Fermented Sipjeondaebo-tang Alleviates Memory Deficits and Loss of Hippocampal Neurogenesis in Scopolamine-induced Amnesia in Mice

Abstract

We investigated the anti-amnesic effects of SJ and fermented SJ (FSJ) on scopolamine (SCO)-induced amnesia mouse model. Mice were orally co-treated with SJ or FSJ (125, 250, and 500 mg/kg) and SCO (1 mg/kg), which was injected intraperitoneally for 14 days. SCO decreased the step-through latency and prolonged latency time to find the hidden platform in the passive avoidance test and Morris water maze test, respectively, and both SCO effects were ameliorated by FSJ treatment. FSJ was discovered to promote hippocampal neurogenesis during SCO treatment by increasing proliferation and survival of BrdU-positive cells, immature/mature neurons. In the hippocampus of SCO, oxidative stress and the activity of acetylcholinesterase were elevated, whereas the levels of acetylcholine and choline acetyltransferase were diminished; however, all of these alterations were attenuated by FSJ-treatment. The alterations in brain-derived neurotrophic factor, phosphorylated cAMP response element-binding protein, and phosphorylated Akt that occurred following SCO treatment were protected by FSJ administration. Therefore, our findings are the first to suggest that FSJ may be a promising therapeutic drug for the treatment of amnesia and aging-related or neurodegenerative disease-related memory impairment. Furthermore, the molecular mechanism by which FSJ exerts its effects may involve modulation of the cholinergic system and BDNF/CREB/Akt pathway.

Figure 1. Schematic overview of the in vivo experimental procedure.

C57BL/6 mice (5-week-old) were administered CON, SCO (1 mg/kg), SJ, or FSJ (125, 250, or 500 mg/kg) for 14 days. To assess newborn cell proliferation (PRO), BrdU (50 mg/kg) injections were given on the final 3 days (day 12–14) of drug administration. For newborn cell survival assessments (SUR), BrdU was injected on the 3 days prior to drug administration. Mice were intracardially perfused on day 15 and brain tissue sections were processed for immunohistochemistry staining. Behavioral tests (passive avoidance test and Morris water maze test) were performed on days 16–23 and mice were sacrificed on day 24 for biochemical studies.

Figure 2. Effects of SJ and FSJ on memory retention and spatial memory in the SCO-induced mouse model of cognitive impairment.

(A) In the passive avoidance test, SCO (1 mg/kg) or the same volume of saline (CON group) was injected in mice 30 min prior to the test. The latency time to cross from the light to dark compartment was measured during training and on test days. Data are presented as the mean ± SEM (n = 6–8 mice/group). ^**p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group; ^##p < 0.01, compared with 500 mg/kg SJ-treated group. In (B–E), the MWM task was performed to determine spatial reference learning and memory in SCO-induced memory deficit mice that were treated with either SJ or FSJ. SCO (1 mg/kg) or the same volume of saline (CON group) was injected in mice 30 min prior to the MWM experiments. Changes in the swimming speed (in B) and total distance moved (in D) during the 6 test days are shown. (C) Representative swimming paths of mice in each treatment group at days 4–6 of the spatial reference trial test are shown. (E) The escape latency time to reach the hidden platform during the 6 days of testing is shown. Data are presented as the mean ± SEM (n = 6–8 mice/group). *p < 0.05, **p < 0.01, compared with CON-treated group; ^#p < 0.05, ^##p < 0.01, compared with SCO-treated group; ^&p < 0.05, ^&&p < 0.01, compared with 500 mg/kg SJ-treated group.

Figure 3. The SCO-induced loss of proliferation and survival of newly-generated BrdU-positive cells in the DG of the hippocampus was prevented by FSJ.

(A) Representative images indicating proliferation of BrdU-positive cells in the DG of each experimental group are shown. In the proliferation study, newly-generated cells existed in the SGZ of the DG. The scale bar = 100 μm. (B) The graph shows the number of BrdU-positive cells in the proliferation study. Data are presented as the mean ± SEM (n = 5 mice/group). **p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group. (C) Representative images indicating survival of BrdU-positive cells in the DG of each experimental group are shown. Surviving newborn cells migrated into the GCL of the DG. The scale bar = 100 μm. (D) The graph shows the number of BrdU-positive cells in the survival study. Data are presented as the mean ± SEM (n = 5 mice/group). **p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group.

Figure 4. The suppressed hippocampal neurogenesis induced by SCO treatment was restored by treatment with FSJ but not SJ.

(A) Representative images of DAB immunostaining for DCX in the hippocampal DG of each 14 day drug administration are shown. DCX-positive cells were highly expressed in the nucleus and neurites in the DG. The scale bar = 100 μm. (B) The graph shows the number and neurite length of DCX-positive cells in the DG. Data are presented as the mean ± SEM (n = 5 mice/group). **p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group. In C and E, representative confocal z-stack images of co-localization of BrdU (green) with DCX (in C, red) or NeuN (in E, red) in brain sections of the survival group are shown. Arrows indicate double positive cells (BrdU⁺/DCX⁺ or BrdU⁺/NeuN⁺). Arrowheads indicate only BrdU⁺ cells. The scale bar = 50 μm. In (D,F) quantitative graphs show the percentage of double-labeled positive cells (BrdU⁺/DCX⁺ (in D) or BrdU⁺/NeuN⁺ (in F)). Data are presented as the mean ± SEM (n = 5 mice/group). **p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group.

Figure 5. Effects of SJ and FSJ on oxidative stress, ACh levels and AChE activity in the hippocampus.

(A) DCFDA dye was used to determine the antioxidant properties of SJ and FSJ in the hippocampus. Data are presented as the mean ± SEM (n = 6–8 mice/group). **p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group. In (B,C) the ACh level and AChE activity in tissue homogenates of hippocampus from CON-treated, SCO-treated, SJ-treated, or FSJ-treated mice were determined using the Amplex red ACh/AChE assay kit. Data are presented as the mean ± SEM (n = 6–8 mice/group). **p < 0.01, compared with CON-treated group; ^#p < 0.05, ^##p < 0.01, compared with SCO-treated group. (D) The effects of SJ and FSJ on expression of ChAT in the hippocampus of mice administered SCO are shown. A representative immunoblot from one of three independent experiments shows the results for the antibodies against ChAT and β-actin. The lower graph shows the quantification of ChAT expressed as the ratio of ChAT/β-actin. Data are presented as the mean ± SEM (n = 6–8 mice/group). **p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group.

Figure 6. FSJ has anti-amnesic effects by preventing the SCO-induced reduction of hippocampal BDNF, CREB, and Akt.

(A) Levels of hippocampal BDNF from each mouse group were determined quantitatively using the ELISA assay. Data are presented as the mean ± SEM (n = 6–8 mice/group). **p < 0.01, compared with CON-treated group; ^#p < 0.05, compared with SCO-treated group. (B) The representative images show DAB immunostaining for pCREB in the hippocampal DG for each 14 day drug treatment. The scale bar = 100 μm. The graph shows the number of pCREB-positive cells in the DG. Data are presented as the mean ± SEM (n = 5 mice/group). **p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group. (C) The effects of SJ or FSJ on pCREB expression in the hippocampus of mice administered SCO are shown. A representative immunoblot from one of three independent experiments shows the results for the antibodies against pCREB, CREB, and β-actin. The lower graph shows the quantification of pCREB expressed as the ratio of pCREB/CREB. Data are presented as the mean ± SEM (n = 6–8 mice/group). **p < 0.01, compared with CON-treated group; ^#p < 0.05, ^##p < 0.01, compared with SCO-treated group. (D) The effects of SJ and FSJ on pAkt expression in the hippocampus in mice treated with SCO are shown. A representative immunoblot from one of three independent experiments shows the results for the antibodies against pAkt, Akt, and β-actin. The lower graph shows the quantification of pAkt expressed as the ratio of pAkt/Akt. Data are presented as the mean ± SEM (n = 6–8 mice/group). **p < 0.01, compared with CON-treated group; ^##p < 0.01, compared with SCO-treated group.

Figure 7. Effects of SJ and FSJ on hippocampal neurogenesis and cognitive function under normal conditions.

(A) The cross-over latency time is shown for the passive avoidance test. (B) The latency time to find the hidden platform is shown for the spatial reference learning and memory test that was performed for 6 days. The values shown are means ± SEM (n = 6). (C) Representative images are shown that indicate proliferation or survival of newly-generated cells in the DG of the hippocampus for CON-treated, 500 mg/kg SJ-treated, or FSJ-treated mice. The scale bar = 100 μm. The graph shows the number of BrdU-positive cells in the proliferation and the survival group. Data are presented as the mean ± SEM (n = 5 mice/group). (D) The level of BDNF, level of ACh, and AChE activity in hippocampus tissue homogenates from CON-treated, 500 mg/kg SJ-treated, or FSJ-treated mice were determined. Data are presented as the mean ± SEM (n = 6–8 mice/group).