Protective Effects of Lacticaseibacillus rhamnosus IDCC3201 on Motor Functions and Anxiety Levels in a Chronic Stress Mouse Model

Male C57BL/6 mice were purchased from Central Lab. Animal (Seoul, Korea) at five weeks old. L. rhamnosus IDCC 3201 (L3201) was provided by Ildong Bioscience (Pyeongtaek, Korea). L3201 were suspended in saline before use for fresh administration. Mice were divided into three groups: control group (CTL group), stress control group (S. CTL group), and stress probiotics (L3201 group). All groups were orally administered saline or L3201 (1.0×10⁹ CFU/mouse/day) from 6 weeks old. We determined the consumption of L3201 based on the results of previous studies (Lee et al., 2016). They were exposed to UCMS from 8 weeks old until sacrifice. Behavioral tests were started at 8 weeks old. Body weight was checked weekly. Mice had ad libitum access to food and water under standard conditions: 21°C–23°C, 45%–55% humidity, and 12 h light/dark cycle. All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of Sejong University (SJ-20230110-01).

UCMS was slightly modified (Mineur et al., 2006; Oh et al., 2020). Stress groups were exposed to stressors such as tube restraint, tail tie-up, food deprivation, water deprivation, illumination, cold object, tilted cage, damp sawdust, sleep cycle change, foreign cage, and cold water bath. Two stressors were used on a randomized schedule in a day.

The EPM was used to measure anxiety-like behavior (Komada et al., 2008). The apparatus is 40 cm above the floor and has a center zone (5 cm×5 cm), two closed arms with walls (30 cm×5 cm×16 cm), and two open arms (30 cm×5 cm×0.5 cm). Mice were placed at the center facing the closed arm and freely explored for 5 min. The tests were recorded and analyzed by Any-maze software version 6.0 (Stoelting, Wood Dale, IL, USA).

The forced swim test was used to measure despair-like behaviors (Porsolt et al., 1977). The apparatus was filled with water (25±1°C) to a depth of 16 cm in a glass beaker (Ø 19×26 cm). Mice were placed in water and forced to swim for 6 min, and the last four minutes of the test were analyzed. All tests were recorded with a camera and analyzed by Any-maze software version 6.0 (Stoelting).

Fresh fecal samples were collected and stored at −80°C prior to metabolic analysis. Gas chromatography–mass spectrometry (GC–MS) analysis was conducted as reported (Thompson et al., 2020; Yoo et al., 2022) with slight modification. Briefly, 40 mg of fecal samples were homogenized in 1 mL of ice-cold methanol by vortexing and centrifuged at 15,000×g for 5 min at 4°C. The supernatant was filtered and thoroughly vacuum-dried. Methoxyamine hydrochloride in pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide were used as derivatization agents. GC–MS analysis was conducted with a Trace 1310 Gas Chromatograph including the ISQ LT single quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The detected metabolites were identified using AMDIS and the NIST Mass database (version 2.0, Gaithersburg, MD, USA). Statistical analysis was performed using Metaboanalyst 5.0 (Eom et al., 2022).

One-way analysis of variance (ANOVA) followed by Duncan’s post hoc test was conducted using SPSS statistics 23 (IBM, Armonk, NY, USA) to analyze the statistical significance between groups. Different superscript letters indicate significant differences. All data are presented as the mean±SEM.

A schematic diagram for the behavioral experiment is illustrated in Fig. 1A. The body weight of the S.CTL group decreased compared to the CTL group during the UCMS period, but the L3201 group did not differ from that of the CTL group (Fig. 1B). In addition, the S.CTL group showed a decrease in weight gain (%), but the L3201 group showed rescued body weight (Fig. 1C). The EPM was conducted to confirm the anti-anxiety effect of oral administration of the L3201 strain (Figs. 1D, E, F, G, H, and I). The EPM showed no difference in the total distance traveled in all groups (Fig. 1D). The S.CTL group spent significantly more time in the closed arms than the CTL group (Fig. 1E). On the other hand, the time spent in the closed arms of the L3201 group was similar to that of the CTL group (Fig. 1E). Additionally, the S.CTL group had a significantly decreased time spent in the open arms compared to the CTL group (Fig. 1F). However, there was no difference in the time spent in the open arms between the CTL and L3201 groups (Fig. 1F). The FST was performed to confirm the antidepressant effect of oral administration of the L3201 strain (Figs. 1H and I). The immobility time of the S.CTL group was significantly increased compared to that of the CTL group (Fig. 1I). The L3201 group tended to have an increased immobility time compared to the CTL group but a decreased immobility time compared to the S.CTL group (Fig. 1I). These results suggest that the administration of L3201 relieves stress-induced anxiety- and despair-like behaviors.

Fig. 1. Oral administration of L3201 relieved anxiety and depression-like behaviors under UCMS. (A) Schematic overview of the in vivo experiment, (B) variations in body weight, (C) percentage of weight gain during UCMS, (D) total distance travelled in the EPM, (E) time in the closed arms in the EPM, (F) time in the open arms in the EPM, (G) the elevated plus maze test apparatus, (H) the forced swimming test apparatus, (I) immobility time in the FST. All data are presented as mean±SEM. ^a,b Different superscript letters indicate statistical difference. UCMS, unpredictable chronic mild stress; CTL, control; S. CTL, stress control; L3201, Lacticaseibacillus rhamnosus IDCC 3201; EPM, elevated plus maze test; FST, forced swimming test.

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Chronic stress induces anxiety- and despair-like behavior in mice (Westfall et al., 2021). EPM is the most frequently performed test in rodents using open time as an anxiety index to identify anxiety-like behavior (Komada et al., 2008). Corticosterone- or UCMS-induced depressed mice spent less time in the open arms than normal mice in EPM (Peng et al., 2021; Zhu et al., 2020). The S.CTL group showed a decrease in open time, which is an anxiety-like behavior caused by stress. Stenman et al. showed the reduction of anxiety-like behaviors in EPM by administering probiotics in a depression model (Stenman et al., 2020). Consistent with previous anxiolytic results, the L3201 group showed a reversal in the reduced open time caused by UCMS, indicating a decrease in anxiety-like behavior.

Increased immobility time in the FST is used to measure a despair indicator (Porsolt et al., 1977). Previous research has shown that immobility is increased in mouse models of depression caused by corticosterone administration or UCMS (Badr et al., 2020; Mineur et al., 2006; Strekalova et al., 2005). Consistent with previous results, the S.CTL group showed increased immobility time, which indicated despair-like behavior caused by UCMS. Murray et al. (2019) and Stenman et al. (2020) showed the effect of reducing despair-like behaviors by reducing the immobility time of FST in a depression model through probiotics administration. Similar to previous studies, in the L3201 group, the increased immobility time induced by UCMS was alleviated, suggesting a stress-relieving effect of L3201 administration.

GC–MS analysis was performed to confirm the effect of oral administration of the L3201 strain (Figs. 2A, B, and C) on fecal metabolomics. As a result, a total of 53 metabolites, including amino acids, fatty acids, organooxygen compounds, and steroids, were detected; among them, 34 metabolites were identified. Multivariate analysis showed different metabolic profiles among the three groups (Fig. 2A). Within the identified metabolites, an upregulation was observed in the expression of 7 metabolites, while a decrease was noted in 26 metabolites, including L-alanine, L-isoleucine, and cholesterol, in the UCMS model compared to the CTL group (Fig. 2B). Conversely, administration of L3201 in the UCMS-induced model increased 13 metabolites, including L-alanine, L-isoleucine, and propanoic acid, while 19 metabolites exhibited a decrease in their levels. Furthermore, among the metabolites downregulated by UCMS induction, D-galactose, α-L-galactofuranoside, L-isoleucine, 4-methylmandelic acid, L-alanine, glycine, 2-ethylhexanoic acid, 3-oxaoct-4-en-2-imine, and propanoic acid were upregulated upon oral administration of L3201. In contrast, L-threose and α-tocopherol, which displayed upregulation in the S.CTL group compared to the CTL group, exhibited a downregulation in response to L3201 treatment.

Fig. 2. Oral administration of L3201 causes changes in fecal metabolites of chronic stress-exposed mice. (A) Principal component analysis plot of fecal metabolomics of the three groups. (B) Log 2-fold change in fecal metabolites in CTL vs. S.CTL group (left) and S.CTL vs. L3201 group (right). (C) A volcano plot for the fecal metabolites in CTL vs. S.CTL group (left) and S.CTL vs. L3201 group (right). (D) Enrichment analysis of the metabolites that were downregulated by UCMS and reinstated upon L3201 treatment. (E, F) Significantly altered metabolic pathways: aminoacyl-tRNA biosynthesis and valine (E), leucine and isoleucine biosynthesis (F) and significantly effected metabolites (red box). PCA, principal component analysis; CTL, control; S.CTL, stress control; L3201, Lacticaseibacillus rhamnosus IDCC 3201; UCMS, unpredictable chronic mild stress.

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The volcano plot visualized the significantly altered metabolic compounds between the two groups (Fig. 2C). L-valine, glycerol, tagatose, oleic acid, stearic acid, cholesterol, L-isoleucine, and L-fucose were significantly reduced in the S.CTL group compared to the CTL group, and oleic acid and β-sitosterol were significantly reduced in the L3201 group compared to the S.CTL group. Interestingly, 4-methylmandelic acid was significantly increased in the L3201 group compared to the S.CTL group. KEGG pathway enrichment analysis showed that metabolites increased upon L3201 treatment under stress are related to aminoacyl-tRNA biosynthesis and valine, leucine, and isoleucine biosynthesis (Figs. 2D, E, and F). These results confirm that the induction of UCMS disrupts gut metabolism and that the administration of L3201 can ameliorate this imbalance.

Recent studies have shown that the gut metabolome is affected by psychological anxiety. A previous study showed that 11 specific metabolic pathways, including amino acid metabolism and lipid metabolism, were altered in the fecal metabolome of multiple depressed animal models under stress (Liu et al., 2022). It has been reported that glutamate, alanine, and L-serine in plasma could represent the extent of depression (Mitani et al., 2006). L-threonine, oxidized proline, serine, tyrosine, alanine, and isoleucine and related amino acid metabolism were found to be altered in the fecal metabolome of rats exposed to chronic stress (Jianguo et al., 2019). Furthermore, the hepatic metabolites of mice transplanted with feces from depressed patients showed significant changes in aminoacyl-tRNA biosynthesis (Li et al., 2018a). Consistent with previous findings, this study found that metabolites involved in aminoacyl-tRNA biosynthesis, such as L-alanine, L-isoleucine, and glycine, were reduced in the stress model compared to the control group.

Specific probiotics, called psychobiotics, have been reported to improve stress and depression. According to Ma et al. (2021), consumption of L. plantarum P-8 has been shown to reduce stress and anxiety in adults, and this has been attributed to an increase in gut microbial neuroactive metabolites upon probiotic intake. Treatment with L. rhamnosus HN001 (HN001) and Bifidobacterium animalis subsp. lactis HN019 (HN019), alone and in combination, improved anxiety-related behavioral indicators and significantly improved inflammatory markers in stress-induced mice (Huang et al., 2022). It was also found that consumption of L. rhamnosus Probio-M9 in stressed adults increased gut microbial diversity and altered intestinal metabolism, including arachidonic acid metabolism and amino acid metabolism (Zheng et al., 2021). Similar to previous studies, the current study showed that the metabolites related to aminoacyl-tRNA biosynthesis and valine, leucine and isoleucine biosynthesis, which were decreased by UCMS induction, were enhanced by the administration of L3201 (Figs. 2E and F). UCMS increases pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-12 like LPS (Reyes and Chandler, 2023; Zhao et al., 2019). In the previous study, IDCC3201 reduced TNF-a and IL-6 among the increased cytokines caused by LPS in cells (Chae et al., 2022). Although this study did not investigate a decrease in cytokine levels by L3201 administration, it is expected to reduce cytokines increased by stress. These results suggest that imbalances in amino acid metabolism play an important role in stress-related psychological disorders and that treatment with L3201 ameliorates these disruptions.