Multifunctional Effects of Mangosteen Pericarp on Cognition in C57BL/6J and Triple Transgenic Alzheimer's Mice

Abstract

Mangosteen- (Garcinia mangostana-) based nutraceutical compounds have long been reported to possess multiple health-promoting properties. The current study investigated whether mangosteen pericarp (MP) could attenuate cognitive dysfunction. First, we found that treatment with MP significantly reduced the cell death and increased the brain-derived neurotrophic factor (BDNF) level in an organotypic hippocampal slice culture (OHSC). We then investigated the effects of age and MP diet on the cognitive function of male C57BL/6J (B6) mice. After 8-month dietary supplementation, the MP diet (5000 ppm) significantly attenuated the cognitive impairment associated with anti-inflammation, increasing BDNF level and decreasing p-tau (phospho-tau S202) in older B6 mice. We further applied MP dietary supplementation to triple transgenic Alzheimer's disease (3×Tg-AD) mice from 5 to 13 months old. The MP diet exerted neuroprotective, antioxidative, and anti-inflammatory effects and reduced the Aβ deposition and p-tau (S202/S262) levels in the hippocampus of 3×Tg-AD mice, which might further attenuate the deficit in spatial memory retrieval. Thus, these results revealed that the multifunctional properties of MP might offer a promising supplementary diet to attenuate cognitive dysfunction in AD.

Figure 1

MP induced neuroprotection through increasing BDNF in adult mouse hippocampal slices. (a) The cell death level of hippocampal slices was characterized with PI staining after treatment with MP (10 μM) or DMSO. The scale bar of PI staining is 500 μm. (b) The quantification of PI staining. Treatment with MP significantly decreased the cell death level. (c) The results of BDNF ELISA in the hippocampal slices. Treatment with MP increased the level of BDNF in the hippocampal slices. Data are expressed as means ± SEM, n = 9–12 slices/group. ^** P < 0.01, compared with the DMSO group.

Figure 2

MP diet attenuated the impairments in spatial learning and memory in older B6 mice. (a) The spatial learning ability of B6 mice at different ages and with dietary supplementation. The older B6 mice showed a poor spatial learning ability as compared with the younger mice, and MP dietary supplementation had no influence on the improvement of spatial learning ability. (b) The spatial learning acquisition of B6 mice at different ages and with dietary supplementation. The MP diet increased the spatial learning acquisition in the older B6 mice. (c) The short-term memory retrieval in mice measured 2 h after the last testing trial. The older B6 mice showed impairment in short-term memory retrieval, and MP dietary supplementation attenuated the impairment in the older B6 mice. (d) The long-term memory retrieval in mice measured 48 h after the last testing trial. The older B6 mice showed impairment in long-term memory retrieval, and MP dietary supplementation showed no influence on the impairment. (e) The swimming velocity of the B6 mice at different ages. The decreased swimming velocity of the older B6 mice was rescued by the MP diet. Data are expressed as mean ± SEM, n = 15/group. ^# P < 0.05 and ^### P < 0.001, comparison between the older and younger groups. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001, comparison between the regular and MP diet groups.

Figure 3

Molecular effects of the MP diet in B6 mice. Representative immunostaining images of BDNF (a), p-tau (S202) (b), activated astrocytes (c), and microglia (d) in the B6 mouse hippocampus. (e) The systemic IL-6 level was determined by ELISA with mouse serum. The IL-6 level was greatly increased in the older B6 mice, which was significantly decreased by the MP diet. All of the deregulations in the older mice were ameliorated after MP treatment. n = 3–5/group. Arrowheads indicate positive staining signals. ^## P < 0.01, comparison between the older and younger groups. ^* P < 0.05, comparison between the regular and MP diet groups.

Figure 4

MP diet attenuated the impairment in spatial memory retrieval in 3×Tg-AD mice. (a) The spatial learning ability was measured in the 3×Tg-AD mice from training days 1~4. The 3×Tg-AD mice showed a poor spatial learning ability at 13 months of age, but the MP diet attenuated the impairment in spatial learning ability. (b) The spatial learning acquisition was measured in the 3×Tg-AD mice. The poor spatial learning acquisition of 13-month-old 3×Tg-AD mice could not be rescued by the MP diet. (c) The retrieval of short-term memory in the 3×Tg-AD mice. The reduced time spent in the target quadrant of the 13-month-old 3×Tg-AD mice was significantly increased by the MP diet. (d) The retrieval of long-term memory in the 3×Tg-AD mice. The MP diet significantly increased the time spent in the target quadrant of the 13-month-old 3×Tg-AD mice. (e) The swimming velocity of the 3×Tg-AD mice. The MP diet had no influence on the reduced velocity of the 13-month-old 3×Tg-AD mice. Data are expressed as means ± SEM. n = 16-17/group. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001, compared with the 13-month-old mice treated with a regular diet.

Figure 5

Neuroprotective effects of the MP diet in 3×Tg-AD mice. Immunostaining images of neurons in the hippocampus (a), calbindin levels in the hippocampus (b), BDNF in the hippocampus (c), ChAT in the MS/DB region (d), TH in the LC region (e), and 5-HT in the raphe nucleus (f). Scale bars are 100 μm in panel (a), 50 μm in panels (b) and (c), and 500 μm in panels (d)~(f). Arrowheads indicate positive staining signals. n = 3–5/group.

Figure 6

Effects of the MP diet on amyloid deposition, p-tau (S202/S262), and NR2A/NR2B in 3×Tg-AD mice. Immunostaining images of APP (a), Aβ ₄₂ (b), and Aβ ₄₂ (c) in the hippocampus of the mice. The levels of BACE1 (d) and NR2A/NR2B ratio (e) in the hippocampus identified by western blot. The MP diet significantly decreased the level of BACE1 and increased the level of the NR2A/NR2B ratio. Immunostaining images of p-tau (S202) (f) in the hippocampus of the mice. (g) The level of p-tau (S262) measured in the hippocampus by western blot. The MP diet greatly decreased the level of p-tau (S262). Scale bar = 100 μm and arrowheads indicate positive staining signals. Data are expressed as means ± SEM. n = 3–5/group. ^* P < 0.05, comparison between the regular and MP diet groups.

Figure 7

The antioxidative and anti-inflammatory activities of MP in 3×Tg-AD mice. (a) The systemic GSH level was determined by ELISA with mouse serum. MP diet significantly increased the GSH level in the mouse serum. (b) The systemic IL-6 level was determined by ELISA with mouse serum. MP diet significantly decreased the IL-6 level in the mouse serum. (c) The level of pp38 was measured in the hippocampus of the mice by western blot. MP diet significantly decreased the level of pp38. (d) The level of COX2 was measured in the hippocampus of the mice by western blot. MP diet significantly decreased the level of COX2. Immunostaining images of activated astrocytes (e) and microglia (f) in the hippocampus. Scale bar = 100 μm and arrowheads indicate positive staining signals. Data are expressed as means ± SEM. n = 3–5/group. ^* P < 0.05 and ^** P < 0.01, comparison between the regular and MP diet groups.