Role of Keap1-Nrf2 signaling in depression and dietary intake of glucoraphanin confers stress resilience in mice

Abstract

The transcription factor Keap1-Nrf2 system plays a key role in inflammation which is involved in depression. We found lower expression of Keap1 and Nrf2 proteins in the prefrontal cortex (PFC), CA3 and dentate gyrus (DG) of hippocampus in mice with depression-like phenotype compared to control mice. Serum levels of pro-inflammatory cytokines in Nrf2 knock-out (KO) mice were higher than those of wild-type mice, suggestive of enhanced inflammation in KO mice. Decreased brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-receptor-kinase B (TrkB) signaling in the PFC, CA3 and DG plays a role in the depression-like phenotype of Nrf2 KO mice. TrkB agonist 7,8-dihydroxyflavone, but not antagonist ANA-12, produced antidepressant effects in Nrf2 KO mice, by stimulating TrkB in the PFC, CA3 and DG. Pretreatment with Nrf2 activator sulforaphane (SFN) prevented the depression-like phenotype induced after repeated social defeat stress. Interestingly, dietary intake of 0.1% glucoraphanin (a precursor of SFN) containing food during juvenile and adolescent stages also prevented the depression-like phenotype evoked in adulthood, after repeated social defeat stress. These findings suggest that Keap1-Nrf2 system plays a key role in depression and that dietary intake of SFN-rich food during juvenile stages and adolescence can confer stress resilience in adulthood.

Figure 1. Potentiation of neurite outgrowth by SFN.

(a) Representative photographs of microtubule-associated protein 2 (MAP-2) immunocytochemistry in PC12 cells. Control: NGF (2.5 ng ml⁻¹) alone, SFN: NGF (2.5 ng ml⁻¹) + SFN (1.0 μM). (b) Effects of SFN on NGF-induced neurite outgrowth in PC12 cells. SFN (0.01, 0.1 or 1.0 μM) potentiated NGF-induced neurite outgrowth in PC12 cells, in a concentration-dependent manner. All data represent the mean ± S.E.M. (n = 6–12) *P < 0.05, ***P < 0.001 as compared with the control group (one-way ANOVA). (c) The potentiating effects of SFN (1.0 μM) on NGF-induced neurite outgrowth were significantly antagonized by treatment with Nrf2 siRNA, but not negative siRNA. Neither Nrf2 siRNA nor negative siRNA alone altered NGF (2.5 ng ml⁻¹)-induced neurite outgrowth. All data represent the mean ± S.E.M. (n = 8). ***P < 0.001 as compared with the SFN (1.0 μM) group (one-way ANOVA). (d) The potentiating effects of SFN (1.0 μM) on Nrf2 protein levels were significantly antagonized by treatment with Nrf2 siRNA, but not the negative siRNA. In contrast, neither Nrf2 siRNA nor negative siRNA alone altered levels of Nrf2 protein in the control (NGF (2.5 ng ml⁻¹)-treated) group. All data represent the mean ± S.E.M. (n = 4). *P < 0.05, **P < 0.01 as compared with the SFN (1.0 μM) group (one-way ANOVA).

Figure 2. Protein levels of Keap1 and Nrf2 in the brain from mice with depression-like phenotype.

(a,b) The levels of Keap1 and Nrf2 proteins in the mouse brain regions from social defeat stress (susceptible) mice and control mice (n = 5 or 6). Data represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 compared with the control group (Student t-test).

Figure 3. Depression-like phenotypes of Nrf2 KO mice and inflammation in Nrf2 KO mice.

(a) The behavior tests in WT and Nrf2 KO mice. Locomotion (LMT); tail-suspension test (TST); forced swimming test (FST), 1% sucrose preference test (SPT) and total fluid intake test (FIT) for SPT. Data represent the mean ± S.E.M (n = 9 or 10). *P < 0.05, **P < 0.01 compared with the WT group (Student t-test). (b,c) Western blot of BDNF, p-TrkB/TrkB, GluA-1 and PSD-95 in WT and Nrf2 KO mice. Data represent the mean ± S.E.M (n= 6 or 7). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the WT group (Student t-test). (d) Serum levels of TNF-α, IL-6, IL-10 and IL-1β in WT and Nrf2 KO mice. Data represent the mean ± S.E.M (n = 9–12). *P < 0.05, **P < 0.01 compared with the WT group (Student t-test).

Figure 4. Antidepressant effect of 7,8-DHF, a TrkB agonist, in Nrf2 KO mice.

(a) 7,8-DHF (10 mg/kg) or vehicle (10 ml/kg) was administered i.p. into WT and Nrf2 KO mice. Behavioral tests were performed 1 hour after injection. Locomotion (LMT); tail-suspension test (TST); forced swimming test (FST), 1% sucrose preference test (SPT) and total fluid intake test (FIT) for SPT. Data represent the mean ± S.E.M. (n = 9–11). *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle-treated KO group (two-way ANOVA). (b) Levels of p-TrkB/TrkB ratio in the mouse brain regions after injection of 7,8-DHF. Data represent the mean ± S.E.M (n = 5 or 6). **P < 0.01, ***P < 0.001 compared with vehicle-treated KO group (two-way ANOVA). (c) ANA-12 did not show antidepressant effect in the Nrf2 KO mice. ANA-12 (0.5 mg/kg) or vehicle (10 ml/kg) was administered i.p. into WT and Nrf2 KO mice. Behavioral tests were performed 1 hour after injection. Locomotion (LMT); tail-suspension test (TST); forced swimming test (FST); 1% sucrose preference test (SPT) and total fluid intake test (FIT) for SPT. Data represent the mean ± S.E.M. (n = 8–11). *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle-treated KO group (two-way ANOVA). (d) SFN did not show antidepressant effect in the Nrf2 KO mice. SFN (10 mg/kg) or vehicle (10 ml/kg) was administered i.p. into WT and Nrf2 KO mice. Behavioral tests were performed 1 hour after injection. Locomotion (LMT); tail-suspension test (TST); forced swimming test (FST), 1% sucrose preference test (SPT) and total fluid intake test (FIT) for SPT. Data represent the mean ± S.E.M. (n = 10–11). **P < 0.01, ***P < 0.001 compared with vehicle-treated KO group (two-way ANOVA).

Figure 5. Pretreatment with SFN prevents depression after repeated social defeat stress.

(a) The schedule of treatment and behavioral evaluations. (b) Social interaction test (SIT): no target, (c) SIT: target. (d) 1% sucrose preference test (SPT), (e) total fluid intake test (FIT) for SPT. Data represent the mean ± S.E.M. (n = 10–14). *P < 0.05, ***P < 0.001 compared with the vehicle-treated stress group (two-way ANOVA). (f,g) Western blot of Keap1, Nrf2 in the mouse brain regions. Data represent the mean ± S.E.M. (n = 5 or 6). *P < 0.01, **P < 0.01, ***P < 0.001 compared with the vehicle-treated stressed group (two-way ANOVA). (h,i) Western blot of BDNF, p-TrkB/TrkB in the mouse brain regions. Data represent the mean ± S.E.M. (n = 5 or 6). *P < 0.01, **P < 0.01, ***P < 0.001 compared with the vehicle-treated stress group (two-way ANOVA).

Figure 6. Dietary intake of 0.1% GP during the juvenile and adolescence prevents depression after repeated social defeat stress.

(a) The schedule of dietary intake of 0.1% glucoraphanin (GP) and behavioral evaluations. (b) Social interaction test (SIT): no target, (c) SIT: target, (d) 1% sucrose preference test (SPT), (e) total fluid intake test (FIT) for SPT. Data represent the mean ± S.E.M. (n = 16 or 17). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the vehicle-treated stressed group (two-way ANOVA).