and serum metabolites were measured by untargeted liquid chromatography tandem-mass spectrometry. At day 7, anxiety-like behaviors, including significant decreases in total movement distance (P = 0.0002) and staying time in the central zone (P = 0.021), were observed. In addition, increased levels of lipopolysaccharide (LPS; P = 0.028) and decreased levels of uridine (P = 0.018) and tryptophan (P = 0.01) were detected in rats after 7 days of SD. After SD, the richness of the gut bacterial community increased, and the levels of Akkermansia muciniphila, Muribaculum intestinale, and Bacteroides caecimuris decreased. The changes in the host metabolism and gut microbiota composition were strongly associated with the anxiety-like behaviors caused by SD. In addition, multi-probiotic supplementation for 14 days modestly improved the anxiety-like behaviors in SD rats but significantly reduced the serum level of LPS (P = 0.045). In conclusion, SD induces changes in the gut microbiota and serum metabolites, which may contribute to the development of chronic inflammatory responses and affect the gut-brain axis, causing anxiety-like behaviors. Probiotic supplementation significantly reduces serum LPS, which may alleviate the influence of chronic inflammation.

Importance: The disturbance in the gut microbiome and serum metabolome induced by SD may be involved in anxiety-like behaviors. Probiotic supplementation decreases serum levels of LPS, but this reduction may be insufficient for alleviating SD-induced anxiety-like behaviors.

Keywords: anxiety; gut microbiota; metabolomics; probiotics; sleep deprivation.

Fig 1

Rats show anxiety-like behaviors after sleep deprivation (SD) and respond to multi-probiotic supplementation. (a) Study protocols, (b) Total distance, (c) average velocity, (d) time in the central zone, (e) upright numbers, and (f) serum LPS levels of rats. Data are expressed as means ± SD. The significance of the comparison was determined using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. BSL, baseline (N = 30); 7d, SD day 7 (N = 29); SA-21d, saline supplementation for 14 days after SD (N = 15); SC-21d, multi-probiotic supplementation for 14 days after SD (N = 14).

Fig 2

Changes in serum metabolites after sleep deprivation and mixed probiotics supplementation. (a) Sparse partial least squares discriminant analysis score plot and (b) loading plot of the serum metabolites. (c) The heatmap shows all serum metabolites in the experimental rats at baseline (BSL), 7 days post-SD (d7), and 14 days of gavage with saline (SA-21d) or mixed probiotics (MP-21d). Four patterns of metabolic changes were classified and indicated as clusters C1–C4. (d) Volcano plot showing the differential metabolites between 7 days post-SD and BSL (FDR <0.05 and fold change >2). Red represents the upregulated differential metabolites at 7 days, and blue represents the downregulated differential metabolites at 7 days. (e) Three metabolites with significantly differential serum levels between the 21d-SA and 21d-MP groups (t-test; P < 0.01 and fold change >2).

Fig 3

Changes in the gut microbiota after sleep deprivation and mixed probiotic supplementation. (a) At the species level, the alpha diversity of the gut microbiota was measured by the Chao1 index (richness) and Shannon index (diversity). The significance of the comparison was determined using the Mann-Whitney U test (*P < 0.05). (b) Beta diversity was visualized using non-metric multidimensional scaling with Bray-Curtis dissimilarity distances at the species level. Permutational multivariate analysis of variance was performed to assess the statistical significance among the four groups. (c) Heatmap showing the 74 different species identified using one-way ANOVA (FDR <0.005). (d) LEfSe analysis identified bacteria species that significantly differed (FDR <0.05 and LDA >3) among samples collected at baseline (BSL), 7 days post-SD (7d-SD), and 14 days of saline (21d-SA) or mixed probiotics (21d-MP) after SD.

Fig 4

The severity of anxiety-like behaviors in rats after sleep deprivation is associated with the changes in serum metabolites and gut microbiota. Heatmaps showing that the (a) serum metabolites and (b) gut bacteria species significantly different from baseline had close relationships with the movement indexes. Significant Spearman correlations are indicated (*P < 0.05, **P < 0.01, and ***P < 0.001). RDA plots show the association of (c) the serum metabolites with four movement indexes and (d) the relationships between the differential taxa from baseline and the movement indexes. TD, total distances; AV, average velocity; UN, upright number; and TICZ, time in the central zone. The arrow length represents the strength of the correlation between the movement indexes and the serum metabolites or microbes.