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. 2022 Sep 7:13:990465.
doi: 10.3389/fpsyt.2022.990465. eCollection 2022.

Combined probiotics attenuate chronic unpredictable mild stress-induced depressive-like and anxiety-like behaviors in rats

Li Huang  1   2 , Xia Lv  1   2 , Xiaolei Ze  3 , Zewei Ma  1   2 , Xuguang Zhang  3 , Ruikun He  3 , Junting Fan  1   2 , Meilin Zhang  1   2 , Boran Sun  2   4 , Fang Wang  5 , Huan Liu  1   2
Affiliations

Combined probiotics attenuate chronic unpredictable mild stress-induced depressive-like and anxiety-like behaviors in rats

Li Huang et al. Front Psychiatry. .

Abstract

Increasing evidence indicated that probiotics can be effective in improving behaviors similar to depression and anxiety disorders. However, the underlying mechanisms remain unclear, as is the effects of single vs. combined probiotics on depression and anxiety. This study aimed to determine whether combined probiotics could attenuate depressive-like and anxiety-like behavior induced by chronic unpredictable mild stress (CUMS) and its potential mechanisms. Rats underwent CUMS treatment and then administered Lactobacillus rhamnosus HN001 (HN001) or Bifidobacterium animalis subsp. lactis HN019 (HN019), alone or in combination. Levels of neurotransmitters, inflammatory factors, and the gut microbiota were measured. HN001 and (or) HN019 treatment improved depressive-like and anxiety-like behavior in rats, including increased moving distance and exploratory behavior (p < 0.05). In addition, altered gut microbiota structure induced by CUMS was amended by HN001 and/or HN019 (p < 0.05). HN001 and/or HN019 intervention also remarkably normalized levels of 5-HT, DA, NE, HVA, DOPAC, HIAA, TNF-α, IL-6, IL-18 and IL-1β in CUMS rats (p < 0.05). Furthermore, the effects of combined probiotics on decreasing inflammation and improved gut microbiota (Chao1 index and ACE index, p < 0.05) were superior to the single probiotics. Moreover, spearman analysis showed a certain correlation between the different microbiota, such as Firmicutes, Bacteroidetes, Verrucomicrobias, Proteobacterias and Actinobacterias, and inflammation and neurotransmitters. These findings suggested that CUMS induced depressive and anxiety-like behaviors can be alleviated by the combination of probiotics, which was possibly associated with the alterations in the gut microbiota composition and increased neurotransmitters and decreased inflammatory factors.

Keywords: Bifidobacterium animalis subsp. lactis HN019; Lactobacillus rhamnosus HN001; chronic unpredictable mild stress; gut microbiota; inflammatory factors; neurotransmitters.

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Conflict of interest statement

Authors XZe, XZh, and RH were employed by BYHEALTH Institute of Nutrition & Health. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The entire experiment producer. CUMS chronic unpredictable mild stress, OFT open-field test, EPM elevated-plus test.
Figure 2
Figure 2
Body weight and depression-like behavior of CUMS rats in different groups during the experiment. (A) Changes of body weight of rat under CUMS. (B) Crossing numbers in open-field test during baseline, 6th and 12th weeks. (C) Numbers of standing in open-field test during baseline, 6th and 12th weeks. (D) Total distance in elevated-plus during baseline, 6th and 12th weeks. (E) Time in the close arms in elevated-plus during baseline, 6 and 12th weeks. Data are expressed as Mean ± SEM (n = 9–10). *p < 0.05 compared with control group. #p < 0.05 compared with CUMS group.
Figure 3
Figure 3
Probiotics supplementation improved the histological variations in rat hippocampi and colon. Representative micrographs of hippocampus, cortex and colon labeled with HE staining (A). Scale bar = 100 μm. Neurons in hippocampus and cortex were assessed by Nissl staining (B). Scale bar = 50 μm (n = 3 brains/group).
Figure 4
Figure 4
Probiotics normalized CUMS-induced changes in neurotransmitters and their metabolite in rat serum and brain. The level of serum and brain 5-HT, DA and NE were detected at the end of the experiment (A,B,D,E,G,H). (C,F,I) showing metabolite of monoamine neurotransmitters in brain. The results are presented as the Mean ± SEM (n = 9-10 in serum, n = 6 in brain). #p < 0.05 compared with CUMS group.
Figure 5
Figure 5
The levels of IL-6, IL-18, IL-1β and TNF-α in serum, brain and colon of CUMS rats in different groups during experiment. These indicators were tested at the end of experiment. The results were presented as the Mean ± SEM (simple in serum was 9-10, simple in brain and colon was 6). *p < 0.05 compared with control group. #p < 0.05 compared with CUMS group.
Figure 6
Figure 6
Four scatter plots were used to describe the correlation of TNF-α (A), IL-6 (B), IL-1β (C) and IL-18 (D) in brain and colon. Rs, represent the spearman correlation coefficient.
Figure 7
Figure 7
Effects of probiotics treatment on gut microbiota changes induced by CUMS. (A) Sobs index, (B) ACE index, (C) chao1 index, (D) Shonnnon index. The results were presented as the Mean ± SEM (n = 7). *p < 0.05 compared with control group. #p < 0.05 compared with CUMS group, &p < 0.05 compared with CUMS-HN001&HN019 group.
Figure 8
Figure 8
Analysis of microbiota flora composition among six groups. (A) unweighted PcoA, (B,D,E) difference of microbiota flora abundance at the phylum, family and genus among six groups. The area of each color represents the abundance of different bacteria in the intestine. The larger the area, the higher the abundance, and vice versa. Data are stated as mean percentage values from each group (n = 7). (C) Comparison of the ratio of Firmicutes to Bacteroidetes among six groups.
Figure 9
Figure 9
Heat map showing the correlation between the phylum (A), family (B), genus (C) and monoamine neurotransmitters and Inflammatory factors. Red and blue colors indicate positive and negative correlations, respectively. *p <0.1, **p < 0.05.
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