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

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 GABA_ARα1, serotonin, and its receptor 5-HT_1AR 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, GABA_ARα1, serotonin, and 5-HT_1AR 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.

Keywords: Bifidobacterium longum P77; GABA; Lactiplantibacillus plantarum P72; depression; serotonin; sleeplessness.

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 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 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), GABA_ARα1 (b), and GABA_ARα2 (c) expression levels and GABA_ARα1⁺ cell population (d). Effects on serotonin (e), 5-HT_1AR (f), and 5-HT_1BR (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 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 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 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), GABA_ARα1 (b), and GABA_ARα2 (c) expression levels. Effects on serotonin (d), 5-HT_1AR (e), and 5-HT_1BR (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 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 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 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), GABA_ARα1 (b), and GABA_ARα2 (c) expression levels. Effects on serotonin (d), 5-HT_1AR (e), and 5-HT_1BR (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 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 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 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-HT_1AR (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.