Gut Microbiota Are Associated With Psychological Stress-Induced Defections in Intestinal and Blood-Brain Barriers

Abstract

Altered gut microbiota has been identified during psychological stress, which causes severe health issues worldwide. The integrity of the intestinal barrier and blood-brain barrier regulates the process of bacterial translocation and can supply the nervous system with real-time information about the environment. However, the association of gut microbiota with psychological stress remains to be fully interpreted. In this study, we established a psychological stress model using an improved communication box and compared the expression of tight junction proteins in multiple regions of the intestinal (duodenum, jejunum, ileum) and blood-brain (amygdala, hippocampus) barriers between model and control rats. We also conducted fecal microbiota analysis using 16S rRNA gene sequencing. Expression levels of the stress-related indicators adrenocorticotropic hormone, NR3C1,2, and norepinephrine were increased in the model group compared to control group. Psychological stress reduced brain and intestinal levels of tight junction proteins, including claudin5, occludin, α-actin, and ZO-1. Microbiota analysis revealed elevated microbial diversity and fecal proportions of Intestinimonas, Catenisphaera, and Globicatella in the model group. Further analysis indicated a negative correlation of Allisonella and Odoribacter, as well as a positive correlation of norank_f__Peptococcaceae, Clostridium_sensu_stricto_1, and Coprococcus_2, with claudin5, occludin, α-actin, and ZO-1. Our use of a rodent model to explore the association between compromised intestinal and blood-brain barriers and altered fecal microbiota under psychological stress improves our understanding of the gut-brain axis. Here, cues converge to control basic developmental processes in the intestine and brain such as barrier function. This study provides new directions for investigating the pathogenesis of emotional disorders and the formulation of clinical treatment.

Keywords: blood–brain barrier; communication box; dysbiosis; gut microbiota; intestinal barrier; psychological stress; tight junction.

FIGURE 1

Equipment for the psychological stress rodent model. (A) Schematic diagram for construction of the modeling device. (a) Wires connected to a small animal stimulator can provide plantar electrical stimulation. (b) Wires fixed at the separator in the direction of rats getting an electric shock can be grasped by model rats to escape electric shock stimulation. (c) Thirty holes with diameter of 1 cm are evenly distributed to facilitate capturing the fear signals of model rats given electric shock. (B) Experimental design. Rats in the middle row received electrical stimulation, while rats in the two adjacent rows received mental stimulation by watching, listening, and smelling the rats given electric shock.

FIGURE 2

Impact of psychological stress stimulation on emotional phenotypes and indicators in rats. (A,B) Development of phenotypic changes after psychological stress. (A) Daily food and water intake in control and model groups (lozenges: control; circles: model; monitoring time: 28 days). (B) Weekly weight of rats in control and model groups (circles: control; squares: model; monitoring time: per week after stimulation, for 4 weeks). (C,D) Emotional phenotype and ACTH level changes after modeling (light gray: control; dark gray: model). (C) Results of the open field test were measured after stimulation for 28 days. Horizontal motion of rats in the open field was calculated (control, n = 12; model, n = 12). (D) ACTH levels in both groups were recorded (n = 6, each group). (E) NE levels in cortex, amygdala, and hippocampus measured by HPLC (n = 6, each group). (F,G) Immunohistochemical analysis of NR3C1 and NR3C2 expression in cortex on day 28 (n = 5, each group). Data shown as mean ± SD; ^∗P < 0.05, ^∗∗P < 0.01, model group vs. control group.

FIGURE 3

Impact of psychological-stress stimulation on tight junction proteins in intestinal and blood–brain barriers. (A,C–E) Decreased expression of tight junction proteins in the amygdala and hippocampus. (A) Representative immunohistochemical staining of α-actin and claudin5 in the amygdala (model and control markers are located in the lower right corner of the image). (C) IOD values for expression levels of four proteins in the amygdala. (D) IOD values for expression levels of four proteins in the hippocampus (light gray: control; dark gray: model; n = 5, each group). (E) Representative electron microscopy pictures of tight junctions of the BBB in the amygdala (tight junction indicated by white arrow; n = 3, each group). (B,F–I) Decreased expression of tight junction proteins in duodenum, jejunum, and ileum. (B) Representative immunohistochemical staining of occludin and ZO-1 in the duodenum (model and control markers are located in the lower right corner of the image). (F) Representative electron microscopy pictures of tight junctions of the intestinal barrier in the duodenum (tight junction indicated by white arrow; n = 3, each group). (G) IOD values for expression levels of four proteins in the duodenum. (H) IOD values for expression levels of four proteins in the jejunum. (I) IOD values for expression levels of four proteins in the ileum (light gray: control; dark gray: model; n = 5, each group). Data shown as mean ± SD; ^∗P < 0.01, model group vs. control group.

FIGURE 4

Effects of psychological-stress stimulation on gut microbiota composition. (A) Plots shown were generated using the abund_jaccard-based PCoA (R² = 0.4058, P < 0.01). The R² value was calculated by Adonis algorithm. (B) The discrete distribution of different groups of samples on PC1 axis. (C) Community abundance in gut microbiota at the phylum level. (D) Differences in gut microbiota composition at the genus level. (E) Results of LEfSe analysis. Nodes with different colors represent microbial groups that are significantly enriched in the corresponding rat groups and have significant effects on the differences between groups; pale yellow nodes represent microbial groups that show no significant differences or no significant effects on the differences between groups. (F) Key taxa found by LDA analysis (multigroup comparison strategy: one-against-all, LDA > 2, P < 0.05). The higher the LDA score, the greater the impact of the representative species abundance on the difference between groups.

FIGURE 5

Correlation between core bacteria and tight junction proteins in multiple regions of the intestinal barrier and BBB at the genus level. (A) Heatmap correlation analysis of brain and intestinal tight junction proteins and core gut microbiota at the genus level. The x-axis represents tight junction proteins in different regions of the brain and intestine. The y-axis represents species at the genus level. R- and P-values were obtained by calculation using the Spearman Grade Coefficient. The R-value is shown in different colors: red, positive correlation; green, negative correlation. The legend on the right shows the color intervals of different R-values; depth of color indicates degree of correlation. ^∗P < 0.05. Cluster trees representing species and environmental factors (left and upper) are shown. ^∗0.01 < P < 0.05, ^∗∗0.001 < P < 0.01, ^∗∗∗P < 0.001. (B–F) R-value histograms showing correlation of five representative genera and tight junction proteins at different sites. The y-axis represents different regions of the brain and intestine. The x-axis represents specific R-values between the flora and four tight junction proteins. Opening of the histogram to the left means that the R-value is negative, and there is a negative correlation. Otherwise, there is a positive correlation (red: claudin5; yellow: occludin; green: ZO-1; blue: α-actin).

FIGURE 6

Roles of defective intestinal barrier and BBB in brain–gut communication. (a) Bidirectional interaction between the intestine and brain via the vagus nerve. (b) Neuroendocrine pathways in brain–gut communication. Psychological stress can activate the HPA axis and release cortisol. Cortisol can increase permeability by directly acting on intestinal mucosa or promoting tryptophan production, and change bacterial composition by influencing the intestinal environment. Increased intestinal permeability induces neurotransmitters and tryptophan-related products produced by intestinal endocrine cells and neurons to enter the blood circulation and react with the brain. (c) Inflammation and immune pathways in brain–gut communication. Psychological stress stimulates inflammatory responses and activates immune cells to release cytokines, which can destroy the integrity of the intestinal barrier and BBB. A defective intestinal barrier promotes more bacterial translocation, allowing bacterial metabolites to act on the brain through the defective BBB. Moreover, cytokines produced during immune activation can also stimulate microglia activation to affect mood.