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Article

Bifidobacterium longum P77 and Lactiplantibacillus plantarum P72 and Their Mix—Live or Heat-Treated—Mitigate Sleeplessness and Depression in Mice: Involvement of Serotonergic and GABAergic Systems

by
Ji-Su Baek
,
Xiaoyang Ma
,
Hee-Seo Park
,
Dong-Yun Lee
and
Dong-Hyun Kim
*
College of Pharmacy, Kyung Hee University, Dongdaemun-gu, Seoul 02447, Republic of Korea
*
Author to whom correspondence should be addressed.
Cells 2025, 14(19), 1547; https://doi.org/10.3390/cells14191547
Submission received: 2 August 2025 / Revised: 26 September 2025 / Accepted: 30 September 2025 / Published: 3 October 2025
(This article belongs to the Special Issue Cellular and Molecular Mechanisms in Neuropsychiatric and Neurological Disorders)
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Abstract

Sleeplessness (insomnia) is a significant symptom associated with stress-induced depression/anxiety. In the present study, we selected Bifidobacterium longum P77, which increased serotonin production in corticosterone-stimulated SH-SY5Y cells, from the fecal bacteria collection of healthy volunteers and examined the effects of B. longum on depression, anxiety, and sleeplessness induced by immobilization stress or by transplantation of cultured fecal microbiota (cFM) from patients with depression. Orally administered B. longum P77 decreased depression/anxiety- and sleeplessness-like behaviors in immobilization stress-exposed mice. B. longum P77 reduced immobilization stress-induced corticosterone, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 expression and the cell population of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)+ in the prefrontal cortex, while the expression levels of immobilization stress-suppressed IL-10, γ-aminobutyric acid (GABA), its receptor GABAARα1, serotonin, and its receptor 5-HT1AR increased. B. longum P77 also alleviated immobilization stress-induced colitis: it decreased TNF-α and IL-6 expression and increased IL-10 expression in the colon. Furthermore, B. longum P77, Lactiplantibacillus plantarum P72, and their combination decreased cFM- or immobilization stress-induced depression-, anxiety-, and sleeplessness-like behaviors. They also decreased cFM-induced, corticosterone, TNF-α, and IL-6 expression levels in the prefrontal cortex and colon, while increasing cFM- or immobilization stress-suppressed GABA, GABAARα1, serotonin, and 5-HT1AR expression levels in the prefrontal cortex. In particular, the combination of B. longum P77 and L. plantarum P72 (P7277) additively or synergistically alleviated depression-, anxiety-, and sleeplessness-like behaviors, along with their associated biomarkers. Heat-killed P7277 also alleviated immobilization stress-induced depression/anxiety- and sleeplessness-like symptoms. These results imply that L. plantarum P72 and/or B. longum P77 can mitigate depression/anxiety and sleeplessness by upregulating GABAergic and serotonergic systems, along with the suppression of NF-κB activation.

1. Introduction

Long-term exposure to stress induces the secretion of adrenal hormones such as corticosterone, aldosterone, and adrenaline as well as proinflammatory cytokines such as tumor necrosis factor (TNF)-α, resulting in anxiety and depression [1,2]. Approximately 90% of patients with depression and anxiety, which are closely associated with decreased serotonin (5-HT) and γ-aminobutyric acid (GABA) levels and elevated interleukin (IL)-6 levels [3,4,5], suffer from sleeplessness [6].
Gut microbiota communicates bidirectionally with the brain through the gut–brain axis [7,8]. Stress-induced gut dysbiosis can contribute to gut inflammation as well as psychiatric disorder, including depression and sleeplessness [9,10,11]. Many probiotics, including lactobacilli and bifidobacteria, modulate immune responses such as inflammation and hypersensitivity and alleviate psychiatric disorders such as depression/anxiety and sleeplessness [12,13,14]. Lactiplantibacillus plantarum D-9, Bifidobacterium longum CCFM687, and B. longum 1714 alleviate stress-induced depression-like behavior in mice [10,15,16]. Lactobacillus rhamnosus GMNL-74 suppresses depression in ampicillin-exposed mice [17]. Bifidobacterium breve CCFM1025 improves sleep disorder in stress-exposed mice [18]. L. plantarum JYLP-326 simultaneously mitigates depression and sleep disorders in stress-exposed mice [18,19]. A mix of Lactobacillus reuteri NK33 and Bifidobacterium adolescentis NK98 (NVP1704) mitigates depression/anxiety- and sleeplessness like behaviors in stress-exposed mice [20]. L. plantarum P8 alleviates anxiety in stressed adults [21]. L. plantarum PS128 improves depression-related symptom in volunteers with self-reported insomnia [22]. B. breve CCFM1025 attenuates major depression disorder in volunteers [23]. NVP1704 mitigates depression- and sleeplessness-related symptoms in healthy volunteers with depression and insomnia symptoms [24]. L. plantarum JYLP-326 also alleviates depression and insomnia in test-anxious college students [19]. B. breve CCFM1025 improves sleep quality in healthy volunteers with stress-induced insomnia [25]. A mix of Lactiplantibacillus plantarum P72 and hempseed oil also synergistically mitigates immobilization stress-induced or cultured fecal microbiota of patients with depression (cFM)-induced depression/anxiety and sleeplessness in mice [26]. Nevertheless, the action mechanism by which probiotics alleviate sleeplessness associated with depression and anxiety remains unclear.
Therefore, we isolated Bifidobacterium longum P77 from the bacteria collection isolated from healthy feces and examined the effects of B. longum P77 and a combination of L. plantarum P72 and B. longum P77 (P7277) on immobilization stress- or cFM-induced depression/anxiety and sleeplessness in mice. Additionally, the effect of heat-killed P7277 (hP7277) on depression/anxiety and sleeplessness was investigated in immobilization stress-exposed mice.

2. Materials and Methods

2.1. Culture of B. longum P77 and L. plantarum P72

B. longum P77 (KCCM13446P) and L. plantarum P72 (KCCM13445P), which were provided by PBLbiolab (Seoul, Korea), were cultured in general media for probiotics such as MRS broth, centrifuged, and freeze-dried, as previously reported [26]. The number of their viable cells was quantified by using a commonly used method such as the culture in the agar plate. Based on the number of live cells, L. plantarum P72 and B. longum P77 were mixed at a ratio of 4:1. This (4:1) mixture is named P7277. Heat-killed P7277 (hP7277) was prepared by (4:1) mixing L. plantarum P72 and B. longum P77 heat-killed at 75 °C for 20 min. hP7277 did not grow on the MRS broth. However, there were no observable external differences between P7277 (live) and hP7277 (heat-killed) when examined visually, under a microscope, and through 16S rRNA gene analysis.

2.2. Culture of the SH-SY5Y Human Neuroblastoma Cell Line

The SH-SY5Y human neuroblastoma cell line, derived from a bone marrow biopsy of a neuroblastoma patient and widely used as a model for neuronal differentiation and neurodegenerative disease research, was cultured at 37 °C in Dulbecco’s Modified Eagle Medium containing 5% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. Cells (1 × 106 cells/mL) were maintained in a humidified incubator with 5% CO2 and 95% air. For experimental treatment, cells were exposed to 300 μM corticosterone (Sigma-Aldrich, Burlington, MA, USA), either alone or in combination with probiotics (1 × 105 colony-forming unit [CFU]/mL), for 24 h. After treatment, culture media were collected, and serotonin concentrations in the supernatants were determined using a commercially available serotonin quantification kit (DLD Diagnostika GmbH, Hamburg, Germany), following the instructions provided by the manufacturer.

2.3. Animals

Male C57BL/6 mice (6 weeks old, 18–20 g) were purchased from Koatech (Pyeongtaek-shi, Republic of Korea). Animals were housed in plastic cages under controlled environmental conditions (temperature: 20–22 °C; humidity: 50 ± 10%; 12 h light/dark cycle) and allowed to acclimate for one week prior to experimentation. During the study period, mice had free access to standard laboratory chow and water. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University (IACC: KHUASP(SE)_23545) and carried out in accordance with the University’s guidelines and ARRIVE guideline [27] and ethical standards for the care and use of laboratory animals.

2.4. Preparation of Mice with Depression/Anxiety and Sleeplessness and Treatment with Test Agents

Mice with depression/anxiety and sleeplessness were prepared by exposing them to immobilization stress or oral gavaging cFM of patients with depression (2 × 108 CFU/0.1 mL/mouse), as previously reported [20].
First, to investigate the effects of B. longum P77 and diphenhydramine against depression/anxiety and sleeplessness, mice were divided into four groups: normal control (NC), immobilization stress-exposed (IM), immobilization stress/B. longum P77-treated (P77), and immobilization stress/diphenhydramine-treated (DH) groups. Each consisted of eight mice. Except for NC, all groups were subjected to immobilization stress daily for 5 days, as previously reported [20]. Thereafter (24 h after the final stress exposure), test agents (IM, vehicle alone; P77, 1 × 109 CFU/mouse of B. longum P77; DH, 20 mg/kg diphenhydramine) were administered once daily for 7 days. NC received saline instead of test agents.
Second, mice were divided into five groups: NC, cFM-exposed (FM), cFM/L. plantarum P72-treated (P72), cFM/B. longum P77-treated (P77), and cFM/P7277-treated (P72P77) groups. Each group consisted of eight mice. cFM (1 × 109 CFU/mouse of the fecal microbiota culture suspended in 0.1 mL saline) was administered orally to all groups except NC for 5 days, as previously described [28]. Twenty-four h after the final cFM treatment, test agents (FM, vehicle; P72, 1 × 109 CFU/mouse of L. plantarum P72; P77, 1 × 109 CFU/mouse of B. longum P77; P72P77, 1 × 109 CFU/mouse of P7277) were orally administered once daily for 7 days. NC received saline.
Third, mice were divided into five groups: NC, immobilization stress-treated (IM), immobilization stress/L. plantarum P72-treated (P72), immobilization stress/B. longum P77-treated (P77), and immobilization stress/P7277-treated (P72P77) groups. Each group consisted of eight mice. Mice, except NC, were exposed to immobilization stress daily for 5 days, as previously reported [29]. Tested agents (IM, vehicle alone; P72, 1 × 109 CFU/mouse of L. plantarum P72; P77, 1 × 109 CFU/mouse of B. longum P77; P72P77, 1 × 109 CFU/mouse of P7277) were orally administered once daily for 7 days from 24 h after the final exposure to immobilization stress. NC was orally gavaged with saline instead of test agents.
Fourth, mice were divided into three groups: NC, immobilization stress-exposed (IM), and immobilization stress/hP7277-treated (P72P77) groups. Each group consisted of eight mice. Mice, except NC, were exposed to immobilization stress once daily for 5 days [29]. Tested agents (IM, vehicle alone; hP72P77, 1 × 109 CFU/mouse of hP7277) were orally administered once daily for 7 days from 24 h after the final exposure to immobilization stress. NC was orally gavaged with saline instead of test agents.
Depression-, anxiety-, and sleeplessness-like behaviors were assessed using multiple behavioral tests, as previously reported [30]. In the open field test, total distance moved, distance traveled in the center area, and time spent in the center area were recorded. In the elevated plus maze test, time spent in the open arms and entry number into the open arms were measured. In the tail suspension test, the immobility time was recorded. Thereafter, sleep latency time and sleep duration were measured (between 1:00 and 5:00 pm) after the exposure to isoflurane in the chamber or the intraperitoneal injection of pentobarbital sodium (40 mg/kg) [26,31]. Detailed protocols are described in Supplementary Methods.
For biomarker analysis, mice were euthanized by CO2 exposure in a closed chamber followed by cervical dislocation. Brain and colon tissues were promptly harvested and stored at −80 °C until further analysis. For immunofluorescence procedures, mice underwent transcardial perfusion with 4% paraformaldehyde, followed by cryoprotection in a 30% sucrose solution. Tissue samples were then processed for immunofluorescence staining according to previously established protocols [29].

2.5. Enzyme-Linked Immunosorbent Assay (ELISA) and Quantitative Polymerase Chain Reaction (qPCR)

Cytokine, serotonin, and GABA levels in the prefrontal cortex and colon were assessed using ELISA kits. Serotonin 1A receptor (5-HT1AR), 5-HT1BR, GABA type A receptor subunit alpha1 (GABAARα1), GABA type A receptor subunit alpha2 (GABAARα2), and 18S rRNA gene levels were accessed, using qPCR, as previously reported [20]. Primers are indicated in Supplementary Table S1. Detailed protocols are described in Supplementary Methods.

2.6. Whole Genome Analysis

The whole genome sequence for B. longum P77 was analyzed, as previously reported [26].

2.7. Statistical Analysis

Experimental data are indicated as mean ± standard deviation (SD) and accessed using GraphPad Prism 9. The significance difference was analyzed using one-way ANOVA with Tukey multiple comparisons test (p < 0.05). The statistical data are indicated in Supplementary Table S2.

3. Results

3.1. B. longum P77 Induced the Release of Serotonin in Corticosterone-Exposed SH-SY5Y Cells

To search serotonin production-inducing gut bifidobacteria, we measured the effects of bifidobacteria, which were selected from the bacteria collection of healthy human feces, on the release of serotonin in corticosterone-exposed SH-SY5Y cells. Among the tested strains, P77 markedly increased serotonin production (Figure 1). P77 was named B. longum P77, based on Gram staining, API kit, and 16S rRNA gene sequencing. The pseudogenome of B. longum P77 was 2,407,312 base pairs (contig 1) with a GC content of 60% (Supplementary Figure S1). The total number of CDS was 2014. The tRNA gene number was 62. The rRNA gene number was 13. The whole genome sequence of B. longum P77 exhibited the highest phylogenetic similarity to B. longum JCM1217 (98.6%), using OrthoANI.

3.2. B. longum P77 Attenuated Depression/Anxiety- and Sleeplessness-Like Symptoms in Immobilization Stress-Exposed Mice

To understand whether a serotonin production-enhancing probiotic B. longum P77 could alleviate psychiatric disorders, we examined their antidepressant effects in mice with immobilization stress-induced depression- and anxiety-like symptoms (Figure 2). Exposure to immobilization stress caused depression/anxiety-like behaviors: it reduced total distance moved, distance traveled in the center, and time spent in the center in the open field test and time spent in the open arms and entry number into the open arms in the elevated plus maze test to 81.0% (F3.28 = 33.92, p < 0.001), 69.5% (F3.28 = 28.94, p < 0.001), 46.8% (F3.28 = 31.16, p < 0.001), 45.6% (F3.28 = 18.72, p < 0.001), and 49.9% (F3.28 = 44.67, p < 0.001) of those observed in NC, respectively, and increased immobility time in the tail suspension test to 143.1% (F3.28 = 46.20, p < 0.001) of that observed in NC.
In contrast, oral administration of B. longum P77 alleviated immobilization stress-induced depression- and anxiety-like behaviors. It recovered immobilization stress-suppressed total distance moved, distance traveled in the center, time spent in the center, time spent in the open arms, and entry number into the open arms to 104.5%, 126.5%, 98.1%, 94.0%, and 67.9% of those observed in NC, respectively. B. longum P77 reduced immobilization stress-induced immobility time to 106.7% of that observed in NC.
Exposure to immobilization stress caused sleeplessness in mice: it increased pentobarbital-induced sleep latency time to 131.3% (F3,28 = 39.31, p < 0.001) of that observed in NC and decreased pentobarbital-induced sleep duration to 44.5% (F3,28 = 63.24, p < 0.001) of that observed in NC. However, B. longum P77 partially recovered sleeplessness in mice. It recovered immobilization stress-increased sleep latency time to 111.4% of that observed in NC and immobilization stress-decreased sleep duration to 72.2% of that observed in NC. Its potency was comparable to that of diphenhydramine.
Exposure to immobilization stress decreased the expression levels of GABA, serotonin, their receptors (GABAARα1, GABAARα2, 5-HT1AR, 5-HT1BR), and IL-10 and increased the expression levels of corticosterone, TNF-α, and IL-6 and population of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-positive cells in the prefrontal cortex (Figure 3, Supplementary Figure S2). However, treatment with B. longum P77 increased immobilization stress-downregulated GABA, serotonin, their receptors, GABAARα1, GABAARα2, 5-HT1AR, 5-HT1BR, and IL-10 expression and the GABAARα1+ cell population in the prefrontal cortex while decreasing immobilization stress-induced corticosterone levels. B. longum P77 decreased immobilization stress-induced TNF-α and IL-6 expression and the NF-κB+CD11c+ cell population and increased immobilization stress-suppressed IL-10 expression in the prefrontal cortex.
Gut dysbiosis-induced gut inflammation is closely involved in the occurrence of psychiatric disorder, including depression [11]. Therefore, we examined whether immobilization stress could cause colitis, a symptom of depression, and B. longum P77 could alleviate immobilization stress-induced colitis. Exposure to immobilization stress caused colitis: it increased myeloperoxidase, TNF-α, IL-1β, and IL-6 expression and decreased IL-10 expression in the colon (Figure 4, Supplementary Figure S3). However, oral administration of B. longum P77 significantly decreased immobilization stress-induced myeloperoxidase, TNF-α, IL-1β, and IL-6 expression and the NF-κB+CD11c+ cell population.

3.3. B. longum P77, L. plantarum P72, and P7277 Alleviated cFM Transplantation-Induced Depression/Anxiety- and Sleeplessness-Like Behaviors and Gut Inflammation in Mice

To determine whether the depression-, anxiety- and sleeplessness-alleviating activity of B. longum P77 could be synergistically enhanced by L. plantarum P72, we examined the effects of B. longum P77, L. plantarum P72, and P7277 on cFM transplantation-induced depression/anxiety- and sleeplessness-like symptoms in mice (Figure 5).
cFM transplantation decreased total distance moved, distance traveled in the center, time spent in the center, time spent in the open arms, and entry number into the open arms to 86.1% (F4.35 = 6.418, p < 0.001), 62.6% (F4.35 = 16.37, p < 0.001), 48.6% (F4.35 = 28.49, p < 0.001), 54.0% (F4.35 = 7.509, p = 0.0002), and 51.4% (F4.35 = 17.94, p < 0.001) of those observed in NC, respectively, and increased immobility time to 124.2% (F4.35 = 13.14, p < 0.001) of that observed in NC. However, orally administered L. plantarum P72, B. longum P77, and P7277 significantly increased cFM-decreased distance traveled in the center to 93.4%, 97.0%, and 92.8% of that observed in NC, respectively, time spent in the center to 77.7%, 78.2% and 80.5% of that observed in NC, respectively, and time spent in the open arms to 110.0%, 92.6%, and 98.6% of that observed in NC, respectively, and entry number into the open arms to109.2%, 91.5%, and 98.2% of that observed in NC, respectively, and decreased cFM-increased immobility time to 79.9%, 92.6%, and 88.6% of that observed in NC, respectively, while FM-decreased total distance moved was significantly increased by treatment with L. plantarum P72 alone.
cFM transplantation caused sleeplessness-like behavior: it increased pentobarbital-induced sleep latency time to 119.5% (F4.35 = 13.14, p < 0.001) of that observed in NC and decreased pentobarbital-induced sleep duration to 50.9% (F4.35 = 9.33, p ≤ 0.001) of that observed in NC. However, oral administration of L. plantarum P72, B. longum P77, and P7277 significantly decreased cFM-increased sleep latency time to 105.4%, 107.3%, and 93.1% of that observed in NC, respectively, and increased cFM-decreased sleep duration to 77.2%, 75.5%, and 92.4% of that observed in NC, respectively.
cFM transplantation decreased serotonin, GABA, GABAARα1, GABAARα2, 5-HT1AR, 5-HT1BR, and IL-10 expression and increased corticosterone and TNF-α expression in the prefrontal cortex (Figure 6). However, oral administration of L. plantarum P72, B. longum P77, and P7277 significantly increased cFM-suppressed GABA, serotonin, GABAARα1, GABAARα2, and IL-10 expression and decreased cFM-induced corticosterone levels. They weakly increased cFM-suppressed 5-HT1AR and 5-HT1BR expression and decreased cFM-induced TNF-α expression.
Next, we examined whether cFM transplantation could cause colitis, a symptom of depression, and L. plantarum P72, B. longum P77, and P7277 could alleviate immobilization stress-induced colitis. cFM transplantation also caused colitis in mice: it increased myeloperoxidase, TNF-α, IL-1β, and IL-6 expression and decreased IL-10 expression in the colon (Figure 7). However, oral administration of L. plantarum P72, B. longum P77, or P7277 significantly suppressed cFM-induced myeloperoxidase, TNF-α, IL-1β, and IL-6 expression. L. plantarum P72 alone significantly increased FM-suppressed IL-10 expression.

3.4. L. plantarum P72, B. longum P77, and P7277 Alleviated Immobilization Stress-Induced Depression/Anxiety- and Sleeplessness-Like Behavior and Systemic Inflammation in Mice

Next, we examined the effects of L. plantarum P72, B. longum P77, and P7277 on immobilization stress-induced depression/anxiety- and sleeplessness-like symptoms in mice (Figure 8). Exposure to immobilization stress significantly decreased total distance moved, distance traveled in the center, time spent in the center, time spent in the open arms, and entry number into the open arms to 85.4% (F4.35 = 15.74, p < 0.001), 55.7% (F4.35 = 7.71, p = 0.001), 41.0% (F4.35 = 14.03, p < 0.001), 37.7% (F4.35 = 21.06, p < 0.001), and 49.3% (F4.35= 18.32, p < 0.001) of those observed in NC, respectively. Immobilization stress increased immobility time to 145.2% (F4.35 = 28.49, p < 0.001) of that observed in NC. However, oral administration of L. plantarum P72, B. longum P77, and P7277 significantly recovered immobilization stress-suppressed total distance moved to 108.5%, 104.2%, and 110% of that observed in NC, respectively, distance traveled in the center to 98.5%, 99.6%, and 106.1% of that observed in NC, respectively, time spent in the center to 92.7%, 93.7 and 99.7% of that observed in NC, respectively, time spent in the open arms to 106.6%, 94.6%, and 110.0% of that observed in NC, respectively, and entry number into the open arms to 82.3%, 75.9%, and 91.7% of that observed in NC, respectively. They decreased immobility time to 108.5%, 104.2%, and 110.0% of that observed in NC, respectively.
Immobilization stress treatment caused sleeplessness-like behavior: it increased isoflurane-induced sleep latency time to 127.3% (F4.35 = 9.67, p < 0.01) of that observed in NC and decreased isoflurane-induced sleep duration to 87.8% (F4.35 = 3.85, p = 0.011) of that observed in NC. However, oral administration of L. plantarum P72, B. longum P77, or P7277 significantly decreased immobilization stress-induced sleep latency time to 97.2%, 102.0%, and 98.4% of that observed in NC, respectively, and increased immobilization stress-decreased sleep duration to 107.2%, 104.7% and 113.9% of that observed in NC, respectively.
Exposure to immobilization stress downregulated GABA, serotonin, and their receptors GABAARα1, GABAARα2, 5-HT1AR, and 5-HT1BR expression and upregulated corticosterone and TNF-α expression in the prefrontal cortex (Figure 9). However, oral administration of L. plantarum P72, B. longum P77, or P7277 increased immobilization stress-suppressed GABA, serotonin, GABAARα1 and GABAARα2, 5-HT1AR, and 5-HT1BR expression in the prefrontal cortex while decreasing the corticosterone level. L. plantarum P72 more potently increased immobilization stress-suppressed GABAARα1 and 5-HT1AR expression than B. longum P77. Immobilization stress-induced TNF-α expression was suppressed by P7277 alone.
Next, we examined whether L. plantarum P72, B. longum P77, and P7277 could alleviate immobilization stress-induced colitis, a symptom of depression, in mice. Exposure to immobilization stress also caused colitis in mice, increasing myeloperoxidase, TNF-α, IL-1β, and IL-6 expression in the colon (Figure 10). However, oral administration of L. plantarum P72, B. longum P77, or P7277 caused a reduction in immobilization stress-induced myeloperoxidase, TNF-α, IL-1β, and IL-6 expression.

3.5. hP7277 Alleviated Immobilization Stress-Induced Depression/Anxiety- and Sleeplessness-Like Behavior and Neuroinflammation in Mice

Next, we examined whether heat-killed P7277 (hP7277) could alleviate depression/anxiety- and sleeplessness-like symptoms in immobilization stress-exposed mice (Figure 11). Immobilization stress treatment also significantly caused depression/anxiety- and sleeplessness-like behaviors: it total distance moved, distance traveled in the center, time spent in the center, time spent in the open arms, and entry number into the open arms to 86.3% (F2,21 = 4.69, p = 0.02), 66.2% (F2,21 = 5.98, p < 0.001), 62.7% (F2,21 = 6.71, p < 0.001), 52.3% (F2,21 = 18.72, p < 0.001), and 49.0% (F2,21 = 30.65, p < 0.001) of those observed in NC, respectively, and increased immobility time to 133.1% (F2,21 = 30.65, p < 0.001) of that observed in NC. However, hP7277 recovered immobilization stress-suppressed total distance moved, distance traveled in the center, time spent in the center, time spent in the open arms, and entry number into the open arms to 98.7%, 80.8%, 82.0%, 95.2%, and 68.7% of those observed in NC, respectively. hP7277 decreased immobilization stress-induced immobility time to 101.1% of that observed in NC. Immobilization stress treatment increased isoflurane-induced sleep latency time to 126.7% (F2,21 = 8.19, p < 0.001) of that observed in NC and decreased isoflurane-induced sleep duration to 82.7% (F2,21 = 7.62, p < 0.001) of that observed in NC. hP7277 reduced immobilization stress-induced sleep latency time to 104.3% of that observed in NC and increased immobilization stress-decreased sleep duration to 101.0% of NC. However, depression/anxiety- and sleeplessness-like behaviors-ameliorating effect of hP7277 was slightly weaker than that of live P7277: total distance moved, distance traveled in the center, time spent in the center, time spent in the open arms, entry number into the open arms, immobility time, sleep latency time, and sleep duration in mice treated with hP7277 were 89.7%, 76.2%, 82.2%, 86.5%, 74.9%, 91.9, 106.0, and 88.7% of those in mice treated with live P7277, respectively.
hP7277 significantly increased immobilization stress-suppressed serotonin and 5-HT1AR expression in the prefrontal cortex and IL-10 expression in the colon, while immobilization stress-induced TNF-α and IL-6 expression in the colon (Figure 12). The immobilization stress-induced corticosterone level in the prefrontal cortex was weakly, but not significantly, decreased.

4. Discussion

Stress causes depression and anxiety, which are accompanied by sleeplessness and gut dysbiosis [32,33]. Gut dysbiosis can cause psychiatric disorders including depression/anxiety [11,34] and systemic inflammation [35]. We also found that exposure to immobilization stress induced depression/anxiety and sleeplessness in mice. Furthermore, exposure to immobilization stress suppressed the production of GABA and serotonin and expression of GABAARα1, GABAARα2, 5-HT1AR, and 5-HT1BR in the brain, while TNF-α and IL-6 expression was induced. Immobilization stress also induced TNF-α and IL-6 expression in the colon. The transplantation of depressive patient-derived fecal microbiota also caused depression/anxiety- and sleeplessness-like behaviors in mice. The transplantation suppressed the expression of GABA, serotonin, and their receptors in the brain. Stress including immobilization stress induces gut dysbiosis, as well as systemic inflammation, which reduces serotonin and GABA production in the brain [29,36,37,38]. The transplantation of gut microbiota from volunteers with depression causes depression/anxiety and neuroinflammation in mice [39,40]. These results imply that exposure to immobilization stress can cause depression/anxiety and sleeplessness through the inflammation-involved suppression of serotonin and GABA systems.
Therefore, we selected serotonin production-inducing B. longum P77 from the bacterial collection of healthy human feces. The dose of B. longum P77 in mouse experiments (1 × 109 CFU/mouse/day) was decided based on the results of animal and in vitro experiments (4 × 108 CFU/mouse/day and 1 × 109 CFU/mouse day in the in vivo experiment and 1 × 109 CFU/mL in the in vitro experiment) previously conducted with L. plantarum P72 [26]. Furthermore, B. longum P77 (1 × 109 CFU/mouse day) alleviated immobilization stress- or cFM-induced depression/anxiety- and sleeplessness-like behaviors. Furthermore, B. longum P77 upregulated immobilization stress- or cFM-suppressed GABA, serotonin, GABAARα1, GABAARα2, 5-HT1AR, and 5-HT1BR expression in the brain. Moreover, B. longum P77 downregulated immobilization stress- or cFM-induced TNF-α, IL-6, and NF-κB+ cell population in the brain and colon. GABA and benzodiazepines (GABAAR agonists) mitigate depression/anxiety and insomnia [41]. 5-HT1AR and HT1BR agonists, such as buspirone, improve depression/anxiety and sleeplessness [42,43,44]. TNF-α and IL-6, which are expressed through the activation of NF-κB [45], suppress the release of GABA from the hippocampus [46]. TNF-α does not suppress serotonin release directly, but it can influence serotonin level, and may play an important role in mood and inflammation [47,48]. Serotonin suppresses TNF-α expression [49,50]. These findings suggest that B. longum P77 can alleviate depression/anxiety and sleeplessness through the regulation of GABA and serotonin signaling, along with NF-κB activation.
Bifidobacterium infantis CCFM687, B. longum 1714, and Bifidobacterium adolescentis NK98 attenuate depression-like behavior in stress-exposed mice [10,16,29]. L. reuteri NK33, L. plantarum R6-3, and L. plantarum P72 alleviate depression-like symptoms in stress-exposed mice [26,29,51]. NVP1704, a combination of L. reuteri NK33 and Bifidobacterium adolescentis NK98, effectively alleviates depression- and insomnia-related symptoms in mice exposed to immobilization stress and healthy volunteers with depression and insomnia symptoms. In the present study, we found that L. plantarum P72 alleviated depression/anxiety- and sleeplessness-like behaviors, as previously reported [26]. L. plantarum P72 also increased immobilization stress- or cFM-suppressed GABA, serotonin, GABAARα1, GABAARα2, 5-HT1AR, and 5-HT1BR expression in the prefrontal cortex, like B. longum P77. Therefore, to understand whether the combination of Lactobacillus species and Bifidobacterium species could show the synergistic or antagonistic effects against depression and SN in vivo, we examined the combined effects of L. plantarum P72 and B. longum P77 on immobilization stress- or cFM-induced depression and SN in mice. When B. longum P77 (0.2 × 109 CFU/mouse/day) was combined with L. plantarum P72 (0.8 × 109 CFU/mouse/day), the combined P7277 (1 × 109 CFU/mouse/day) alleviated additively or synergistically immobilization stress- or cFM-induced depression/anxiety- and sleeplessness-like behaviors in mice. P7277, a mix of L. plantarum P72 and B. longum P77, more potently, but not significantly, alleviated immobilization stress- or cFM-induced depression/anxiety- and sleeplessness-like behaviors than B. longum P77 (0.2 × 109 CFU/mouse/day) or L. plantarum P72 (0.2 × 109 CFU/mouse/day) alone. P7277 upregulated immobilization stress- or cFM-suppressed GABA, serotonin, GABAARα1, GABAARα2, 5-HT1AR, and 5-HT1BR expression in the brain more potently, but not significantly, than L. plantarum P72 or B. longum P77 alone. Moreover, P7277 also downregulated immobilization stress- orc cFM-induced corticosterone, TNF-α, IL-6, and NF-κB+ cell population in the brain and colon more potently, but not significantly, than L. plantarum P72 or B. longum P77 alone. These results suggest that the combination of B. longum P77 and L. plantarum P72 may additively or synergistically alleviate depression and sleeplessness and could therefore offer greater therapeutic benefits for the treatment of depression, anxiety, and sleeplessness than either probiotic alone.
hP7277, a heat-killed P7277, also alleviated depression/anxiety- and sleeplessness-like behaviors. hP7277 also upregulated immobilization stress-suppressed GABA, serotonin, GABAARα1, GABAARα2, 5-HT1AR, and 5-HT1BR expression in the brain and downregulated immobilization stress-induced corticosterone, TNF-α and IL-6 levels in the brain and colon. However, its efficacy was weaker than that of live P7277. These results suggest that, although the reduced efficacy of hP7277 may be because it lacks the ability to proliferate or adhere in the intestine, it can also be useful as an alternative therapeutic option of depression/anxiety and sleeplessness in place of live P7277.
Approximately 90% of patients with depression suffer from insomnia [6,52]. Nevertheless, insomnia can sometimes be an early warning sign that precedes the development of depression. Therefore, combing standard antidepressant medication with sleep-focused interventions has shown significant success in improving both depression and insomnia [53]. Based on these findings, P7277 may be beneficial for the therapy of patients with depression and sleeplessness (insomnia).
In the present study, we were not able to investigate whether B. longum P77, L. plantrum P72, and P7277 are effective as a single-dose treatment or whether their effects differ by sex. Therefore, further studies including these aspects are necessary to clarify the specific mechanisms through which the components of L. plantarum P72 and B. longum P77 exert their effects on depression and sleeplessness.

5. Conclusions

B. longum P77 alleviated depression/anxiety- and sleeplessness-like symptoms by inducing GABA and serotonin production and suppressing the expression of proinflammatory cytokines. P7277, a mix of L. plantarum P72 and B. longum P77, additively or synergistically alleviated depression/anxiety- and sleeplessness-like symptoms. P7277 and hP7277, which is a heat-killed P7277, may improve depression/anxiety and sleeplessness through the activation of GABA and serotonin systems, along with the suppression of NF-κB activation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells14191547/s1, Figure S1. Taxonomic classification by genome-wide comparative analysis of B. longum P77; Figure S2. Effects of B. longum P77 and diphenhydramine on GABAARα1+ (a) and NF-κB+CD11c+ cell population (b) in the prefrontal cortex of mice with immobilization stress-induced depression/anxiety and sleeplessness; Figure S3. Effects of B. longum P77 and diphenhyramine on colonic NF-κB+CD11c+ cell population in mice with immobilization stress-induced colitis; Table S1: Primers used in this study; Table S2. Statistic data in the present study. Methods: Preparation of mice with immobilization stress and behavioral tasks.

Author Contributions

J.-S.B.: Writing—original draft, software, validation, supervision, methodology, formal analysis, conceptualization. X.M.: Writing—original draft, software, validation, methodology, formal analysis. H.-S.P.: Writing—original draft, validation, methodology, formal analysis. D.-Y.L.—methodology, investigation. D.-H.K.: Writing—original draft, writing—review and editing, project administration, supervision, resources, conceptualization. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Commercialization Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korea government (MSIT) (RS-2024-00486802) and the Medical Research Program (2017R1A5A2014768) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, Korea.

Institutional Review Board Statement

All animal experiments were ethically approved by The Committee for the Care and Use of Laboratory Animals in the Kyung Hee University (Approval Code, IACC: KHUASP(SE)_23545; Approval date, 4 December 2023) and conducted according to the Ethical Policies and Guidelines of the University for Laboratory Animals Care and Use and ARRIVE guideline.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data generated in this study are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Cells 14 01547 g001
Figure 1. Effect of B. longum P77 on the serotonin production in corticosterone (Cort)-stimulated SH-SY5Y cells. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. Cort.
Figure 1. Effect of B. longum P77 on the serotonin production in corticosterone (Cort)-stimulated SH-SY5Y cells. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. Cort.
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Figure 2. Effects of B. longum P77 and diphenhydramine on immobilization stress-induced depression/anxiety- and sleeplessness-like behaviors in mice. Effects on total distance moved (a), distance traveled in the center (b), time spent in the center (c), and track path (d) in the open field test. Effects on time spent in the open arms (e) and entry number into the open arms (f) in the elevated plus maze test and immobility time in the tail suspension test (g). Effects on sleep latency time (h) and sleep duration (i). Test agents (IM, vehicle; P77, 1 × 109 CFU/mouse/day of B. longum P77; DH, 20 mg/kg of diphenhydramine) were treated in immobilization stress/pentobarbital-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Figure 2. Effects of B. longum P77 and diphenhydramine on immobilization stress-induced depression/anxiety- and sleeplessness-like behaviors in mice. Effects on total distance moved (a), distance traveled in the center (b), time spent in the center (c), and track path (d) in the open field test. Effects on time spent in the open arms (e) and entry number into the open arms (f) in the elevated plus maze test and immobility time in the tail suspension test (g). Effects on sleep latency time (h) and sleep duration (i). Test agents (IM, vehicle; P77, 1 × 109 CFU/mouse/day of B. longum P77; DH, 20 mg/kg of diphenhydramine) were treated in immobilization stress/pentobarbital-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
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Figure 3. Effects of B. longum P77 and diphenhydramine on the expression of depression/anxiety- and sleeplessness-related biomarkers in the prefrontal cortex of mice exposed to immobilization stress. Effects on GABA (a), GABAARα1 (b), and GABAARα2 (c) expression levels and GABAARα1+ cell population (d). Effects on serotonin (e), 5-HT1AR (f), and 5-HT1BR (g) expression levels. Effects on corticosterone (h), TNF-α (i), IL-6 (j) and IL-10 (k) expression and NF-κB+CD11c+ cell population (l). Test agents (IM, vehicle; P77, 1 × 109 CFU/mouse/day of B. longum P77; DH, 20 mg/kg of diphenhydramine) were treated in immobilization stress/pentobarbital-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Figure 3. Effects of B. longum P77 and diphenhydramine on the expression of depression/anxiety- and sleeplessness-related biomarkers in the prefrontal cortex of mice exposed to immobilization stress. Effects on GABA (a), GABAARα1 (b), and GABAARα2 (c) expression levels and GABAARα1+ cell population (d). Effects on serotonin (e), 5-HT1AR (f), and 5-HT1BR (g) expression levels. Effects on corticosterone (h), TNF-α (i), IL-6 (j) and IL-10 (k) expression and NF-κB+CD11c+ cell population (l). Test agents (IM, vehicle; P77, 1 × 109 CFU/mouse/day of B. longum P77; DH, 20 mg/kg of diphenhydramine) were treated in immobilization stress/pentobarbital-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
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Figure 4. Effects of B. longum P77 and diphenhydramine on immobilization stress-induced colitis in mice. Effects on myeloperoxidase (a), TNF-α (b), IL-1β (c), IL-6 (d), and IL-10 expression (e) and NF-κB+CD11c+ cell population (f) in the colon. Test agents (IM, vehicle; P77, 1 × 109 CFU/mouse/day of B. longum P77; DH, 20 mg/kg of diphenhydramine) were treated in immobilization stress/pentobarbital-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Figure 4. Effects of B. longum P77 and diphenhydramine on immobilization stress-induced colitis in mice. Effects on myeloperoxidase (a), TNF-α (b), IL-1β (c), IL-6 (d), and IL-10 expression (e) and NF-κB+CD11c+ cell population (f) in the colon. Test agents (IM, vehicle; P77, 1 × 109 CFU/mouse/day of B. longum P77; DH, 20 mg/kg of diphenhydramine) were treated in immobilization stress/pentobarbital-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
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Cells 14 01547 g005
Figure 5. Effects of L. plantarum P72, B. longum P77, and P7277 on cFM-induced depression/anxiety- and sleeplessness-like behaviors in mice. Effects on total distance moved (a), distance traveled in the center (b), time spent in the center (c), and track path (d) in the open field test. Effects on time spent in the open arms (e) and entry number into the open arms (f) in the elevated plus maze test and immobility time in the tail suspension test (g). Effects on sleep latency time (h) and sleep duration (i). Test agents (FM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in cFM/pentobarbital-exposed mice. NC was treated with saline in cFM-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. FM.
Figure 5. Effects of L. plantarum P72, B. longum P77, and P7277 on cFM-induced depression/anxiety- and sleeplessness-like behaviors in mice. Effects on total distance moved (a), distance traveled in the center (b), time spent in the center (c), and track path (d) in the open field test. Effects on time spent in the open arms (e) and entry number into the open arms (f) in the elevated plus maze test and immobility time in the tail suspension test (g). Effects on sleep latency time (h) and sleep duration (i). Test agents (FM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in cFM/pentobarbital-exposed mice. NC was treated with saline in cFM-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. FM.
Cells 14 01547 g005
Cells 14 01547 g006
Figure 6. Effects of L. plantarum P72, B. longum P77, and P7277 on the expression of depression/anxiety- and sleeplessness-related biomarkers in the prefrontal cortex of mice exposed to cFM. Effects on GABA (a), GABAARα1 (b), and GABAARα2 (c) expression levels. Effects on serotonin (d), 5-HT1AR (e), and 5-HT1BR (f) expression levels. Effects on corticosterone (g), TNF-α (h) and IL-10 expression (i). Test agents (FM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in cFM/pentobarbital-exposed mice. NC was treated with saline in cFM-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. FM.
Figure 6. Effects of L. plantarum P72, B. longum P77, and P7277 on the expression of depression/anxiety- and sleeplessness-related biomarkers in the prefrontal cortex of mice exposed to cFM. Effects on GABA (a), GABAARα1 (b), and GABAARα2 (c) expression levels. Effects on serotonin (d), 5-HT1AR (e), and 5-HT1BR (f) expression levels. Effects on corticosterone (g), TNF-α (h) and IL-10 expression (i). Test agents (FM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in cFM/pentobarbital-exposed mice. NC was treated with saline in cFM-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. FM.
Cells 14 01547 g006
Cells 14 01547 g007
Figure 7. Effects of L. plantarum P72, B. longum P77, and P7277 on cFM-induced colitis in mice. Effects on myeloperoxidase (a), TNF-α (b), IL-1β (c), IL-6 (d), and IL-10 expression (e) in the colon. Test agents (FM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in cFM-exposed mice. NC was treated with saline in cFM/pentobarbital-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. FM.
Figure 7. Effects of L. plantarum P72, B. longum P77, and P7277 on cFM-induced colitis in mice. Effects on myeloperoxidase (a), TNF-α (b), IL-1β (c), IL-6 (d), and IL-10 expression (e) in the colon. Test agents (FM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in cFM-exposed mice. NC was treated with saline in cFM/pentobarbital-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. FM.
Cells 14 01547 g007
Cells 14 01547 g008
Figure 8. Effects of L. plantarum P72, B. longum P77, and P7277 on immobilization stress-induced depression/anxiety- and sleeplessness-like behaviors in mice. Effects on total distance moved (a), distance traveled in the center (b), time spent in the center (c), and track path (d) in the open field test. Effects on time spent in the open arms (e) and entry number into the open arms (f) in the elevated plus maze test and immobility time in the tail suspension test (g). Effects on sleep latency time (h) and sleep duration (i). Test agents (IM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Figure 8. Effects of L. plantarum P72, B. longum P77, and P7277 on immobilization stress-induced depression/anxiety- and sleeplessness-like behaviors in mice. Effects on total distance moved (a), distance traveled in the center (b), time spent in the center (c), and track path (d) in the open field test. Effects on time spent in the open arms (e) and entry number into the open arms (f) in the elevated plus maze test and immobility time in the tail suspension test (g). Effects on sleep latency time (h) and sleep duration (i). Test agents (IM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Cells 14 01547 g008
Cells 14 01547 g009
Figure 9. Effects of L. plantarum P72, B. longum P77, and P7277 on the expression of depression/anxiety- and sleeplessness-related biomarkers in the prefrontal cortex of mice exposed to immobilization stress. Effects on GABA (a), GABAARα1 (b), and GABAARα2 (c) expression levels. Effects on serotonin (d), 5-HT1AR (e), and 5-HT1BR (f) expression levels. Effects on corticosterone (g) and TNF-α (h) expression. Test agents (IM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Figure 9. Effects of L. plantarum P72, B. longum P77, and P7277 on the expression of depression/anxiety- and sleeplessness-related biomarkers in the prefrontal cortex of mice exposed to immobilization stress. Effects on GABA (a), GABAARα1 (b), and GABAARα2 (c) expression levels. Effects on serotonin (d), 5-HT1AR (e), and 5-HT1BR (f) expression levels. Effects on corticosterone (g) and TNF-α (h) expression. Test agents (IM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Cells 14 01547 g009
Cells 14 01547 g010
Figure 10. Effects of L. plantarum P72, B. longum P77, and P7277 on immobilization stress-induced colitis in mice. Effects on myeloperoxidase (a), TNF-α (b), IL-1β (c) and IL-6 (d) expression in the colon. Test agents (IM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Figure 10. Effects of L. plantarum P72, B. longum P77, and P7277 on immobilization stress-induced colitis in mice. Effects on myeloperoxidase (a), TNF-α (b), IL-1β (c) and IL-6 (d) expression in the colon. Test agents (IM, vehicle; P72, 1 × 109 CFU/mouse/day of L. plantarum P72; P77, 1 × 109 CFU/mouse/day of B. longum P77; P72P77, 1 × 109 CFU/mouse/day of P7277) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
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Cells 14 01547 g011
Figure 11. Effect of heat-killed P7277 (hP72P77) on immobilization stress-induced depression/anxiety- and sleeplessness-like behaviors in mice. Effect on total distance moved (a), distance traveled in the center (b), time spent in the center (c), and track path (d) in the open field test. Effects on time spent in the open arms (e) and entry number into the open arms (f) in the elevated plus maze test and immobility time in the tail suspension test (g). Effect on sleep latency time (h) and sleep duration (i). Test agents (IM, vehicle; hP72P7, 1 × 109 CFU/mouse/day of heat-killed L. plantarum P72 and B. longum P77 [4:1] mix) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Figure 11. Effect of heat-killed P7277 (hP72P77) on immobilization stress-induced depression/anxiety- and sleeplessness-like behaviors in mice. Effect on total distance moved (a), distance traveled in the center (b), time spent in the center (c), and track path (d) in the open field test. Effects on time spent in the open arms (e) and entry number into the open arms (f) in the elevated plus maze test and immobility time in the tail suspension test (g). Effect on sleep latency time (h) and sleep duration (i). Test agents (IM, vehicle; hP72P7, 1 × 109 CFU/mouse/day of heat-killed L. plantarum P72 and B. longum P77 [4:1] mix) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Cells 14 01547 g011
Cells 14 01547 g012
Figure 12. Effect of hP7277 on the expression of depression/anxiety- and sleeplessness-related biomarkers in the prefrontal cortex and colon of mice exposed to immobilization stress. Effect on serotonin (a), 5-HT1AR (b), and corticosterone (c) levels in the prefrontal cortex. Effects on TNF-α (d), IL-6 (e), and IL-10 expression (f) in the colon. Test agents (IM, vehicle; hP72P77, 1 × 109 CFU/mouse/day of heat-killed L. plantarum P72 and B. longum P77 [4:1] mix) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
Figure 12. Effect of hP7277 on the expression of depression/anxiety- and sleeplessness-related biomarkers in the prefrontal cortex and colon of mice exposed to immobilization stress. Effect on serotonin (a), 5-HT1AR (b), and corticosterone (c) levels in the prefrontal cortex. Effects on TNF-α (d), IL-6 (e), and IL-10 expression (f) in the colon. Test agents (IM, vehicle; hP72P77, 1 × 109 CFU/mouse/day of heat-killed L. plantarum P72 and B. longum P77 [4:1] mix) were treated in immobilization stress/isoflurane-exposed mice. NC was treated with saline in immobilization stress-untreated mice. n = 8. # p < 0.05 vs. NC. * p < 0.05 vs. IM.
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MDPI and ACS Style

Baek, J.-S.; Ma, X.; Park, H.-S.; Lee, D.-Y.; Kim, D.-H. Bifidobacterium longum P77 and Lactiplantibacillus plantarum P72 and Their Mix—Live or Heat-Treated—Mitigate Sleeplessness and Depression in Mice: Involvement of Serotonergic and GABAergic Systems. Cells 2025, 14, 1547. https://doi.org/10.3390/cells14191547

AMA Style

Baek J-S, Ma X, Park H-S, Lee D-Y, Kim D-H. Bifidobacterium longum P77 and Lactiplantibacillus plantarum P72 and Their Mix—Live or Heat-Treated—Mitigate Sleeplessness and Depression in Mice: Involvement of Serotonergic and GABAergic Systems. Cells. 2025; 14(19):1547. https://doi.org/10.3390/cells14191547

Chicago/Turabian Style

Baek, Ji-Su, Xiaoyang Ma, Hee-Seo Park, Dong-Yun Lee, and Dong-Hyun Kim. 2025. "Bifidobacterium longum P77 and Lactiplantibacillus plantarum P72 and Their Mix—Live or Heat-Treated—Mitigate Sleeplessness and Depression in Mice: Involvement of Serotonergic and GABAergic Systems" Cells 14, no. 19: 1547. https://doi.org/10.3390/cells14191547

APA Style

Baek, J.-S., Ma, X., Park, H.-S., Lee, D.-Y., & Kim, D.-H. (2025). Bifidobacterium longum P77 and Lactiplantibacillus plantarum P72 and Their Mix—Live or Heat-Treated—Mitigate Sleeplessness and Depression in Mice: Involvement of Serotonergic and GABAergic Systems. Cells, 14(19), 1547. https://doi.org/10.3390/cells14191547

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