Mussel oligopeptides ameliorate cognition deficit and attenuate brain senescence in d -galactose-induced aging mice

Food and Chemical Toxicology 59 (2013) 412–420

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Mussel oligopeptides ameliorate cognition deficit and attenuate brain senescence in D-galactose-induced aging mice Yue Zhou a, Ying Dong a,⇑, Qinggang Xu b, Yuanqing He a, Shilei Tian a, Shuyun Zhu a, Ying Zhu a, Xiuping Dong c a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China c School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China b

a r t i c l e

i n f o

Article history: Received 20 February 2013 Accepted 9 June 2013 Available online 22 June 2013 Keywords: Mussel oligopeptides D-Galactose Oxidative damage PI3K/Akt/NOS signal Lactate level

a b s t r a c t Dietary supplementation exerts beneficial effects in reducing incidence of chronic neurodegenerative diseases. The purpose of this study was to examine protective effects of mussel (Mytilus edulis) oligopeptides supplementation on brain function in D-galactose induced aging mice. Sixty female 8-month-old mice were randomly divided into five groups: vehicle control, D-galactose, and D-galactose combined with 200, 500, 1000 mg/kg mussel oligopeptides. The results showed that mussel oligopeptides could improve cognitive learning and memory ability and protect the hippocampal neurons. In addition, GSH, SOD and GSH-pX activities were increased and MDA level was significantly decreased in mice fed with mussel oligopeptides. It was also found that mussel oligopeptides supplementation prevented D-galactose-induced elevations of iNOS activity and NO production and lactate acid levels in brain. Moreover, PI3K and Akt genes were up-regulated by mussel oligopeptides supplementation. These findings suggest that mussel oligopeptides are able to enhance exercise capacity and protect against oxidative damage caused by Dgalactose in aging model mice through regulating oxidation metabolism and PI3K/Akt/NOS signal pathway. Therefore, mussel oligopeptides are good materials for future development of healthcare products to combat age-related brain dysfunction and to improve healthy life span. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Due to the increase of population and a prolonged life span, the aging related issues have brought great attention to the world. Aging is a progressive physiological functional impairment process involving various organs and tissues. In mammals, deterioration of the central nervous and brain retrograde are particularly universal aspects of the aging process. Brain aging shows several phenotypes and age-related losses occur to sensation, cognition, memory, and motor control. Age-associated cognitive decline and neuronal loss are present in old-age neurodegenerative disease like dementia of Alzheimer’s disease (Cole et al., 2005) and Parkinson (Navarro and Boveris, 2010). Learning and memory ability is one of the basic functions in brain, so aging is typically accompanied by impairments in these cognitive abilities (Erickson and Barnes, 2003). In addition, in the aged brain, neurogenesis is dramatically decreased (Rao et al., 2006), which is closely related to cognitive decline. Aging appears to be a phenotypic event like any other diseases, which is influenced by inherited, metabolic and environmental ⇑ Corresponding author. Tel.: +86 51188797202; fax: +86 51188780201. E-mail address: [email protected] (Y. Dong). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.06.009

factors including endocrine signaling, stress responses, metabolism, and telomeres (Kenyon, 2005). Oxidative stress resulting in macromolecule damage is thought to be one of the main causative factors of aging (Colavitti and Finkel, 2005; Lopez-Torres and Barja, 2008; Perez et al., 2009). The strong correlation between increasing brain aging and the accumulation of oxidative damage has largely supported the hypothesis of aging, because the brain is believed to be particularly susceptible to oxidative stress (Coyle and Puttfarcken, 1993). D-Galactose is a reducing sugar, which could induce accelerated aging showing neurological impairment and decreased activity of anti-oxidant enzymes (Song et al., 1999). Researchers report that administration of D-galactose in mice may lead to neurodegeneration by inhibiting neurogenesis, enhancing caspase-mediated apoptosis and increasing oxidative damage and mitochondrial dysfunction, as a result of cognitive dysfunction and memory deficit (Cui et al., 2006; Kumar et al., 2010; Zhang et al., 2005). Some interventions were found to extend longevity and postpone the process of aging. Dietary supplementation exerts beneficial effects in reducing the incidence of chronic neurodegenerative diseases (Farooqui and Farooqui, 2009). Studies have shown that the population eating some marine food every day has low

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mortality and low risk for age-associated disease (Albert et al., 1998; Chernoff, 2004; Kaushik et al., 2008; Park et al., 2009). The marine mussels have been widely used all over the world as health marine food source. A preparation rich in peptide, amino acid, polysaccharide from mussels has been reported to possess various biological activities, such as antimicrobial (Gerdol et al., 2012), anti-oxidation (Jung et al., 2007; Xu et al., 2008), anti-inflammatory (Whitehouse et al., 1997), and anti-hypertensive properties (Je et al., 2005). Emerging evidence indicates that mussel peptides are believed to possess an improved medical efficacy which can delay the aging process and increase lifespan. Based on the knowledge of broad activities of mussel oligopeptides and the lack of studies on their anti-aging functions up to now, we investigated the protective effect of mussel oligopeptides on impaired exploratory behavior, learning, and memory in the Dgal-treated mouse and discussed the underlying neuron-protective mechanism of mussel oligopeptides to mouse brain. 2. Materials and methods 2.1. Reagents and drugs D-Galactose was purchased from Beijing Chemical-Regent Company (Beijing, China and dissolved in 0.9% saline at concentrations of 20 mg/ml. Commercial kits used for determination of glutathione (GSH),superoxide dismutase (SOD), glutathione peroxidases (GSH-pX), malondialdehyde (MDA), nitric oxide (NO), inducible NO synthase enzymes (iNOS) and lactate levels were purchased from Jiancheng Institute of Biotechnology (Nanjing, China). Phenylmethanesulfonyl fluoride (PMSF) and RIPA Lysis Buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China).

2.2. Materials processing Mussel (Mytilus edulis) meat was manually removed from the shells, collected and freeze dried. The lyophilized powder was mixed with ice distilled water (1:20, wt: vol) and adjusted to pH 12.0. After pH adjustment, the homogenate was allowed to stand for 60 min at 4 °C and centrifuged at 12,000g for 20 min at 4 °C. The resultant supernatant was adjusted to pH 5.2 and then centrifuged at 12,000g for 20 min at 4 °C. The precipitated substances were adjusted to a pH of 7.0 for enzymatic hydrolysis using neutrase and flavourzyme at enzyme/substrate ratio of 1/60 (w/w) and 52.5 °C for 2 h. The hydrolyzate was clarified by centrifugation (8000g, 20 min at 4 °C) to remove the residue. The resultant supernatant was fractionated through ultra-filtration membranes with a range of molecular weight (MW) cut-off of 10 kDa, 3 kDa and 1 kDa. The composition of mussel oligopeptides by MW was as follows: MW less than 1 kDa, 77.42%; 1–3 kDa, 8.91%; 3–10 kDa, 4.45%; 10 kDa and higher, 7.88%. Fractionates of below 1 kDa were lyophilized and stored at 20 °C until used. 2.3. Animals and administration Middle-aged (8-month-old; n = 60) female ICR strain mice weighing 47–51 g were obtained from the Experimental Animal Center of Yangzhou University (Yangzhou, China) under the license number SCXK (SU) 2009–0002 and SYXK (SU) 2008–0024. Animals were given free access to water and normal diet. All experimental procedures were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the university committees for animal experiments. After a week of adaptation, middle-aged mice were divided randomly into five groups. Group 1 served as vehicle control with injection of saline (vehicle control group), and the other groups of mice received daily intraperitoneal injection of D-galactose (D-gal, aged model group) at dose of 200 mg/(kg day) for 2 months, respectively. At the same time, mice in groups 3–5 received daily mussel oligopeptides (PP, 200, 500 and 1000 mg/kg/day, respectively) for 2 months, and the mice of groups 1 and 2 were given saline orally at the same dose. The body weight of each mouse was monitored on a weekly basis to provide an index of general health. After the behavioral testing, mice were sacrificed and brain tissues were immediately collected for experiments or stored at 70 °C for later use.

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During the trial training, after habituation to the dark compartment for 3 min, the mouse was placed in the light compartment with its back to the door and allowed to enter the dark compartment. Upon entry, the door was closed, and the animal remained in the dark compartment for 30 s. Thereafter, the mouse was placed back in its home cage. Three such training trails were also run on the other day, and the time (step-through latency, STL) taken by the mouse to enter the dark compartment was measured and scored. Once the mouse entered the dark compartment, an inescapable scrambled foot-shock (2.0 mA for 2 s) was then delivered through the grid floor. The mouse was removed after receiving the foot shock and returned to the light compartment by the experimenter. The door was re-opened 10 s later to start the next trial. Training continued in this manner until the mouse stayed in the light compartment for more than 180 s in a single trial. Twenty-four hours later, each animal was placed in the light compartment, and the step-through latency and number errors were recorded until 300 s had elapsed (retention trial). If the mouse had still not entered the dark compartment, the wrong times records was 0, the time of step-through latency records is 300 s (Sato et al., 2004). 2.5. Morris water maze The ability of mice to learn and remember the location of a hidden platform was determined by Morris water maze (MWM) test. The MWM test was conducted as previously described (Moy et al., 2007). The water maze consisted of a circular, 180-cm diameter and 50-cm deep, black-painted tank filled with 22–24 °C water clouded with prepared Chinese ink to a depth of 30 cm. A circular 10 cm diameter escape transparent plexiglass platform was placed in the pool 1–1.5 cm below the surface of the water and maintained at a constant position. There were numerous extra-visual cues in the edge of the pool. Animals were always tested in the same order, using the same maze and spatial environment at approximately the same time each day. Mice received four trials per day over four consecutive days. For each trial, the mice were placed into the water facing the wall, with the start locations varying pseudo-randomly (NE, SE, NW, or W). The mice were then permitted to swim until they reached the escape platform. A maximum of 60 s was allowed before the mice were placed on the platform. Once on the platform, the mice remained there for 5 s before being removed for an inter-trial interval of 30 min. To assess the spatial memory retention following a period of learning, probe trials were conducted on day 5. The hidden platform was then removed from the pool and each mouse was allowed to swim for 60 s starting from the northwestern position. Mice that failed to reach the visible platform were excluded from the analysis. Daily escape latencies to the hidden platform, as well as percent time spent and distance traveled in the target quadrant during the probe trial, along with swim speed, were measured with the tracking system and water maze software (Ethovision, version 3.1, Noldus, the Netherlands) via a video camera mounted directly above the water maze center. Mice were dried under a heating lamp after each trial, and all experiments were started at the same time every day. 2.6. Rota rod test The Rota rod test was used to assess motor coordination and balance in rodents. Mice have to keep their balance on a rotating rod (Rota rod Version XZY-4B, Institute of Medical Science, Shandong, China). The Rota rod treadmill had a 3 cm diameter rod divided into five equal sections with a non-slippery surface. The rod was 50 cm above the base (trip plate). The rod turned at 6 rotations per min throughout the test. A mouse was placed onto the rod and a timer was started when its tail was released. Fall latency was recorded by the experimenter, unless the mouse remained atop the rod for the trial limit of 3 min on day 1, 5 min on day 2 and 3 min on day 5. Pre-training on day 1 was concluded when the mouse remained on the rod for a full 3 min or after 4 training trials. 2.7. Preparation of brain tissues After behavioral tests, all mice were fasted overnight and then sacrificed by decapitation. Brains were removed carefully and quickly to 0.9% cold saline, weighed. The left hemisphere was dissected with a blunt technique into the hippocampus and the cortex. The hippocampus was fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin. Hippocampal damage was evaluated by counting the number of surviving neurons per millimeter length of the CA1 pyramidal cell layer under light microscopy. The right hemispheres were homogenized with ice-cold saline and stored at 70 °C for biochemical analysis. On the day of assay, a 10% (w/v) tissue homogenate was prepared in 50 mM phosphate-buffered saline (pH 7.4) using glass homogenizer. The homogenate was centrifuged at 4000 rpm for 10 min at 4 °C and the supernatant was used.

2.4. Passive avoidance task 2.8. Analytical procedures The mice were trained in a step-through passive avoidance task. The apparatus consisted of a dark and an illuminated compartment (19  19  12 cm) with a steel-rod grid floor. One of the light compartments was equipped with a 20 W lamp located centrally at a height of 30 cm. The two compartments were connected by a small door that allowed the mice pass through.

The supernatants were used to estimate the concentrations of SOD, MDA, GSH, GSH-pX, NO, NOS and lactate according to the manufacturer’s instructions with the kits, respectively. Each sample was analyzed in triplicate. For the superoxide dismutase (SOD) assay, the supernatant was further centrifuged at 8000g for 20 min. The

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activities of total nitric oxide synthase in the brain were spectrophotometrically measured as previously described (Yang et al., 2006) based on the oxidation of oxyhaemoglobin to methaemoglobin by nitric oxide. The inducible NOS (iNOS, calcium-independent) activity was measured by adding ethylene glycol-bis-(2-aminoethyl)-N,N,N0 ,N0 -tetraacetic acid (EGTA) to chelate free Ca2+ in the reaction mixture. Lactate concentration was determined with the colorimetric Lactate Assay Kit. Optical density (OD) was measured at 530 nm.

the PP group than that in aged model group (P < 0.01) and similar (P > 0.05) to that in the vehicle control group (Fig. 1A). In the acquisition trial, the step-through latencies did not differ among the experimental groups (P > 0.05) (Fig. 1B). However, for 24 h-retention trials, the step-through latency time (seconds) of the D-galactose-treated aged model mice was significantly shorter than that of the vehicle control mice. After 500 and 1000 mg/kg PP administration, step-through latency was significantly longer than that of the D-gal-group (P < 0.05), and was not significantly different (P > 0.05) from the vehicle control mice.

2.9. Western blot analysis About 100 mg brain tissue was homogenized in 1 ml lysis buffer solution containing 20 mol/l PMSF. The homogenate was centrifuged at 12,000g for 20 min. The proteins were separated by 10% SDS–PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Gels were blotted onto polyvinylidenfluoride membrane, washed with Tris-buffered saline (pH7.5) containing 0.1% (vol/vol) Tween-20 (TBS-T), blocked for 2 h at room temperature and incubated with primary antibodies diluted in Blocking Buffer with 0.1% Tween-20 overnight at 4 °C. Primary antibodies comprised: rabbit anti-PI3K (1:2000, Santa Cruz Biotechnology), rabbit anti-b-actin (1:1500, Santa Cruz Biotechnology), and rabbit anti-Akt (1:1000). After incubation with the first antibody, membranes were washed four times for 5 min with TBS-T, and incubated with the appropriate secondary HRP-coupled antibodies against rabbit (1:2000; Santa Cruz Biotechnology) in Rotiblock for 2 h at room temperature. Membranes were washed with TBS-T four times for 5 min. Immunoreactive bands were detected with 3, 30-diaminobenzidine (DAB) staining. B-actin was served as protein loading control.

A life-long daily administration of D-gal had no significant effect (P > 0.05) on body weight (Table 1). Body weight was the maximum in vehicle control mice and mussel oligopeptides (PP, 1000 mg/kg) slightly reduced body weight. However, the PP diet had a barely significant effect (P > 0.05) on body weight compared with aged model group. It is indicated that the life-long administration of mussel oligopeptides did not have any adverse effect on food consumption and animal’s body weight.

3.2.2. Morris water maze test Morris water maze consists of a series of spatial learning acquisition training and spatial accuracy memory in probe trial and so has been regarded as one of the most frequently used laboratory tools in spatial learning and memory. Fig. 2 showed that the escape latency became progressively shorter in all groups in a day-dependent manner and the alleviated effects of mussel oligopeptides (PP) on impairment of spatial learning and memory ability of animals (two-way ANOVA, effect of treatment: F (4, 16) = 25.62, p < 0.001; effect of day: F (4,175) = 33.87, p < 0.001). Compared with the vehicle control group, the D-galactose-treated aged model group markedly spent longer time (P < 0.05) in finding the platform on days 3–5. However, the prolonged escape latency was significantly reduced (P < 0.05) by long-term administration of PP at high dose compared with the D-gal-treated aged model on days 4–5 (Fig. 2A). In the probe test (Fig. 2B), the aged model group made fewer (P < 0.05) platform crossings than the vehicle control group, and the PP at dose of 1000 mg/kg/day treatments could significantly increase (P < 0.05) the number of times of crossing over the platform site compared with the D-gal-treated aged model. Furthermore, as shown in Fig. 2C, the aged model group had a decreased (P < 0.05) spatial preference for the target quadrant compared with the vehicle control group, and significant difference (P < 0.05) was observed between the aged model group and the PP high dose group. At the swimming speed during probe trial session, no significant difference (P > 0.05) was observed among groups (Fig. 2D). Consequently, the results indicate that PP did not show any marked effects on the swimming speed and decreased times to crossing over the platform were not due to impaired motor function in D-gal-treated mice. Taken together, these results showed that PP improved spatial learning and memory in D-gal-treated aged model mice.

3.2. Effect of mussel oligopeptides in memory function in D-galactose treated mice

3.3. Effect of mussel oligopeptides in Rota rod tests in D-galactosetreated mice

3.2.1. Passive avoidance test In training session in the avoidance test, D-galactose-treated aged model mice showed an initial increase in error-number, while the group treated with 1000 mg/kg PP showed a significant decrease in the error-number compared with aged model mice (P < 0.05). Following training, the error-number was much less in

The Rota-rod test was performed to test whether impaired motor coordination caused lower locomotor activity. As shown in Fig. 3, D-galactose induced aged model mice exhibited a significant loss of motor coordination activity as compared with young adult animals (P < 0.05). The high dose of PP treatment group remained significantly more time (P < 0.001) on the Rota rod than the aged model group.

2.10. Statistical analysis All the presented data were expressed as means ± SD unless otherwise indicated. A two-way analysis of variance (ANOVA) with repeated measures was used for analyzing group differences in the escape latency during the MWM training task and in body weight measurement; the factors used were treatment and time. Oneway ANOVA was used for statistically analyzing all other data and P < 0.05 was considered statistically significant. The differences in the means of various groups were analyzed following Duncan’s method of the least significant ranges (LSR) using SPSS software.

3. Results 3.1. Body weight

Table 1 Effects of increasing age and different treatments on the body weights (g) of ICR mice. Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

D-gal

46.7 ± 2.0 46.3 ± 2.1

46.5 ± 2.9 44.5 ± 2.3

47.2 ± 2.2 44.2 ± 3.9

47.2 ± 2.8 44.7 ± 3.4

47.5 ± 2.5 46.2 ± 2.6

47.3 ± 2.2 46.2 ± 2.7

47.8 ± 2.4 46.5 ± 3.7

48.2 ± 3.3 46.9 ± 4.0

+PP(1000) +PP(500) +PP(200)

46.2 ± 2.5 46.8 ± 2.3 47.3 ± 3.8

46.3 ± 2.4 45.7 ± 3.5 47.1 ± 4.5

45.5 ± 2.3 46.2 ± 2.7 47.6 ± 4.2

44.5 ± 3.2 46.5 ± 2.5 47.2 ± 3.5

44.3 ± 3.0 47.1 ± 2.3 47.6 ± 4.1

44.2 ± 2.6 47.2 ± 4.3 47.7 ± 3.8

44.3 ± 3.5 47.6 ± 3.5 47.9 ± 4.1

44.1 ± 2.5 47.8 ± 4.2 47.9 ± 4.6

Vehicle control

Results are given as mean body weights ± standard deviation. n = 12 for each animal group. Statistical analysis (two-way ANOVA). There is not a significant difference (P > 0.05) between any groups. Vehicle control, animals receiving saline; D-gal, animals receiving D-galactose; PP, animals receiving D-galactose and mussel oligopeptides.

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Fig. 1. Effects of mussel oligopeptides on the performance of mice in the step-down passive avoidance test. Each mouse was trained for 5 min to ‘‘remember’’ the system. Twenty-four hours later, mice were placed in the same cage and latency time (A) and the number of errors (B) within 5 min were recorded. Data were expressed as means ± SD number errors (n = 8). P < 0.05 and P < 0.01 compared with the D-galactose-induced aged model groups; #P < 0.05 compared with the vehicle control group. Vehicle control, mice were treated with saline; D-gal, mice were treated with D-galactose; PP, mice were treated with D-galactose and mussel oligopeptides. The same in Figs. 2–6.

3.4. Effects of mussel oligopeptides on histological pathology in the hippocampus Extensively damaged neurons in the hippocampus CA1 region were observed and the number of surviving neurons was reduced in the D-gal-induced aged model group compared with the vehicle control group. Pyramidal neuronal shrinkage and chromatin condensation of nuclei were also observed in aged model group. However, significantly higher normal surviving neurons (P < 0.001) in the pyramidal cell layer were observed in the PP (1000 mg/kg) group compared with the aged model group (Fig. 4). 3.5. Mussel oligopeptides supplementation reduces oxidative damage in aged mice SOD and GSH-pX are two important enzymes in the antioxidant defense system. As compared with vehicle control group, D-gal treatment significantly decreased SOD (Fig. 5A) and GSH-pX

(Fig. 5D) activities (P < 0.05). PP treatments obviously inhibited the reduction of SOD and GSH-pX induced by D-galactose; and very significant elevation (P < 0.001) was shown at the higher dose (1000 mg/kg). MDA is often measured as a major marker of lipid peroxidation in the aging tissue. MDA level was significantly increased (P < 0.05) in D-gal-treated aged model group mice as compared to vehicle control group. PP treatment showed significant decrease (P < 0.05) in MDA level as compared to the aged model group (Fig. 5B). There was also a significant reduction (P < 0.05) of GSH level in the D-galactose treated aged model group as compared to the vehicle control group. The treatment of PP showed significant increase (P < 0.05) in GSH level as compared to the aged model group (Fig. 5C). In brain, lactate levels were significantly increased in D-gal-induced aged model mice compared with the vehicle control group (P < 0.001). The lower levels of lactate were maintained

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Fig. 2. Effects of mussel oligopeptides on the spatial learning and memory of mice in the Morris water maze test. (A) Mean latency in the hidden platform test during five consecutive days of training. A two-way ANOVA was used and the factors were treatment and day, effect of treatment: F (4, 16) = 25.62, p < 0.001; effect of day: F (4,175) = 33.87, p < 0.001. (B) The number of crossings over the exact location of the former platform on day 6. (C) Comparison of time spent in the target quadrant on day 6. (D) The swimming speed on day 6. All values are expressed as means ± SD (n = 8). P < 0.05 compared with the D-galactose-induced aged model group; #P < 0.05 compared with the vehicle control group.

D-galactose-induced

aged model group (Fig. 6B). Since it is known that excessive NO is derived by NOS, our results also showed that iNOS activity in brain tissue of D-galactose administrated mice was elevated compared to the vehicle control, but was inhibited by the administration of mussel oligopeptides (Fig. 6A). From these results, we conclude that mussel oligopeptides enhance the function of iNOS which was impaired by oxidative stress under high D-galactose condition. The results demonstrated that iNOS activity and NO generation occurred with an increase in superoxide production.

Fig. 3. Effect of mussel oligopeptides in mice on the reversal from D-galactoseinduced impairment of motor balanced activity. Data were expressed as means ± SD number errors (n = 6). means P < 0.05 and means P < 0.001 compared with the Dgalactose-induced aged model groups. #P < 0.05 and ###P < 0.001 compared with the vehicle control groups.

(P < 0.05) in mice with PP administration compared with the aged model group (Fig. 5E). 3.6. Mussel oligopeptides supplementation regulates NO production and NOS activity D-Galactose significantly increased (P < 0.001) the NO generation compared to the vehicle control group, and the effect was abolished by mussel oligopeptides. A significant lower (P < 0.01) level of NO generation was observed under PP condition compared to the

3.7. PI3K/Akt signaling pathway is involved in mussel oligopeptides effects on brain senescence We further examined the effect of mussel oligopeptides on the expressions of PI3K and Akt in D-galactose treated mice. Fig. 6C and D showed that the expressions of PI3K and Akt were significantly decreased by D-galactose. After the mice were fed with mussel oligopeptides, the expressions of PI3K and Akt were significantly increased. These findings indicated that PI3K/Akt signaling pathway played a role in oxidative stress under D-galactose and it was involved in the actions of mussel oligopeptides against iNOS in brain cells of mice. 4. Discussion The consequences of the aging process are characterized by an accelerated decline of functional performance including a decline

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Fig. 4. Histopathology in the hippocampus CA1 region of mice. Light microphotographs of the hippocampus with hematoxylin-eosin stain in the vehicle control group (A and B), D-galactose-induced aged model group (C and D), and mussel oligopeptides (PP, 1000 mg/kg)-treated group (E and F). Extensively damaged neurons in CA1 were observed in the D-gal-induced aged model group and the number of surviving neurons was reduced compared with the vehicle control group. (D) Pyramidal neuronal shrinkage and chromatin condensation of nuclei were observed. (E) PP treatment increased the number of neurons and (F) numerous normal surviving neurons were observed in the pyramidal cell layer. Arrows indicate the area examined with a high power micrograph. (G) The number of surviving neurons in the hippocampus CA1 region. Bar graph of results of neuronal counting expressed as number of surviving neurons/mm CA1 pyramidal layer. Data are expressed as mean ± SD (n = 6). ###p < 0.001 vs. with the vehicle control group; p < 0.001 vs. D-galactose-induced aged model group.

in physiological function and reduced physical activity (Carter et al., 2009). Therefore, the term ‘‘functional age’’ is a reliable index to define older individuals. In both humans and rodents, intellectual ability (Shaw et al., 2006) and behavioral control of executive functions (Ernst et al., 2009) are the most representative measurements in functional aging. D-Galactose-induced functional aging has been used as an experimental animal model for studying brain aging and to develop suitable preventive strategy (Kumar et al., 2009; Wei et al., 2005). In this study, it was also clearly shown that D-galactose-treated middle-aged mice showed worse executive function with aging assessed by passive avoidance, water maze and Rota rod tests. D-galactose showed memory retention and a more sensitive behavioral characterization in motor coordination and balance deficits. We also observed two distinct effects of mussel oligopeptides to ameliorate cognitive deficits in D-galactose-treated mice. The first was to attenuate the decline in motor learning and memory. Results showed that oral administration of mussel oligopeptides significantly contributed to memory retention by reducing stepthrough latencies in the avoidance experiments, and increasing spatial learning and memory in performance across the platform in the MWM tests. The second was to ameliorate the decline in motor coordination and balance. In concert with these behavioral changes, mussel oligopeptides supplementation significantly ameliorated the D-galactose-in-

duced increase in neuronal damage. It is reported that D-galactose can produce neuronal injury in brain and reduces the survival of new neurons. In particular, pyramidal cells of Hippocampal are very sensitive to D-galactose (Cui et al., 2006). Our results also showed that extensively damaged neurons in the CA1 region of the hippocampus were observed after D-galactose treatment, and mussel oligopeptides significantly ameliorated pathological injury of the hippocampus. These results provide direct evidence that mussel oligopeptides can confer marked histopathological protection against D-galactose. The neuronal injury associated with D-galactose is accompanied by generation of reactive oxygen species and progressive deficit in learning and memory. A lot of reports have shown that the accelerated aged animals induced by the chronic administration of Dgalactose are associated with accumulation of oxidative stress (Hsia et al., 2011; Kumar et al., 2010). Diminished activity of antioxidant enzymes and increase in Lipid peroxidation are biomarkers of oxidative stress (Cui et al., 2006; Hsia et al., 2011). Oxidative stress plays a pivotal role in the age-associated cognitive decline in neurodegenerative disease like Alzheimer’s (Veerendra Kumar and Gupta, 2003) and Parkinson (Kaur et al., 2011). The damage caused by the oxidative stress is due to neuronal membranes easily damaged by free radicals. MDA is an important indicator of oxidative damage under conditions of oxidative stress (Elia et al., 2002). In the current study, aged mice induced by D-galactose showed a

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Fig. 5. The effect of mussel oligopeptides on the activity of SOD (A), MDA (B), GSH (C), GSH-pX (D) and lactate levels (E) in brain. Each value is expressed as means ± SD (n = 6).  P < 0.05, P < 0.01 and P < 0.001 compared with the D-galactose-induced aged model groups; #P < 0.05, ##P < 0.01 and ###P < 0.001 compared with the vehicle control groups.

significant increase in MDA level in the brain, indicating an elevation in oxidative stress during aging. Mussel oligopeptides that decreased the MDA level in D-gal-treated mice further demonstrated that they ameliorated the oxidative stress in the brain induced by D-galactose. There was a simultaneous significant increase in the reduced glutathione levels in mussel-treated mice. Glutathione is an endogenous defense antioxidant mainly in the reduced form by directly reacting with ROS. So, GSH level parallels the antioxidant defense capacity in the brain. In the present study, the restored reduced glutathione levels may be due to the electron and H+ donating capacity of reducing components present in mussel oligopeptides. In addition, mussel oligopeptides have a direct effect on antioxidant enzymes such as SOD and GSH-pX. SOD can catalyze the conversion of radical superoxide (O2) into H2O2 (Escobar et al., 1996). Furthermore, GSH-pX converts H2O2 to H2O and O2 related to the production of abnormal structures (Mahieu et al., 2005). Mussel oligopeptides may have different functional property such as scavenging of reactive oxygen species, inhibition of the generation of free radicals and chain-breaking activity. Besides the effects on scavenging of reactive oxygen species or intervention of lipid peroxidation, treatment with mussel oligopeptides may influence secretion of lactate. A recent paper

provides evidence that during aging, the oxidative stress increased and thus induced the increment of the lactate level throughout the brain (Nelson et al., 2009). High brain lactate levels were regarded as a marker in aging (Ross et al., 2010). The increased brain lactate levels have been accounted under other pathological circumstances or neuronal damage, such as intoxication (Lawson-Smith et al., 2011), stroke (Woo et al., 2010) and Alzheimer disease (AD) (von Pfostl et al., 2012). In the present study, we found that lactate levels measured in brain of D-gal-treated mice were significantly increased. In contrast, mice with oral administration of mussel oligopeptides had significantly decreased lactate levels. Therefore, lactate in brain appeared to be degraded through the reduction of oxidative stress after supplementation with mussel oligopeptides. Under the oxidative stress, the overproduction of NO contributes to neurotoxicity (Prast and Philippu, 2001) and appropriate level of NO plays important roles in neuroprotection (Holscher, 1997). Nitric oxide (NO) is generated from L-arginine by a family of NO synthase enzymes (NOS), an enzyme that exists in 3 isoforms: neuronal NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). Inducible NOS can be induced by various stimulates. The overproduction of NO by iNOS has been implicated in various pathophysiological processes. NO and O2 can produce a more

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Fig. 6. Mussel oligopeptides attenuate brain senescence through suppressing NO and activating PI3K/Akt level. (A) iNOS activity. (B) NO contents. (C) Western blot assay on expressions of PI3K and Akt. (D) The quantitative protein expressions of PI3K and Akt were summarized in bar graphs presented by mean values of 3 independent experiments (n = 3). Error bars represent the SD. Compared with that of vehicle control group; there was less PI3K and Akt expressions in aging model mice. After being treated with mussel oligopeptides, expression levels of these two proteins were remarkably increased compared with the aging model mice. means P < 0.05, P < 0.01 and  P < 0.001 compared with the D-galactose-induced aged model group; #P < 0.05, ##P < 0.01 and ###P < 0.001 compared with the vehicle control groups.

aggressive agent, the peroxynitrite radical (ONOO), which can cause the brain damage, as found in the pathological observations in this study. So NO plays a role in maintaining ‘‘antioxidant homeostasis’’ and impaired NO bioactivity is a pathogenic factor in aging (Shi et al., 2010). The depletion of the brain GSH results in the increase of nitric oxide synthase (NOS) (Heales et al., 1996). We observed that accompanying with the decrease of GSH level in the mouse brain, the activities of iNOS and NO were significantly elevated by D-galactose. These changes indicated that D-galactose would stimulate the increase in the activity of iNOS and excessively release NO, and then the mussel oligopeptides mobilized its protective mechanism to inhibit the overproduction of NO in the mouse brain. Multiple signs of aging are evident in the mammalian brain. Down-regulation of neurotrophic factors including brain-derived neurotrophic factor (BNDF), vascular endothelial growth factor (VEGF) and insulin/IGF-1 could contribute to the damaging effects of stress during aging (Duman, 2005). Free-radical generators significantly suppress the amount of BDNF release and some molecules, including cholecystokinin and glutamate, promote BDNF production in neurons. Expression of particular genes such as PI3K and NOS are important to ascribe neuroprotective activities of BDNF (Gokce et al., 2009). Akt could function in mammals very similar to the insulin signaling pathway in regulation of longevity by modulating energy metabolism, stress resistance and regenerative capacity (Mattson et al., 2002). Activation of PI3K/Akt pathway enhanced learning and memory (Lu et al., 2010; Minichiello et al., 1999) and was thought to be involved in neuronal survival (Lee et al., 2011). NO was reported to be downstream of Akt in VEGF signaling. Akt also could regulate nitric oxide (NO) production by direct phosphorylation and activation of inducible NO synthase (iNOS) (Park et al., 2003). In the present study, NO, produced by iNOS in brain, showed the change pattern equivalent with iNOS. In-

creased iNOS activities and NO levels confirmed NO pathway modifications in this model, which are presumably related to increased oxidative stress. Modifications in the NO pathway and the PI3K/Akt pathways may play a major role in mussel oligopeptides-mediated neuroprotection. In accordance with the above results, our finding suggests that PI3K/Akt/iNOS pathways may be one kind of pathway contributing to induction of brain aging by D-galactose. Also, mussel oligopeptides up-regulate neuroprotective activities via a pathway that includes PI3K, Akt iNOS and NO, resulting in the amelioration of brain deficits.

5. Conclusions The present study revealed that oral administration of mussel oligopeptides could significantly prevent the cognitive impairment, protect hippocampal neurons and attenuate the oxidative damage induced by D-galactose. In addition, the age-related lactate level in brain was significantly decreased. The protein expression profiles demonstrated that mussel oligopeptides suppressed overproduction of NO and iNOS activity in brain via PI3k/Akt pathway. It is reasonable to conclude that the protective effect of mussel oligopeptides is due to suppression of oxidative stress mediated brain aging. These observations collectively suggest that mussel oligopeptides could target many biochemical pathways in reducing oxidative damage, improving the age-related functional decline, and preserving physical performance.

Conflict of Interest The authors declare that there are no conflicts of interest.

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Acknowledgments This work was supported by the National Hi-Technology Research & Development (863) Program (No. 2011AA100803) and a project Funded by the Priority Academic Program Development of Jiangsu higher education institutions. References Albert, C.M., Hennekens, C.H., O’Donnell, C.J., Ajani, U.A., Carey, V.J., Willett, W.C., Ruskin, J.N., Manson, J.E., 1998. Fish consumption and risk of sudden cardiac death. JAMA 279, 23–28. Carter, C.S., Leeuwenburgh, C., Daniels, M., Foster, T.C., 2009. Influence of calorie restriction on measures of age-related cognitive decline: role of increased physical activity. J. Gerontol. 64, 850–859. Chernoff, R., 2004. Protein and older adults. J. Am. Coll. Nutr. 23, 627S–630S. Colavitti, R., Finkel, T., 2005. Reactive oxygen species as mediators of cellular senescence. IUBMB Life 57, 277–281. Cole, G.M., Lim, G.P., Yang, F., Teter, B., Begum, A., Ma, Q., Harris-White, M.E., Frautschy, S.A., 2005. Prevention of Alzheimer’s disease: omega-3 fatty acid and phenolic anti-oxidant interventions. Neurobiol. Aging 26 (Suppl 1), 133–136. Coyle, J.T., Puttfarcken, P., 1993. Oxidative stress, glutamate, and neurodegenerative disorders. Science, vol. 262. New York, NY, pp. 689–695. Cui, X., Zuo, P., Zhang, Q., Li, X., Hu, Y., Long, J., Packer, L., Liu, J., 2006. Chronic systemic D-galactose exposure induces memory loss, neurodegeneration, and oxidative damage in mice: protective effects of R-alpha-lipoic acid. J. Neurosci. Res. 83, 1584–1590. Duman, R.S., 2005. Neurotrophic factors and regulation of mood: role of exercise, diet and metabolism. Neurobiol. Aging 26 (Suppl 1), 88–93. Elia, A.C., Waller, W.T., Norton, S.J., 2002. Biochemical responses of bluegill sunfish (lepomis macrochirus, rafinesque) to atrazine induced oxidative stress. Bull. Environ. Contam. Toxicol. 68, 809–816. Erickson, C.A., Barnes, C.A., 2003. The neurobiology of memory changes in normal aging. Exp. Gerontol. 38, 61–69. Ernst, M., Romeo, R.D., Andersen, S.L., 2009. Neurobiology of the development of motivated behaviors in adolescence: a window into a neural systems model. Pharmacol. Biochem. Behav. 93, 199–211. Escobar, J.A., Rubio, M.A., Lissi, E.A., 1996. Sod and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radical Biol. Med. 20, 285–290. Farooqui, T., Farooqui, A.A., 2009. Aging: an important factor for the pathogenesis of neurodegenerative diseases. Mech. Ageing Dev. 130, 203–215. Gerdol, M., De Moro, G., Manfrin, C., Venier, P., Pallavicini, A., 2012. Big defensins and mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis. Dev. Comp. Immunol. 36, 390–399. Gokce, O., Runne, H., Kuhn, A., Luthi-Carter, R., 2009. Short-term striatal gene expression responses to brain-derived neurotrophic factor are dependent on MEK and ERK activation. PLoS ONE 4, e5292. Heales, S.J., Bolanos, J.P., Clark, J.B., 1996. Glutathione depletion is accompanied by increased neuronal nitric oxide synthase activity. Neurochem. Res. 21, 35–39. Holscher, C., 1997. Nitric oxide, the enigmatic neuronal messenger: its role in synaptic plasticity. Trends Neurosci. 20, 298–303. Hsia, C.H., Wang, C.H., Kuo, Y.W., Ho, Y.J., Chen, H.L., 2011. Fructo-oligosaccharide systemically diminished D-galactose-induced oxidative molecule damages in BALB/cJ mice. Br. J. Nutr., 1–6. Je, J.Y., Park, P.J., Byun, H.G., Jung, W.K., Kim, S.K., 2005. Angiotensin I converting enzyme (ACE) inhibitory peptide derived from the sauce of fermented blue mussel, Mytilus edulis. Bioresour. Technol. 96, 1624–1629. Jung, W.K., Qian, Z.J., Lee, S.H., Choi, S.Y., Sung, N.J., Byun, H.G., Kim, S.K., 2007. Free radical scavenging activity of a novel antioxidative peptide isolated from in vitro gastrointestinal digests of Mytilus coruscus. J. Med. Food 10, 197–202. Kaur, H., Chauhan, S., Sandhir, R., 2011. Protective effect of lycopene on oxidative stress and cognitive decline in rotenone induced model of Parkinson’s disease. Neurochem. Res. 36, 1435–1443. Kaushik, S., Wang, J.J., Flood, V., Liew, G., Smith, W., Mitchell, P., 2008. Frequency of fish consumption, retinal microvascular signs and vascular mortality. Microcirculation 15, 27–36. Kenyon, C., 2005. The plasticity of aging: insights from long-lived mutants. Cell 120, 449–460. Kumar, A., Dogra, S., Prakash, A., 2009. Effect of carvedilol on behavioral, mitochondrial dysfunction, and oxidative damage against D-galactose induced senescence in mice. Naunyn. Schmiedebergs Arch. Pharmacol. 380, 431–441. Kumar, A., Prakash, A., Dogra, S., 2010. Naringin alleviates cognitive impairment, mitochondrial dysfunction and oxidative stress induced by D-galactose in mice. Food Chem. Toxicol. 48, 626–632. Lawson-Smith, P., Olsen, N.V., Hyldegaard, O., 2011. Hyperbaric oxygen therapy or hydroxycobalamin attenuates surges in brain interstitial lactate and glucose; and hyperbaric oxygen improves respiratory status in cyanide-intoxicated rats. Undersea Hyperb. Med. 38, 223–237. Lee, C., Park, G.H., Jang, J.H., 2011. Cellular antioxidant adaptive survival response to 6-hydroxydopamine-induced nitrosative cell death in C6 glioma cells. Toxicology 283, 118–128.

Lopez-Torres, M., Barja, G., 2008. Calorie restriction, oxidative stress and longevity. Rev. Esp. Geriatr. Gerontol. 43, 252–260. Lu, J., Wu, D.M., Zheng, Y.L., Hu, B., Zhang, Z.F., 2010. Purple sweet potato color alleviates D-galactose-induced brain aging in old mice by promoting survival of neurons via PI3K pathway and inhibiting cytochrome C-mediated apoptosis. Brain Pathol. 20, 598–612. Mahieu, S., Millen, N., Gonzalez, M., Contini Mdel, C., Elias, M.M., 2005. Alterations of the renal function and oxidative stress in renal tissue from rats chronically treated with aluminium during the initial phase of hepatic regeneration. J. Inorg. Biochem. 99, 1858–1864. Mattson, M.P., Duan, W., Maswood, N., 2002. How does the brain control lifespan? Ageing Res. Rev. 1, 155–165. Minichiello, L., Korte, M., Wolfer, D., Kuhn, R., Unsicker, K., Cestari, V., Rossi-Arnaud, C., Lipp, H.P., Bonhoeffer, T., Klein, R., 1999. Essential role for TrkB receptors in hippocampus-mediated learning. Neuron 24, 401–414. Moy, S.S., Nadler, J.J., Young, N.B., Perez, A., Holloway, L.P., Barbaro, R.P., Barbaro, J.R., Wilson, L.M., Threadgill, D.W., Lauder, J.M., Magnuson, T.R., Crawley, J.N., 2007. Mouse behavioral tasks relevant to autism: phenotypes of 10 inbred strains. Behav. Brain Res. 176, 4–20. Navarro, A., Boveris, A., 2010. Brain mitochondrial dysfunction in aging, neurodegeneration, and Parkinson’s disease. Front Aging Neurosci., 2. Nelson, V.M., Dancik, C.M., Pan, W., Jiang, Z.G., Lebowitz, M.S., Ghanbari, H.A., 2009. PAN-811 inhibits oxidative stress-induced cell death of human Alzheimer’s disease-derived and age-matched olfactory neuroepithelial cells via suppression of intracellular reactive oxygen species. J. Alzheimers. Dis. 17, 611–619. Park, D.W., Kim, J.R., Kim, S.Y., Sonn, J.K., Bang, O.S., Kang, S.S., Kim, J.H., Baek, S.H., 2003. Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression. J. Immunol. 170, 2093–2099. Park, S.K., Tucker, K.L., O’Neill, M.S., Sparrow, D., Vokonas, P.S., Hu, H., Schwartz, J., 2009. Fruit, vegetable, and fish consumption and heart rate variability: the Veterans Administration Normative Aging Study. Am. J. Clin. Nutr. 89, 778–786. Perez, V.I., Bokov, A., Van Remmen, H., Mele, J., Ran, Q., Ikeno, Y., Richardson, A., 2009. Is the oxidative stress theory of aging dead? Biochim. Biophys. Acta 1790, 1005–1014. Prast, H., Philippu, A., 2001. Nitric oxide as modulator of neuronal function. Prog. Neurobiol. 64, 51–68. Rao, M.S., Hattiangady, B., Shetty, A.K., 2006. The window and mechanisms of major age-related decline in the production of new neurons within the dentate gyrus of the hippocampus. Aging Cell 5, 545–558. Ross, J.M., Oberg, J., Brene, S., Coppotelli, G., Terzioglu, M., Pernold, K., Goiny, M., Sitnikov, R., Kehr, J., Trifunovic, A., Larsson, N.G., Hoffer, B.J., Olson, L., 2010. High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio. Proc. Nat. Acad. Sci. USA 107, 20087–20092. Sato, T., Tanaka, K., Ohnishi, Y., Teramoto, T., Irifune, M., Nishikawa, T., 2004. Effects of steroid hormones on (Na+, K+)-ATPase activity inhibition-induced amnesia on the step-through passive avoidance task in gonadectomized mice. Pharmacol. Res. 49, 151–159. Shaw, P., Greenstein, D., Lerch, J., Clasen, L., Lenroot, R., Gogtay, N., Evans, A., Rapoport, J., Giedd, J., 2006. Intellectual ability and cortical development in children and adolescents. Nature 440, 676–679. Shi, Y., Camici, G.G., Luscher, T.F., 2010. Cardiovascular determinants of life span. Pflugers. Arch. 459, 315–324. Song, X., Bao, M., Li, D., Li, Y.M., 1999. Advanced glycation in D-galactose induced mouse aging model. Mech. Ageing Dev. 108, 239–251. Veerendra Kumar, M.H., Gupta, Y.K., 2003. Effect of Centella asiatica on cognition and oxidative stress in an intracerebroventricular streptozotocin model of Alzheimer’s disease in rats. Clin. Exp. Pharmacol. Physiol. 30, 336–342. von Pfostl, V., Li, J., Zaldivar, D., Goense, J., Zhang, X., Serr, N., Logothetis, N.K., Rauch, A., 2012. Effects of lactate on the early visual cortex of non-human primates, investigated by pharmaco-MRI and neurochemical analysis. Neuroimage 61, 98–105. Wei, H., Li, L., Song, Q., Ai, H., Chu, J., Li, W., 2005. Behavioural study of the Dgalactose induced aging model in C57BL/6J mice. Behav. Brain Res. 157, 245– 251. Whitehouse, M.W., Macrides, T.A., Kalafatis, N., Betts, W.H., Haynes, D.R., Broadbent, J., 1997. Anti-inflammatory activity of a lipid fraction (lyprinol) from the NZ green-lipped mussel. Inflammopharmacology 5, 237–246. Woo, C.W., Lee, B.S., Kim, S.T., Kim, K.S., 2010. Correlation between lactate and neuronal cell damage in the rat brain after focal ischemia: an in vivo 1H magnetic resonance spectroscopic (1H-MRS) study. Acta Radiol. 51, 344–350. Xu, H., Guo, T., Guo, Y.F., Zhang, J., Li, Y., Feng, W., Jiao, B., 2008. Characterization and protection on acute liver injury of a polysaccharide MP-I from Mytilus coruscus. Glycobiology 18, 97–103. Yang, Y.J., Zhao, J.L., You, S.J., Wu, Y.J., Jing, Z.C., Yang, W.X., Meng, L., Wang, Y.W., Gao, R.L., 2006. Different effects of tirofiban and aspirin plus clopidogrel on myocardial no-reflow in a mini-swine model of acute myocardial infarction and reperfusion. Heart 92, 1131–1137. Zhang, Q., Huang, Y., Li, X., Cui, X., Zuo, P., Li, J., 2005. GM1 ganglioside prevented the decline of hippocampal neurogenesis associated with D-galactose. NeuroReport 16, 1297–1301.