Lactobacillus johnsonii BS15 Prevents Psychological Stress-Induced Memory Dysfunction in Mice by Modulating the Gut-Brain Axis

Abstract

Researchers are attempting to harness the advantages of the gut-brain axis to prevent neurocognitive disorders by enhancing intestinal health. In this study, four groups of ICR mice were orally gavaged with either phosphate-buffered saline (control and CW groups) or the probiotic strain Lactobacillus johnsonii BS15 (P and PW group; daily amounts of 2 × 10⁸ colony-forming units) for 28 days. From days 22 to 28, the mice in the CW and PW groups were subjected to water-avoidance stress (WAS). The issue of whether psychological stress-induced memory dysfunction can be prevented via L. johnsonii BS15 pretreatment to modulate the gut-brain axis was investigated. Results show that L. johnsonii BS15 enhanced gut development by increasing villus height in the jejunum and ileum as well as villus height:crypt depth ratio in the ileum. L. johnsonii BS15 increased the activities of digestive enzymes, including trypsin and lipase in the jejunum and ileum. The intestinal goblet cell number was also increased by L. johnsonii BS15 pretreatment. Moreover, L. johnsonii BS15 balanced the gut microbiota by increasing the log₁₀ DNA gene copies of Lactobacillus spp. and L. johnsonii and decreasing that of Enterobacteriaceae in the cecum. L. johnsonii BS15 also exerted preventive effects on intestinal permeability WAS by modulating diamine oxidase and _D-lactate levels in the serum and mRNA expression levels of the tight junction proteins claudin-1, occludin, and ZO-1 in the jejunum and ileum. L. johnsonii BS15 pretreatment modulated inflammatory factors, specifically tumor necrosis factor-alpha, interferon-gamma, and interleukin-10. L. johnsonii BS15 pretreatment improved their performance in two behavioral tests, namely the novel object and T-maze tests. This result indicates that psychological stress-induced memory dysfunction possibly could be prevented through the gut-brain axis. In addition, L. johnsonii BS15 exerted beneficial effects on the hippocampus by modulating memory-related functional proteins, especially those related to synaptic plasticity, such as brain-derived neurotrophic factor and stem cell factor. Moreover, L. johnsonii BS15 recovered antioxidant capacity and exerted protective effects on mitochondrion-mediated apoptosis in the hippocampus. Collectively, the modulation of the gut-brain axis by L. johnsonii BS15 could be considered a promising non-invasive treatment modality for psychological stress-induced memory dysfunction.

Keywords: Lactobacillus; gut microbiota; gut–brain axis; memory dysfunction; probiotic.

FIGURE 1

Effects of L. johnsonii BS15 on villus height (Vh), crypt depth (Cd), and Vh/Cd ratio in jejunum and ileum. (A) Vh and Cd; (a) Vh/Cd ratio. Data are presented with the means ± SD (n = 6). *Difference is significant at the 0.05 level (P < 0.05); **difference is significant at the 0.01 level (P < 0.01).

FIGURE 2

Effects of L. johnsonii BS15 on digestive enzyme activity in the jejunum and ileum. (A) Enzymatic activity of amylase; (B) enzymatic activity of trypsin; (C) enzymatic activity of lipase. Data are presented as the means ± SD (n = 6). *Difference is significant at the 0.05 level (P < 0.05); **difference is significant at the 0.01 level (P < 0.01).

FIGURE 3

Goblet cell number in the jejunum and ileum under original magnification (×400). (A) Jejunum in the control group; (B) jejunum in the P group; (C) ileum in the control group; (D) ileum in the P group. In the P group, the number of goblet cells in the jejunum (4B) and ileum (4D) was higher, respectively, compared with that of the control group in the jejunum (4A) and ileum (4C).

FIGURE 4

Effects of L. johnsonii BS15 on goblet cell number in the jejunum and ileum. Data are presented as the means ± standard deviation (n = 6). ***Difference is significant at the 0.001 level (P < 0.001).

FIGURE 5

Microbial populations in the cecum as quantified by quantitative PCR. Data are presented as mean ± SD. NS, not significant (P > 0.05); *difference is significant at the 0.05 level (P < 0.05); **difference is significant at the 0.01 level (P < 0.01); ***difference is significant at the 0.001 level (P < 0.001). (A–F) Log₁₀ DNA gene copies of total bacteria, Firmicutes, Bacteroidetes, Lactobacillus spp., Lactobacillus johnsonii, and Enterobacteriaceae.

FIGURE 6

Effect of L. johnsonii BS15 on gut integrity and permeability. Data are presented as the means ± SD (n = 6). NS, not significant (P > 0.05); *difference is significant at the 0.05 level (P < 0.05); **difference is significant at the 0.01 level (P < 0.01); ***difference is significant at the 0.001 level (P < 0.001). (A,B) Levels of DAO and _D-Lactate in the serum. (C–H) mRNA expression levels of tight junction protein (Claudin-1, Occludin, and ZO-1, respectively) in the jejunum and ileum.

FIGURE 7

mRNA-expression levels of inflammatory factors in the ileum. Data are presented as the means ± standard deviation (n = 6). NS, not significant (P > 0.05); **difference is significant at the 0.01 level (P < 0.01); ***difference is significant at the 0.001 level (P < 0.001). (A–F) mRNA-expression levels and protein contents of TNF-α, IFN-γ, IL-1β, IL-6, IL-4, and IL-10, respectively. TNF-α, tumor necrosis factor-alpha; INF-γ, interferon-gamma.

FIGURE 8

Protein contents of inflammatory factors in the ileum. Data are presented as the means ± SD (n = 6). NS, not significant (P > 0.05); *difference is significant at the 0.05 level (P < 0.05); **difference is significant at the 0.01 level (P < 0.01). (A–F) protein contents of TNF-α, IFN-γ, IL-1β, IL-6, IL-4, and IL-10, respectively. TNF-α, tumor necrosis factor-alpha; INF-γ, interferon-gamma.

FIGURE 9

Effects of L. johnsonii BS15 on the correct times with both 0 s (A) and 1 min (B) of retention interval of the T-maze test. Data are presented as the means ± SD (n = 8). NS, not significant (P > 0.05); *difference is significant at the 0.05 level (P < 0.05); **difference is significant at the 0.01 level (P < 0.01).

FIGURE 10

Effects of L. johnsonii BS15 on the exploration ratio of the novel object test. Data are presented as the means ± standard deviation (n = 10). NS, not significant (P > 0.05); ^∗difference is significant at the 0.05 level (P < 0.05); ^∗∗∗difference is significant at the 0.001 level (P < 0.001).

FIGURE 11

mRNA-expression levels of memory-related functional proteins in the hippocampus. Data are presented as the means ± standard deviation (n = 6). NS, not significant (P > 0.05); ^∗difference is significant at the 0.05 level (P < 0.05); ^∗∗difference is significant at the 0.01 level (P < 0.01); ^∗∗∗difference is significant at the 0.001 level (P < 0.001). (A–F) Relative expression of BDNF, CREB, NCAM, SCF, c-Fos, and NMDAR, respectively. BDNF, brain-derived neurotrophic factor; CREB, cyclic amp response element binding protein; NCAM, neural cell adhesion molecule; SCF, stem cell factor; NMDAR, N -methyl-D-aspartate receptor.

FIGURE 12

Antioxidant indexes in the hippocampus. Data are presented as the means ± SD (n = 6). NS, not significant (P > 0.05); *difference is significant at the 0.05 level (P < 0.05); **difference is significant at the 0.01 level (P < 0.01); ***difference is significant at the 0.001 level (P < 0.001). (A–F) Activities or contents of T-AOC, SOD, CAT, GSH-Px, MDA, and GSH, respectively. T-AOC, total anti-oxidation capacity; SOD, superoxide dismutase; CAT, catalase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; GSH, glutathione.

FIGURE 13

Apoptosis-related functional protein contents and mRNA-expression levels in the hippocampus. Data are presented as the means ± SD (n = 6). NS, not significant (P > 0.05); ^∗difference is significant at the 0.05 level (P < 0.05); ^∗∗difference is significant at the 0.01 level (P < 0.01); ^∗∗∗difference is significant at the 0.001 level (P < 0.001). (A–F) mRNA-expression levels of Bax, bcl-2, bcl-xl, Bad, caspase-9, and caspase-3, respectively, (G,H) protein contents of Bax and bcl-2.