Ameliorative effect of lotus seedpod proanthocyanidins on cognitive impairment and brain aging induced by d -galactose

Experimental Gerontology 74 (2016) 21–28

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Ameliorative effect of lotus seedpod proanthocyanidins on cognitive impairment and brain aging induced by D-galactose Yu-Shi Gong a, Juan Guo a, Kun Hu a, Yong-Qing Gao a, Bi-Jun Xie b,⁎, Zhi-Da Sun b, Er-Ning Yang b, Fang-Li Hou a a b

School of Food Science, Guangdong Pharmaceutical University, Zhongshan 528458, China Natural Product Laboratory, Food Science and Technology Department, Huazhong Agriculture University, Wuhan 430070, China

a r t i c l e

i n f o

Article history: Received 1 June 2015 Received in revised form 10 November 2015 Accepted 30 November 2015 Available online 30 November 2015 Keywords: Lotus seedpod proanthocyanidins D-Galactose Free radicals Learning and memory Brain aging

a b s t r a c t This study mainly investigated the ameliorative effect of lotus seedpod proanthocyanidins (LSPC) and the mechanism underlying such effect on cognitive impairment and brain aging induced by D-galactose. Aging mice induced by D-galactose (150 mg/kg, sc injection daily for 6 weeks) were chosen for the experiment. LSPCs (30, 60, and 90 mg/kg, ig) were provided after D-galactose injection. Learning and memory functions were detected by Y-maze and step-down avoidance tests. Then, some biochemical indexes related to cognitive ability and aging were measured. Histopathological feature and P53 protein expression in the hippocampus were observed. Results showed that the three different doses of LSPC could significantly ameliorate the learning and memory abilities impaired by D-galactose. LSPC significantly reduced the levels of malondialdehyde and nitric oxide (i.e. 90 mg/kg LSPC group vs. model group, P = 0.008), reduced the content of β-amyloid peptide 1–42 (i.e. 90 mg/kg LSPC group vs. model group, P = 0.009), decreased the activities of acetylcholinesterase, monoamine oxidase B, total nitric oxide synthase (i.e. 90 mg/kg LSPC group vs. model group, P = 0.006), and neuronal nitric oxide synthase and synchronously increased the activities of superoxide dismutase and glutathione peroxidase in the brain. Furthermore, LSPC could prevent neuron damage and could lessen the expression of P53 protein in the hippocampus. These findings demonstrated that LSPC effectively attenuated cognitive damage and improved parameters related to brain aging in senescent mice induced by D-galactose, and may be used to treat Alzheimer's disease. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Proanthocyanidins are widely distributed in common foods including cereals, fruits, vegetables, and wines and belong to a ubiquitous group of plant polyphenols (Ronald and Liwei, 2005). Proanthocyanidins exhibit potent antioxidant capacity and possible protective effects on human health. They reduce the risk of chronic diseases, such as cardiovascular diseases and cancers (Ronald and Liwei, 2005). In addition, proanthocyanidins also exhibit vasodilatory, anti-allergic, antiinflammatory, antibacterial, cardioprotective, immune-stimulating, anti-viral, and estrogenic activities (Debasis et al., 2000). The lotus seedpod is the inedible part of Nelubo nucifera Gaertn (Ling et al., 2005). Lotus is an industrial crop that is widespread in China, and its cultivated area was N 40,000 ha in 1999. Lotus seedpod proanthocyanidins (LSPC) have been first isolated and characterized in Natural Product Laboratory, Food Science and Technology Department, Huazhong Agriculture University (Wuhan, China) (Ling et al., 2005).

⁎ Corresponding author. E-mail addresses: [email protected] (Y.-S. Gong), [email protected] (B.-J. Xie).

http://dx.doi.org/10.1016/j.exger.2015.11.020 0531-5565/© 2015 Elsevier Inc. All rights reserved.

Lotus seedpod is likely to be another important source of proanthocyanidins besides grape seed. LSPC exhibits excellent antioxidant activity (Ling and Xie, 2002a, 2002b; Duan and Xie, 2003; Ling et al., 2005; Duan et al., 2005; Gong et al., 2008; Xu et al., 2009, 2010a). Furthermore, LSPC can improve the cognitive deficits of senescence-accelerated mice (SAMP8) and aged rats because LSPC has the ability to scavenge oxygen free radicals, to stimulate antioxidant enzyme activity, to rejuvenate cholinergic system and to reverse the decreased phosphorylation of adenosine 3′,5′-monophosphate (cAMP) response element-binding protein in the hippocampus (Gong et al., 2008; Xu et al., 2009, 2010b, 2011). Progressive neurological dysfunction is a key aspect associated with human aging. Animal models for the study of disorders that manifest late in life are difficult to develop (García et al., 2011). A large number of studies have demonstrated that long-term injection of D-galactose can mainly lead to excessive reactive oxygen species (ROS) formation, antioxidant enzyme activity decrease, neuronal damage, and cognitive impairment in rats or mice (Ho et al., 2003; Wei et al., 2005; Chen et al., 2006; Cui et al., 2006; Banji et al., 2014; Hao et al., 2014). Furthermore, this mimetic aging model has age-related advanced glycation end products (AGEs) that may enhance oxidative stress damage and abnormal phosphorylation and affect learning and memory functions

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(Reddy et al., 2002; Tsai et al., 2011). D-Galactose can impair neurogenesis in the dentate gyrus through a process similar to the natural aging in mice (Ho et al., 2003; Zhang et al., 2005). Thus, D-galactose injection has been gradually accepted as a tool for establishing an aging model in brain aging studies or in anti-aging pharmacological research (Zhang et al., 2008; Li et al., 2010, 2015; Banjia et al., 2013; Wei et al., 2014). Cognitive deficits are characteristics of aging and age-related neurodegenerative disorders that lead to a progressive loss of cognitive function, especially in spatial memory (Barnes et al., 1980). Since oxidative damage may play a role in the aging process, including the associated cognitive decline, many researchers believe that antioxidant supplements may alleviate age-related impairment in spatial learning and memory functions and delay the aging process. Therefore, we investigated the effect of LSPC on learning and memory impairment and brain aging and the mechanism underlying such effect in animal models induced by 6-week subcutaneous injection of D-galactose. A certain amount of data was provided to support that LSPC could attenuate Alzheimer's disease (AD) development.

2. Materials and methods 2.1. Reagents and drugs D-Galactose was purchased from Shanghai Bo'ao Biological Technology Co., Ltd. (Shanghai, China) and dissolved in 0.9% saline at concentration of 1.5%. Lotus seedpod proanthocyanidins were separated by our lab and dissolved in physiological saline. Commercial kits used for determining malondialdehyde (MDA), nitric oxide (NO), and protein levels, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), nitric oxide synthase (NOS), acetylcholinesterase (AChE), and monoamine oxidase B (MAO-B) activities were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). β-Amyloid peptide 1–42 (Aβ1–42) ELISA assay kit was purchased from the Genetics Co. (Schlieren, Switzerland). P53 antibody and paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Strept AvidinBiotin Complex (SABC) and 3,3V-diaminobenzidine tetrachloride (DAB) immunohistochemistry kits were purchased from Wuhan Boster Bio-engineering Limited Company (Wuhan, China).

2.2. Preparation of lotus seedpod proanthocyanidins (LSPC) Lotus seedpods of N. nucifera Gaertn were collected in Honghu Lake (Hubei, China). This variety of N. nucifera Gaertn is named Number 2 Wuhan plant and was authenticated by the Department of Botany, Wuhan Plant Institute of the Chinese Academy of Science. LSPCs were isolated by using the method of Ling et al. (2005). LSPCs were extracted with Me2CO/H2O and purified by Sephadex LH-20 column chromatography with a purity of 98%, as measured by the method reported by Porter et al. (1986). ESI-MS analysis indicates that the extract contains monomers, dimers, and tetramers of proanthocyanidins, in which the amounts of dimmers are greatest, and catechin and epicatechin are the base units. 2.3. Animals and drug administration Kunming mice were used. The numbers of males and females were equal. The mice were aged 3 months old, weighed 27 ± 2 g, and were obtained from Center for Laboratory Animal Sciences, Southern Medical University (Guangdong, China). In this experiment, a total of 50 mice were used. Animals were housed five per cage under conditions free of specific pathogens at a temperature of 25 ± 1 °C and relative humidity of 55%–60% and exposed to a daily cycle of 12 h light/dark (07:00 on and 19:00 off). A normal solid diet and water were provided ad libitum, and animals were allowed free access to food and water.

The mice were randomly divided into five groups consisting of 10 animals each: control, model, and three LSPC groups. Except for the control group, the mice were subcutaneously injected with D-galactose at a dose of 150 mg/kg body weight once daily for 6 weeks, whereas mice in the control group were treated with the same volume of physiological saline. At the same time, LSPC groups' mice were provided with LSPC dissolved in physiological saline at three doses of 30, 60, and 90 mg/kg body weight respectively by oral gavage after injection of D-galactose. Mice in the control and model groups were administered with same volume of physiological saline. 2.4. Behavioral tests 2.4.1. Y-maze test To evaluate the learning and memory performances of the mice, a Ymaze (MG-2, manufactured by Zhangjiagang biomedical instrument plant of Jiangsu province, China) test was performed at the end of the treatment. The procedure used was a modified version of Heyser's method (Heyser et al., 1999). The mice were put into the Y-maze separately. The test was performed in a sound-isolated and dark room. The Y-maze apparatus consisted of three identical arms (three compartments of 30 cm × 15 cm × 10 cm with connector 10 cm × 6 cm × 10 cm), connected into a Y shape. There was a lamp at the end of each arm and the bottom of each arm was covered with electric net. However, the lamp of only one of the three arms was turned on. At this moment, there was no electric current at the bottom of this arm, which was considered as a safe area. The lamps in the other two arms were not turned on, and there was electric current (50–70 V) on the base (considered as unsafe area). Safe and unsafe areas were alternated randomly. At the beginning of the experiment, no lamp was turned on. Each mouse was given 2–3 min to be acclimatized to the circumstances and subsequently subjected to electric shock with the same intensity and duration. Then, one of the three lamps was switched and the mouse was placed in this safe area, where the mouse adapted to the condition for 10 s. Afterward, the other lamp was switched to force the mouse to run to the safe area. Such a trial process was continued. During the experiment, if the mouse ran to the safe arm directly, it was a correct response. Otherwise, it was an incorrect response. Nine correct responses out of ten consecutive trials were considered as learning criterion. Trial continued until each mouse reached the learning criterion. The total number of trials needed to reach the criterion was recorded to show the learning ability of the mouse. The fewer the total number, the stronger the learning ability of the mouse. After 24 h, the electric shock test was repeated 10 times for each mouse, and the incorrect responses were recorded to reflect the memory ability of the mouse. The fewer the incorrect responses, the stronger the memory ability of the mouse. 2.4.2. Step-down avoidance test The apparatus consisted of five paralleled 12 cm × 12 cm × 30 cm plastic boxes. The floors of these boxes were made of parallel bronze bars. The five boxes were separated by opaque black plastic. A high and diameter of 4.5 cm insulating platform as a safety platform to avoid electric shock for mice was placed at the right rear in each box. In the training session, each mouse was put into the box for adapting to 3 min and then was put on the grid floor with its back against the platform. A continuous electric shock (36 V) was delivered to the grid floor by an isolated stimulator. When the electric shock was delivered, mice escaped from the grid floor back onto the platform. In this session, the number of repeated step-down in 300 s was counted as errors. At 24 h after training, mice were placed on the platform for the retention test. Step-down latency (time of mice stepping from platform to grid floor for the first time) and the number of errors were recorded with improved retention, as reflected by a long latency and a reduction in errors. The electric shocks were still delivered for 300 s.

Y.-S. Gong et al. / Experimental Gerontology 74 (2016) 21–28

2.5. Preparation of whole brain homogenates After behavioral tests, all mice were sacrificed by decapitation. Whole brains were excised immediately, weighed and immersed in ice-cold physiological saline. A 10% homogenate of the whole brains was prepared. The homogenate was centrifuged at 4000 r/min for 10 min at 4 °C to remove cell debris and nuclei. The resulting supernatant was stored at −80 °C for various biochemical assays. 2.6. Biochemical assays MDA, NO, and protein levels, as well as SOD, GSH-Px, NOS, AChE, and MAO-B activities, were determined by using commercially available kits. The level of Aβ1–42 was quantified using a commercial ELISA assay kit. All procedures completely complied with the manufacturer’s instructions. 2.7. Hematoxylin and eosin staining 2.7.1. Tissue preparation After behavioral tests, all mice were sacrificed by decapitation. Hippocampus was excised immediately, fixed by 10% neutral formaldehyde solution and then embedded by paraffin. Slices (5 μm) were cut with a microtome. These free-floating sections were mounted on a clean slide glass, and then toasted at 60 °C for 2 h. Dimethylbenzene handled was followed for 20 min per time. Each slice was treated with ethanol, 95% ethanol, 80% ethanol and distilled water in this order until it became transparent. 2.7.2. Hematoxylin and eosin staining The sections were sequentially treated with hematoxylin stain for 5 min and 10% hydrochloric acid in ethanol differentiating for 30 s and then washed by distilled water for 15 min. Slices were stained by eosin for 2 min followed by dehydration, transparentizing, and mounting in the usual manner. Finally, histological features of apoptotic cells in the hippocampus revealed by hematoxylin and eosin (HE) staining were observed under an optical microscope. 2.8. Immunohistochemistry for P53 2.8.1. Tissue preparation After behavioral tests, all mice were sacrificed by decapitation. Hippocampus was excised immediately, fixed by 4% paraformaldehyde in 0.01 mol/L phosphate buffer, and embedded by paraffin. Slices 5 μm in size were cut with a microtome. Slice was mounted on a clean slide glass, and then toasted at 60 °C for 2 h. Dimethylbenzene treatment followed for 20 min. Slice was treated with ethanol, 95% ethanol, 80% ethanol, and distilled water in this order until the slice became transparent.

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followed by a Tukey's test. Differences among means were determined by the least significance difference test with significance defined at P b 0.05. 3. Results 3.1. Effect of LSPC on body weight ANOVA indicated that no significant changes were found for body weight among all the groups throughout the whole experiment (Fig. 1). Therefore LSPC had no effect on the changes of mice body weight, and all mice grew normally. 3.2. Behavioral effect of LSPC 3.2.1. Y-maze test ANOVA indicated that certain doses of oral LSPC significantly improved learning and memory abilities of D-galactose-treated mice for Y-maze (Table 1). Trials to reach criterion and incorrect responses after 24 h of D-galactose-treated mice were significantly more than those of control group mice (P b 0.01). Mice that received LSPC (30, 60, and 90 mg/kg) took significantly fewer trials to reach criterion than those that only received D-galactose (P b 0.05, P b 0.01 and P b 0.01, respectively), and had a dose dependent manner. Mice that had received LSPC at the doses of 60 and 90 mg/kg significantly took the least incorrect responses after 24 h (P b 0.01 and P b 0.01, respectively). Mice that received LSPC at 90 mg/kg took the least number of trials and the least number of incorrect responses after 24 h among the three groups that received LSPC. 3.2.2. Step-down avoidance test The results showed that some doses of oral LSPC significantly ameliorated cognitive ability of D-galactose-treated mice for step-down avoidance test (Table 2). The model group mice had more error times and shorter latency for retention than those of control group mice (P b 0.05, P b 0.01, and P b 0.05, respectively). LSPC administration at 60 and 90 mg/kg contributed to decreased number of mistakes (P b 0.01) during the training session. In the retention session, 90 mg/kg LSPC group mice showed longer latency and less mistakes compared with the model group mice (P b 0.05). Mice treated with 60 mg/kg LSPC only showed fewer mistakes than the model group mice (P b 0.05).

2.8.2. SABC immunohistochemistry The sections were placed in 0.3% hydrogen peroxide (H2O2) in PBS for 30 min, then pre-incubated with normal goat serum to prevent nonspecific binding at 4 °C for 30 min and incubated with a primary polyclonal rabbit antibody against rat P53 at 37 °C for 1 h or overnight at 4 °C. After washing thrice for 10 min with PBS, the sections were incubated sequentially, in goat anti-mouse or anti-rabbit IgG followed by avidin, biotin horseradish peroxidase solution. Between incubations, the tissues were washed with PBS 3 times for 10 min each. The sections were visualized using DAB and mounted on gelatin-coated slides. Immunoreactions were observed under an optical microscope. 2.9. Statistics Statistical analysis was performed using Origin Pro 7.5. All data in the text were expressed as mean ± S.D. and analyzed by one-way ANOVA

Fig. 1. Effect of LSPC on body weight of D-galactose-treated mice. Values are expressed as mean ± SD. n = 10.

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Table 1 Effect of LSPC on learning and memory abilities of D-galactose-treated mice in Y-maze test.## Groups

Mean trials to criterion

Mean incorrect responses after 24 h

Control group Model group LSPC 30 mg/kg LSPC 60 mg/kg LSPC 90 mg/kg

14.0 ± 2.8 21.1 ± 4.0## 16.9 ± 2.9⁎ 15.8 ± 3.2⁎⁎ 14.7 ± 2.8⁎⁎

1.2 ± 1.2 5.3 ± 2.0## 3.5 ± 1.4 3.2 ± 1.7⁎⁎ 1.8 ± 1.1⁎⁎

Values are expressed as mean ± SD. n = 10. ## P b 0.01 compared with the control group. ⁎⁎ P b 0.01 compared with the model group. ⁎ P b 0.05 compared with the model group.

3.3. Effect of LSPC on Aβ1–42 level in brain ANOVA indicated that LSPC could significantly reduce Aβ1–42 content in the brain of D-galactose-treated aging mice, and a certain relationship with dose existed (Fig. 2). Aβ1–42 level from model group mice was significantly higher than that of control group mice (P b 0.01). Three doses of LSPC administration reduced Aβ1–42 level, whereas the 60 and 90 mg/kg LSPC groups exhibited significantly lower level when compared with the model group (P b 0.05 and P b 0.01, respectively). 3.4. Effects of LSPC on AChE and MAO-B activities in whole brain ANOVA indicated that LSPC could significantly lower AChE and MAO-B activities in whole brain of aging mice induced by D-galactose (Table 3). AChE and MAO-B activities from model group mice were significantly higher than that of control group mice (P b 0.01 and P b 0.01, respectively). Groups treated with 60 and 90 mg/kg LSPC exhibited less AChE activity compared with model group (P b 0.05 and P b 0.01, respectively). LSPC administration at the dose of 30, 60, and 90 mg/kg made the MAO-B activity in brain significantly reduced (P b 0.01, P b 0.01, and P b 0.01, respectively). In addition, MAO-B activity in mice receiving 90 mg/kg LSPC was less than that in control group mice. 3.5. Effect of LSPC on MDA content and SOD and GPx activities in brain Activities of SOD, GPx were increased and MDA content was reduced in the brain homogenates from D-galactose-treated mice after supplementation with LSPC (Table 4). MDA concentration increased significantly in D-galactose-treated mice brain when compared with control group mice (P b 0.01). SOD and GSH-Px activities were found to be significantly decreased in the model group when compared with the control group (P b 0.01 and P b 0.01, respectively). Administration of LSPC at the dose of 30, 60, and 90 mg/kg all decreased MDA content when compared with the model group, but only 90 mg/kg LSPC exhibited significant (P b 0.01). A significant increase of SOD activity was observed in LSPC-administered (60 and 90 mg/kg) groups when compared with

Fig. 2. Effect of LSPC on Aβ1–42 level in the brain of D-galactose-treated mice. Values are expressed as mean ± SD. n = 10. ##P b 0.01 compared with the control group; ⁎⁎P b 0.01, ⁎P b 0.05 compared with the model group.

the model group (P b 0.01 and P b 0.05, respectively). As to GSH-Px activity, it could be significantly elevated by 30, 60, and 90 mg/kg dose of LSPC (P b 0.01, P b 0.01, and P b 0.01, respectively). 3.6. Effect of LSPC on NO level and NOS activity in brain ANOVA indicated that NO level and NOS activity in D-galactosetreated mice brain could be significantly modified by chronic oral LSPC administration (Table 5). Marked increase in the activities of tNOS and inducible nitric oxide synthase (iNOS) was observed in D-galactosetreated mice brain when compared with control group mice (P b 0.01 and P b 0.05, respectively). Supplementation of LSPC (60 and 90 mg/kg) significantly reduced the tNOS activity when compared with the model group (P b 0.05 and P b 0.01, respectively). Three doses of LSPC treatment could reduce nNOS activity in the brain of D-galactose-treated mice, but only the effect of 90 mg/kg LSPC administration was significant (P b 0.01). As for iNOS activity when compared with the model group, LSPC could not significantly lower its activity. NO level in D-galactose-treated mice brain was significantly higher than in the control group mice brain (P b 0.01). LSPC administration at doses of 30 and 90 mg/kg significantly reduced the NO level (P b 0.01 and P b 0.01, respectively). 3.7. Histopathological observation of the neurons in the hippocampus HE staining features of cell loss in the hippocampus are shown in Fig. 3. Mice in the control group had full hippocampus neurons, which were arranged tightly and morphologically intact. Pyramidal neurons presented round and large nuclei and clear nucleoli (Fig. 3A).

Table 2 Effect of LSPC on learning and memory abilities of D-galactose-treated mice in step-down test.## Groups

Mistakes for training

Mistakes for retention

Latency for retention (s)

Control group Model group LSPC 30 mg/kg LSPC 60 mg/kg LSPC 90 mg/kg

3.2 ± 1.1 5.0 ± 1.7# 3.5 ± 1.1 3.0 ± 0.8⁎⁎ 2.9 ± 1.2⁎⁎

1.3 ± 0.8 3.3 ± 1.5## 2.0 ± 1.1 1.7 ± 1.2⁎ 1.7 ± 1.0⁎

257.2 ± 39.0 170.9 ± 71.7# 222.3 ± 47.8 244.2 ± 68.1 259.1 ± 56.8⁎

Values are expressed as mean ± SD. n = 10. ## P b 0.01 compared with the control group. # P b 0.05 compared with the control group. ⁎⁎ P b 0.01 compared with the model group. ⁎ P b 0.05 compared with the model group.

Y.-S. Gong et al. / Experimental Gerontology 74 (2016) 21–28 Table 3 Effect of LSPC on AChE and MAO-B activities in the brain of D-galactose-treated mice.## Groups

AChE (U/mg prot)

MAO-B (U/mg prot)

Control group Model group LSPC 30 mg/kg LSPC 60 mg/kg LSPC 90 mg/kg

0.598 ± 0.055 1.052 ± 0.160## 0.951 ± 0.062 0.890 ± 0.090⁎ 0.743 ± 0.069⁎⁎

11.37 ± 1.15 15.22 ± 1.64## 12.72 ± 1.13⁎⁎ 11.57 ± 0.49⁎⁎ 9.68 ± 0.28⁎⁎

Values are expressed as mean ± SD. n = 10. ## P b 0.01 compared with the control group. ⁎⁎ P b 0.01 compared with the model group. ⁎ P b 0.05 compared with the model group.

Widespread damage was visible in the hippocampus of model group mice treated with D-galactose (Fig. 3B). Intercellular space increased, and cells were loosely arranged. Pyramidal neurons either presented a densely stained shrunken appearance with minimal cytoplasm or had disappeared. Compared with that of model group (Fig. 3B), the majority of the neurons were rescued in the hippocampus of mice treated with three different doses of LSPC (30, 60, and 90 mg/kg) (Fig. 3C–E). Neurons in the hippocampus of mice treated with 60 and 90 mg/kg LSPC almost appeared normal (Fig. 3D and E). 3.8. P53 immunohistochemistry in the hippocampus Note: A control group; B model group; C 30 mg/kg LSPC group; D 60 mg/kg LSPC group; E 90 mg/kg LSPC group. After immunohistochemical staining, tan or brown particles could be seen in the positive neurons where P53 protein was expressed (Fig. 4). Few P53-positive neurons were found in the control group (Fig. 4A). A great quantity of P53-positive neurons was observed in the hippocampus of senescent mice induced by D-galactose, thereby indicating that P53 protein was expressed in model mice brain (Fig. 4B). In contrast, administration of LSPC at three different doses (30, 60, and 90 mg/kg) markedly reduced the number of P53-positive neurons in the hippocampus (Fig. 4C–E). 4. Discussion This D-galactose-mimetic aging study suggests that neuroprotective effects of LSPC are achieved at least partly by improving learning and memory functions, attenuating oxidative damage, inhibiting Aβ1–42 overproduction, enhancing the function of acetylcholine system and monoaminergic system, and reducing histological abnormalities and cell loss of hippocampus neurons. These results imply that LSPC may be used in the therapy or nutritional intervention of aging and agingassociated diseases like AD. In this study, significant differences between the control and Dgalactose-treated mice were observed in a Y-maze task and stepdown avoidance test, thereby indicating that chronic administration of D -galactose impaired cognitive function of mice. The data also

Table 4 Effect of LSPC on MDA content, SOD and GPX activities in the brain of D-galactose-treated mice.## Groups

MDA (nmol/mg prot)

SOD (U/mg prot)

GSH-Px (U/mg prot)

Control group Model group LSPC 30 mg/kg LSPC 60 mg/kg LSPC 90 mg/kg

1.10 ± 0.06 1.71 ± 0.09## 1.54 ± 0.14 1.51 ± 0.18 1.25 ± 0.13⁎⁎

113.43 ± 6.76 83.58 ± 6.57## 97.61 ± 6.89 104.64 ± 8.81⁎⁎ 103.74 ± 12.27⁎

63.07 ± 2.28 27.29 ± 1.89## 52.98 ± 1.20⁎⁎ 54.99 ± 5.68⁎⁎ 49.14 ± 4.37⁎⁎

Values are expressed as mean ± SD. n = 10. ## P b 0.01 compared with the control group. ⁎⁎ P b 0.01 compared with the model group. ⁎ P b 0.05 compared with the model group.

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indicated that LSPC supplement was capable of reversing the behavioral impairment induced by D-galactose. Oxidative stress is considered as an implicating factor in the genesis of central nervous system disorders during aging (Poon et al., 2004; Banjia et al., 2013). Reactive oxygen species (ROS), its intermediate products (MDA and 4-hydroxynonenal) and reactive nitrogen species (RNS) play a pivotal role in facilitating the onset of neurodegenerative disorders in aging by oxidizing macro-molecules like proteins, DNA, and lipids (Banjia et al., 2013). Oxidative damage to proteins, DNA, and lipids is responsible for cellular dysfunction and age-associated functional deterioration in many organs, such as deterioration of learning and memory abilities in the brain (Hayakawa et al., 1992; Stadtman, 1992; Varadarajan et al., 2000). Under normal physiological conditions, NO is a biological unconventional messenger in the brain and plays crucial roles in some physiological processes, such as neuromodulation, neurotransmission, and synaptic plasticity (Clement et al., 2003; Cahuana et al., 2004; Gow et al., 2004). However, under the pathology conditions, excessive NO has neurotoxicity and induces cell apoptosis as well as sleep deprivation. In addition, NO can be implicated in the pathogenesis of several degenerative neurologic disorders that occur with age according to its inflammation and free radical properties. These disorders include Parkinson's disease and AD (Liu et al., 2002; Colas et al., 2005, 2006). Excessive production of NO, as a consequence of NOS induction in activated glia, has been attributed to participate in neurodegeneration, impairing the brain function such as cognitive ability and damaging the structure of neurons culminating in various brain pathologies (Calabrese et al., 2000; Liu et al., 2002). There are numerous endogenous antioxidants (SOD, GSH, GSH-Px, vitamin E, and vitamin C) that constitute the body's own natural defense systems and keep a certain balance with ROS and RNS for limiting free radical damage in neuronal tissues (Ogawa, 1994). The increased oxidative damage during aging might be due to the insufficiency of antioxidants (Reiter, 1995). This study found that LSPC could reduce MDA and NO levels and tNOS and nNOS activities, which were up-regulated by D-galactose. At the same time, LSPC could enhance antioxidant activities, such as the activities of SOD and GSH-Px. Thus, LSPC had anti-aging effect and may be a candidate for treating aging-related diseases. AD is a progressive neurodegenerative disorder and the leading cause of dementia (Querfurth and Laferla, 2010). It is characterized by memory loss and learning loss (Spires and Hyman, 2005). The senile plaques found in the brain of AD patients are composed primarily of β-amyloid (Aβ) peptide. According to the amyloid hypothesis, Aβ plays a critical pathogenic role in AD as it initiates a deleterious cascade in brain and ultimately leads to cognitive impairment (Jiang et al., 2014). Aβ is a protein produced by the proteolytic processing of the amyloid precursor protein by β and γ secretases, releasing fragments having 40 amino acids to 42 amino acids in length (Muñoz et al., 2015). In early stages of AD, changes in behavior and progressive loss of memory become evident (Wuwongse et al., 2013), possibly because the overproduction of Aβ leads to Aβ-associated free-radical production and cell death (Satheesh and Nisha, 2014). It has been documented that Aβ deposition in brain tissue causes the generation of free radicals, which in turn results in Aβ aggregation and fibrosis consequently leads to neuronal cells apoptosis (Varadarajan et al., 2000; Butterfield and Lauderback, 2002; Butterfield, 2003). The development of new drugs for treatment of AD has now been largely focused on compounds that promote clearance of Aβ (Jiang et al., 2014). D-galactose can reportedly induce agingrelated and/or AD-like pathological changes, including the increase of ROS and the decrease of antioxidant enzyme activity in the brain (Luo et al., 2009; Hsieh et al., 2009). In our present study, data indicated that D-galactose treatment increased Aβ1–42 level in the brain. LSPC could suppress Aβ1–42 overproduction and alleviate its neurotoxicity. AChE and MAO-B are respectively relevant enzymes to acetylcholine and monoaminergic neurotransmitters, which are related to learning and memory (Schetinger et al., 1999). AChE is an important regulatory enzyme that controls the transmission of nerve impulses

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Table 5 Effect of LSPC on NO level and NOS activity in the brain of D-galactose-treated mice.## Groups

tNOS (U/mg prot)

iNOS (U/mg prot)

nNOS (U/mg prot)

NO (μmol/g prot)

Control group Model group LSPC 30 mg/kg LSPC 60 mg/kg LSPC 90 mg/kg

1.196 ± 0.044 1.545 ± 0.131## 1.388 ± 0.085 1.341 ± 0.098⁎ 1.146 ± 0.123⁎⁎

0.645 ± 0.030 0.858 ± 0.094# 0.890 ± 0.153 0.847 ± 0.068 0.889 ± 0.097

0.551 ± 0.062 0.686 ± 0.175 0.498 ± 0.162 0.494 ± 0.151 0.257 ± 0.160**

0.358 ± 0.105 0.633 ± 0.116## 0.389 ± 0.105⁎⁎ 0.490 ± 0.071 0.348 ± 0.081⁎⁎

Values are expressed as mean ± SD. n = 10. ## P b 0.01 compared with the control group. # P b 0.05 compared with the control group. ⁎⁎ P b 0.01 compared with the model group. ⁎ P b 0.05 compared with the model group.

across cholinergic synapses and its activity is considered a good indicator of cholinergic activity (Kaizer et al., 2004). AChE hydrolyzes acetylcholine to choline and acetate (Kaizer et al., 2004). Increased AChE was associated with amyloid plaques in AD brain (Chauhan and Siegel, 2007). MAO is an important enzyme in the catabolism of a wide range of monoamine neurotransmitters, including noradrenaline, dopamine, and 5-hydroxytryptamine. MAO exists in two forms, A and B (Yu et al., 2002). Most studies have reported no change or a decrease of MAO-A, along with an increase of MAO-B in the aging brain (Fowler, 1980; Chen et al., 2007). In the present study, increased AChE and MAO-B activities were found in the

brain of mice afflicted with senescence induced by D -galactose, whereas LSPC could lower the activities of AChE and MAO-B. Thus, LSPC's neuroprotective effects were achieved by modulating the activities of enzymes related to neurotransmitter metabolism and by normalizing the function of cognitive system. P53 is an important regulatory gene for promoting apoptosis. It belongs to the receptor family of tumor necrosis factor/nerve growth factor. Expression of P53 protein transmits apoptotic signals. When the DNA of cell is damaged, P53 protein prevents DNA replication, to provide enough time for DNA repair. If DNA repair is unsuccessful, P53 protein leads to apoptosis and cell cycle arrests (Bennett et al., 1998;

Fig. 3. Protective effect of LSPC on cell loss in the hippocampus of D-galactose-treated mice revealed by hematoxylin and eosin (HE) staining (×200). Note: A control group; B model group; C 30 mg/kg LSPC group; D 60 mg/kg LSPC group; E 90 mg/kg LSPC group.

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Fig. 4. Effect of LSPC on P53 expression in the hippocampus of D-galactose-treated mice by immunohistochemical staining (×200).

Culmsee et al., 2003; Leker et al., 2004). The results showed that hippocampus neurons of aging mice induced by D-galactose were less, and abnormal cells appeared in the hippocampus. Even some neurons presented apoptosis features. P53 protein promoting apoptosis was expressed in the brain of D-galactose-treated mice. LSPC could inhibit nerve cell apoptosis and reduce the expression of P53 protein, even reaching normal level. This further offered the evidence from the perspective of pathology that LSPC improved learning and memory injury of aging mice induced by D-galactose. 5. Strengths and limitations LSPCs have been isolated for the first time and characterized in our laboratory, and lotus seedpod is likely to be another important source of proanthocyanidins besides grape seed. The effects of LSPC on cognitive deficits and brain aging induced by D-galactose, as well as the mechanism underlying such effects, have been first established in this research. Clear findings regarding the effect of LSPC on many factors related to brain aging and function have been obtained. LSPCs are capable of reversing oxidative damage and preventing Aβ overproduction, which play a critical role in the pathogenesis of AD. This result will be helpful for anti-aging and AD research. Furthermore, LSPC can reduce the NO level, which is related to inflammation and sleep–wake cycle (Clement et al., 2003; GautierSauvigne et al., 2005; Kumar and Singh, 2008) in the brain of senescent mice. This finding suggests that LSPC can ameliorate the learning and memory abilities probably by improving sleep, which is a potentially

important mechanism that needs attention. However, this conclusion needs research support. More studies on Aβ aggregation and distribution in different area of hippocampus need to be carried out.

Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (NSFC, No. 31071633).

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