Leucine-Histidine Dipeptide Attenuates Microglial Activation and Emotional Disturbances Induced by Brain Inflammation and Repeated Social Defeat Stress

Abstract

The number of patients with mental illnesses is rapidly increasing, and daily lifestyle is closely associated with the development of symptoms. It is suggested that inflammatory molecules derived from microglia play crucial roles for the pathophysiology of depression. In the present study, we discovered that leucine-histidine (LH) dipeptide suppressed activation of primary microglia. The effects of LH dipeptide orally administered were measured using tail suspension test (TST) in mice injected with lipopolysaccharide and social interaction test in mice received social defeat stress. LH dipeptide reduced pro-inflammatory cytokines upon stimulation in microglia. Orally administered LH dipeptide was delivered to the brain and suppressed the production of pro-inflammatory cytokines in the brain and concomitant depression-like behavior in the TST. Moreover, oral administration of LH dipeptide suppressed the induction of depression- and anxiety-like behaviors induced by repeated social defeat stress. These results indicate that LH dipeptide suppressed the activation of microglia and ameliorated depression-associated emotional disturbances. Further, we found that LH dipeptide was abundant in various fermented products. Together with previous epidemiological reports that daily intake of these fermented foods is negatively associated with the incidence of psychiatric diseases, our findings suggest that food rich in LH dipeptide may improve mental health.

Keywords: antidepressant; cytokine; dipeptide; inflammation; microglia.

Figure 1

Effects of leucine–histidine (LH) dipeptide on the activation of primary microglia. The effects of in vitro LH dipeptide treatment on inhibiting microglial activation. (A), Amount of TNF-α in the supernatant of microglia treated with 0, 5, 10, or 50 μM LH dipeptide followed by treatment with 5 ng/mL lipopolysaccharide (LPS) and 0.5 ng/mL interferon-γ (IFN-γ). (B), Amount of TNF-α in the supernatant of microglia treated with 0, 10, or 50 μM LH dipeptide, leucine (Leu), or histidine (His) and followed by treatment with 5 ng/mL LPS and 0.5 ng/mL IFN-γ. (C,D), The expression of CD86 and percentages of CD206-positive/CD11b-positive microglia treated with 0, 10, or 50 μM LH dipeptide followed by treatment with 5 ng/mL LPS and 0.5 ng/mL IFN-γ, respectively. M.F.I. means mean fluorescent intensity. Columns and bars represent the mean and SE values of three independent wells per sample, respectively. The p values shown were calculated using the Student t test (LPS [−] versus [+], 0 μM LH dipeptide) and one-way ANOVA followed by Dunnett’s test. * p < 0.05 and ** p < 0.01.

Figure 2

Effects of LH dipeptide on neuronal inflammation and depression-like behavior induced by LPS. (A), ICR mice were orally administered 0 or 50 mg/kg of LH dipeptide for seven days and then intracerebroventricularly injected with PBS or 0.5 mg/kg LPS 1 h after the last administration. The mice were subjected to the tail suspension test (TST) and open field test 24 h after the administration of LPS. B-E, Amounts of TNF-α in frontal cortex (B) and hippocampus (D) and IL-1β in the frontal cortex (C) and hippocampus (E) 24 h after LPS injection, respectively. (F), Immobility timed in the TST. (G), Total distances (cm) in the open field test. H, Immobility times in the TST of sham-operated mice. Data represent the mean ± SE of 10 mice per group. The p values were calculated using one-way ANOVA followed by the Tukey–Kramer test. *p < 0.05 and **p < 0.01.

Figure 3

Effects of intragastrically administered LH dipeptide on repeated social defeat stress (R-SDS). C56BL/6N mice were orally administered 50 mg/kg LH dipeptide and subjected to R-SDS daily for 10 consecutive days. (A), On days 0, 2, and 4, mice were subjected to social interaction with ICR mice in chamber. (B), Times in the interaction zone on days 2 of R-SDS. (C), Times in the avoidance zone on days 2 of R-SDS. Data represent the mean ± SE of 8–10 mice per group. The p values were calculated by one-way ANOVA followed by the Tukey–Kramer test. * p < 0.05 and ** p < 0.01.

Figure 4

Effects of dietary LH dipeptide on R-SDS. C56BL/6N mice were supplied with a diet containing 0.15% LH dipeptide and subjected to R-SDS daily for 10 consecutive days. (A), On days 0, 2, and 6, mice were subjected to social interaction with ICR mice in the chamber. (B), Times in the interaction zone on days 6. (C), Times in the avoidance zone on days 6. After 11 days, mice were subjected to the elevated plus maze for 5 min, and times spent in the open arms were measured. C, The percentage of time in open arms. Data represent the mean ± SE of four to eight mice per group. The p values were calculated using the Student t test to evaluate the significance of the difference between defeat (−) and (+) with LH (−) or without LH (+). * p < 0.05.