Role of oligomeric proanthocyanidins derived from an extract of persimmon fruits in the oxidative stress-related aging process

Abstract

Many researchers have focused on the oligomeric form of proanthocyanidins with a lower level of polymerization found in foodstuffs such as grape seeds and blackberries. The present study indicated that the oral administration of oligomers isolated from persimmon fruits extended the lifespan of senescence-accelerated mouse prone/8 (SAMP8), a murine model of accelerated senescence. On the other hand, oligomer-treated SAMP8 did not show stereotypical behavior. We also revealed that the oral administration of oligomers improved spatial and object recognition memory in SAMP8. The density of axons in the hippocampal CA1 was significantly increased by oligomer administration. Moreover, the administration of oligomers increased the phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in the hippocampal CA3, hypothalamus, and choroid plexus. We speculate that memory improvement accompanied by histological changes may be induced directly in the hippocampus and indirectly in the hypothalamus and choroid plexus through VEGFR-2 signaling. In the present study, we elucidated the protective effect of oligomers against memory impairment with aging. VEGFR-2 signaling may provide a new insight into ways to protect against memory deficit in the aging brain.

Figure 1

Fractionation of persimmon oligomeric proanthocyanidins.

Figure 2

Effects of oligomers on lifespan of SAM. Forty-five or forty-six-week-old SAMP8 mice were administered vehicle (Veh, water p.o., n = 10), while another two groups were administered oligomers orally at doses of 1 or 10 mg/kg body weight/day (n = 10) using a stomach tube until death. For the remaining group of mice, the mean food intake was restricted to 60% until death (n = 10). SAMR1 mice (45–46 weeks old, n = 10) were used as a control group. (A) Effects of oligomers on survival of SAMP8. (B) Lifespan index based on survival data. Open circle: SAMR1; closed circle: SAMP8 (Veh); open triangle: SAMP8 (oligomers at 1 mg/kg B.W./day); closed triangle: SAMP8 (oligomers at 10 mg/kg B.W./day); gray square: SAMP8 (60% food restriction).

Figure 3

Effects of oligomers on Sirt1 expression in the brain of SAM. Forty-five-week-old SAMP8 were administered vehicle (Veh, water p.o., n = 5), oligomers (O (50), 50 mg/kg B.W./day, p.o., n = 5), or resveratrol (Res (90), 90 mmol/kg B.W./ day, p.o., n = 5). After 5 weeks of administration, brain lysates were immunoblotted with antibodies for Sirt1. Sirt 1 expression intensities were divided by β-actin expression. SAMR1 mice were used as a control (Cont, n = 4).

Figure 4

Effects of oligomers on stereotypical behaviors. Forty-five or forty-six-week-old SAMP8 mice were administered vehicle (Veh, water p.o., n = 4), while another two groups were administered oligomers orally at doses of 10 mg/kg body weight/day (O (10), n = 4) using a stomach tube until death. For the remaining group of mice, the mean food intake was restricted to 60% until death (FR, n = 4). SAMR1 mice (Cont, 45–46 weeks old, n = 4) were used as a control group. One hundred and thirty-nine days after the start of administration, actions of rearing up on the hindlimbs and jumping from the bottom of the cage were counted for 15 min (A, B). The time spent hanging from the lid was measured for 10 min (C). Administration was continued during the tests. ^a p = 0.0034 (Student’s t-test); * p < 0.05 (One-way ANOVA, post-hoc Bonferroni’s test).

Figure 5

Effects of oligomers on motor function. Forty-five or six-week-old SAMP8 mice were administered vehicle (Veh, water p.o., n = 4), while another two groups were administered oligomers orally at doses of 10 mg/kg body weight/day (O (10), n = 4) using a stomach tube until death. For the remaining group of mice, the mean food intake was restricted to 60% until death (FR, n = 4). SAMR1 mice (Cont, 45-46 weeks old, n = 4) were used as a control group. One hundred and thirty-nine days after the start of administration, the time spent on the inclined surface without dropping was measured (A). The number of rotations measured for 30 min (B). Administration was continued during the tests. ^a p = 0.0159 (A: Student’s t-test).

Figure 6

Effects of oligomers on spatial memory deficit in SAMP8. Eighteen-week-old SAMP8 were administered vehicle (Veh, water p.o., n = 6; closed circles) or oligomers (10 mg/kg body weight/day, p.o., n = 6; open squares or 100 mg/kg body weight/day, p.o., n = 5; closed squares) for 5 weeks. SAMR1 were used as a control (Cont, n = 5; open circles). Fifteen days after the start of administration, memory acquisition tests were continued for 6 days in a Morris water maze. Administration was continued during the tests. Escape latencies to a hidden platform were measured (A). The swimming velocities of mice in the memory acquisition test are shown (B). Thirty-eight-week-old SAMP8 were administered vehicle (Veh, water p.o., n = 7) or oligomers (O (10), 10 mg/kg body weight/day, p.o., n = 7 or O (50), 50 mg/kg body weight/day, p.o., n = 7). Age-matched SAMR1 were used as a control (Cont, n = 7). Twenty-eight days after administration started, an object location test was performed. The preference index was defined as the number of times a mouse made contact with any one of the objects (training session) or the moved object (test session) out of the total number of times the mouse made contact with both objects (%) (C). * p < 0.05 vs. Veh. (A and B: Repeated measures two-way ANOVA followed by Dunnett’s or Bonferroni’s post-hoc test); * p < 0.05 (C: One-way ANOVA followed by Bonferroni’s post-hoc test); ^a p = 0.0005; ^b p = 0.0213 (C: paired t-test).

Figure 7

Effects of oligomers on object recognition memory deficit in SAMP8. Eighteen-week-old SAMP8 were administered vehicle (Veh, water p.o., n = 6) or oligomers (O (10), 10 mg/kg body weight/day, p.o., n = 6 or O (100), 100 mg/kg body weight/day, p.o., n = 5). Age-matched SAMR1 were used as a control (Cont, n = 5). Twenty-four days after the start of administration, a novel object recognition test was performed (A). Thirty-eight-week-old SAMP8 were administered vehicle (Veh, water p.o., n = 7) or oligomers (O (10), 10 mg/kg body weight/day, p.o., n = 7 or O (50), 50 mg/kg body weight/day, p.o., n = 7). Age-matched SAMR1 were used as a control (Cont, n = 7). Twenty-three days after the start of administration, a novel object recognition test was performed (B). The preference index was defined as the number of times a mouse made contact with any one of the objects (training session) or the novel object (test session) out of the total number of times the mouse made contact with both objects (%). * p < 0.05 (One-way ANOVA followed by Bonferroni’s post-hoc test); ^a p = 0.0174; ^b p = 0.0014 (paired t-test).

Figure 8

Effects of oligomers on the decrease of axons, dendrites, and synapses in the hippocampus. Fifty-nine-week-old SAMP8 were administered vehicle (Veh, water p.o., n = 3) or oligomers (O (50), 50 mg/kg body weight/day, p.o., n = 3). After seven days of administration, brain slices were immunostained with p-NF-H (A), MAP2 (B), and synaptophysin antibodies (C). The intensities of immuno-positive areas in the hippocampus were quantified. ^a p = 0.0243; ^b p = 0.0344 (Student’s t-test).

Figure 9

Effects of oligomers on phosphorylated VEGFR-2 (p-VEGFR-2) and VEGFR-2 expressions. Fifty-nine-week-old SAMP8 were administered vehicle (Veh, water p.o.) or oligomers (O (50), 50 mg/kg body weight/day, p.o.). After seven days of administration, brain lysates were immunoblotted with antibodies for p-VEGFR-2 (A) or VEGFR-2 (B). Expression intensities were divided by β-actin expressions to calculate ratios. ^a p = 0.0481 vs. O (50) (Student’s t-test).

Figure 10

Effects of oligomers on p-VEGFR-2 and VEGFR-2 expression in various brain regions. Fifty-nine-week-old SAMP8 were administered vehicle (Veh, water p.o., n = 3) or oligomers (O (50), 50 mg/kg B.W./day, p.o., n = 3). After seven days of administration, brain slices were immunostained with p-VEGFR-2 (A) and VEGFR-2 (B) antibodies.

Figure 11

Intensities of p-VEGFR-2- (A) and VEGFR-2 (B)-positive areas were quantified in the cerebral cortex, hypothalamus and choroid plexus, and the CA1, CA3, and DG of the hippocampus. ^a p = 0.0429 vs. O (50) (Student’s t-test).

Figure 12

Effects of oligomers and VEGFR-2 on memory. Male ddY mice (6 weeks old) were administered oligomers (O (50), 50 mg/kg body weight/day, p.o., n = 4) for 7 days. Then, the vehicle (Veh, 5% DMSO in 0.9% NaCl) was injected intracerebroventricularly at 60 min after the final administration of oligomers. Five days after vehicle injection, SU1498(5 nmol/μL, solution is 5% DMSO in 0.9% NaCl) was injected intracerebroventricularly at 60 min after the final administration of oligomers. ^a p = 0.0392 (Student’s t-test).