Sublingual AKBA Exerts Antidepressant Effects in the Aβ-Treated Mouse Model

Abstract

The 3-O-acetyl-11-keto-β-boswellic acid (AKBA) is the most active compound of Boswellia serrata proposed for treating neurodegenerative disorders, including Alzheimer's disease (AD), characterized in its early phase by alteration in mood. Accordingly, we have previously demonstrated that an intracerebroventricular injection of soluble amyloid beta _1-42 (Aβ) peptide evokes a depressive-like phenotype in rats. We tested the protective effects of AKBA in the mouse model of an Aβ-induced depressive-like phenotype. We evaluated the depressive-like behavior by using the tail suspension test (TST) and the splash test (ST). Behavioral analyses were accompanied by neurochemical quantifications, such as glutamate (GLU), kynurenine (KYN) and monoamines, and by biochemical measurements, such as glial fibrillary acid protein (GFAP), CD11b and nuclear factor kappa B (NF-kB), in mice prefrontal cortex (PFC) and hippocampus (HIPP). AKBA prevented the depressive-like behaviors induced by Aβ administration, since we recorded a reduction in latency to initiate self-care and total time spent to perform self-care in the ST and reduced time of immobility in the TST. Likewise, the increase in GLU and KYN levels in PFC and HIPP induced by the peptide injection were reverted by AKBA administration, as well as the displayed increase in levels of GFAP and NF-kB in both PFC and HIPP, but not in CD11b. Therefore, AKBA might represent a food supplement suitable as an adjuvant for therapy of depression in early-stage AD.

Keywords: 3-O-acetyl-11-keto-β-boswellic acid; beta amyloid; depressive-like behavior; glutamate; kynurenine; sublingual route of administration.

Figure 1

Mass spectrum (full scan mode) of 3-O-acetyl-11-keto-β-boswellic acid (AKBA).

Figure 2

Cerebral AKBA quantification after sublingual or intranasal administration. (A) Cerebral AKBA levels (mg AKBA/mg of brain) at 5, 15, and 30 min after sublingual (black square, n = 4, 15 n = 4, 30 n = 5), or intranasal (reverse empty triangle, 5 n = 5, 15 n = 5, 30 n = 5) administration. (B) Cerebral AUCs of sublingual (black bar, n = 5), and intranasal (empty bar, n = 5) administration. (C) Plasmatic AKBA levels (mg AKBA/mg of brain) at 5, 15, and 30 min after sublingual (black square, n = 4, 15 n = 4, 30 n = 5), or intranasal (reverse empty triangle, 5 n = 5, 15 n = 5, 30 n = 5) administration. (D) Plasmatic AUCs of sublingual (black bar, n = 5), and intranasal (empty bar, n = 5) administration. Two-way ANOVA followed by Bonferroni multiple comparisons test * p < 0.05 sublingual vs. intranasal at 5 min; *** p < 0.001 sublingual vs. intranasal. One-way ANOVA followed by Tukey’s multiple comparisons test, # p < 0.05 5 vs. 30 min for sublingual.

Figure 3

Behavioral effects of AKBA administration in Aβ-treated mice by using the splash test and the tail suspension test. (A) Latency to leak (sec) in the splash test of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (SHAM n = 9, Aβ n = 9, Aβ+AKBA n = 10). (B) Time spent performing self-care (sec) in the splash test of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (SHAM n = 9, Aβ n = 9, Aβ+AKBA n = 10). (C) Immobility time (sec) in the tail suspension test of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (SHAM n = 12, Aβ n = 9, Aβ+AKBA n = 6). One-way ANOVA followed by Tukey’s multiple comparisons test, ** p < 0.01, * p < 0.05 Aβ versus SHAM, and ## p < 0.01, # p < 0.05 Aβ+AKBA versus Aβ.

Figure 4

Effects of AKBA administration on NA and GLU content in PFC and HIPP of Aβ-treated mice. (A) NA (fmol/mg of tissue, SHAM n = 8, Aβ n = 6, Aβ+AKBA n = 6) and (B) GLU (uM/mg of tissue, SHAM n = 5, Aβ n = 5, Aβ+AKBA n = 6) levels in the PFC of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar). (C) NA ( fmol/mg of tissue, SHAM n = 6, Aβ n = 6, Aβ+AKBA n = 6) and (D) GLU (uM/mg of tissue, SHAM n = 6, Aβ n = 6, Aβ+AKBA n = 5) levels in the HIPP of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar). One-way ANOVA followed by Tukey’s multiple comparison test, * p < 0.05 Aβ versus SHAM; * p < 0.05, *** p < 0.001 Aβ+AKBA versus SHAM; # p < 0.05, ## p < 0.01 Aβ+AKBA versus Aβ.

Figure 5

Effects of AKBA administration on KYN and 5-HT content in PFC and HIPP of Aβ-treated mice. (A) KYN (fmol/mg of tissue, SHAM n = 4, Aβ n = 5, Aβ+AKBA n = 5) and (B) 5-HT (fmol/mg of tissue, SHAM n = 7, Aβ n = 4, Aβ+AKBA n = 6) levels in the PFC of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar). (C) KYN (fmol/mg of tissue, SHAM n = 6, Aβ n = 6, Aβ+AKBA n = 5) and (D) 5-HT (fmol/mg of tissue, SHAM n = 6, Aβ n = 6, Aβ+AKBA n = 6) levels in the HIPP of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar). One-way ANOVA followed by Tukey’s multiple comparison test, * p < 0.0, ** p < 0.01 Aβ versus SHAM; *** p < 0.001 Aβ+AKBA versus SHAM; ## p < 0.01 Aβ+AKBA versus Aβ.

Figure 6

Effects of AKBA administration on cortical GFAP, CD11b and NFkB-p65 levels. Representative image of Western blotting of (A) GFAP, (B) CD11b and (C) NFkB-p65. (D) Quantification of the optical density of GFAP bands normalized to the actin protein value in the PFC of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (SHAM n = 5, Aβ n = 4, Aβ+AKBA n = 4). (E) Quantification of the optical density of CD11b bands normalized to the actin protein value in the PFC of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (SHAM n = 4, Aβ n = 4, Aβ+AKBA n = 4). Quantification of the optical density of NFkB-p65 bands normalized to the actin protein value in the PFC of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (F) SHAM n = 4, Aβ n = 4, Aβ+AKBA n = 4). One-way ANOVA followed by Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001 Aβ versus SHAM; # p < 0.05, ### p < 0.001 Aβ+AKBA versus Aβ.

Figure 7

Effects of AKBA administration on hippocampal GFAP, CD11b and NFkB-p65 levels. Representative image of Western blotting of (A) GFAP, (B) CD11b and (C) NFkB-p65. (D) Quantification of the optical density of GFAP bands normalized to the actin protein value in the HIPP of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (SHAM n = 5, Aβ n = 3, Aβ+AKBA n = 5). (E) Quantification of the optical density of CD11b bands normalized to the actin protein value in the PFC of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (SHAM n = 4, Aβ n = 5, Aβ+AKBA n = 4). (E) Quantification of the optical density of NFkB-p65 bands normalized to the actin protein value in the PFC of mice 7 days after icv injection of vehicle (SHAM, 5 µL, white bar), Aβ (Aβ, 4 µM, black bar), and Aβ+AKBA (Aβ, 4 µM + sublingual AKBA, 5 mg/kg, gray bar), (F) SHAM n = 3, Aβ n = 4, Aβ+AKBA n = 4). One-way ANOVA followed by Tukey’s multiple comparison test, ** p < 0.01 Aβ versus SHAM; * p < 0.05 Aβ+AKBA versus SHAM; # p < 0.05 Aβ+AKBA versus Aβ.