Reversible effects of vitamins C and E combination on cognitive deficits and oxidative stress in the hippocampus of melamine-exposed rats

Pharmacology, Biochemistry and Behavior 132 (2015) 152–159

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Reversible effects of vitamins C and E combination on cognitive deficits and oxidative stress in the hippocampus of melamine-exposed rats Lei An a,b, Jingxuan Fu a, Tao Zhang a,⁎ a b

College of Life Sciences and Key Laboratory of Bioactive Materials Ministry of Education, Nankai University, Tianjin 300071, PR China Max-Planck Institute for Neurological Research, Cologne 50931, Germany

a r t i c l e

i n f o

Article history: Received 31 December 2014 Received in revised form 9 March 2015 Accepted 11 March 2015 Available online 20 March 2015 Keywords: Melamine Oxidative damage Rats Spatial cognition Vitamin

a b s t r a c t Previous studies showed that the spatial cognitive deficits of rats were induced by chronic melamine exposure, which was associated with the hippocampal oxidative damage. Currently, we examined the antioxidative effect of vitamins C and E combination on cognitive function in melamine-treated rats. Melamine was oral administrated to male adolescent Wistar at a dosage of 300 mg/kg/day for 28 days. After that, animals received vitamins C and E at a dose of 150 and 200 mg/kg, respectively, intraperitoneally for the next 7 days. Cognitive behaviors were investigated using the Morris water maze test. The biochemical indexes were detected in the hippocampal homogenate. The treatment with vitamin complex significantly ameliorated cognitive deficits induced by melamine. ROS, MDA, and NO contents were almost back to normal, while SOD, CAT, GSH-Px, and NOS activities were improved as well. The neural apoptosis in the hippocampus were ameliorated by regulating the expression of anti-apoptotic protein (Bcl-2) and caspase-3. Additionally, histological observation showed that vitamin complex effectively alleviated the injuries of hippocampal neurons. These results suggest that the potential therapeutic for oxidative damage induced neuronal apoptosis after treatment of vitamins C and E combination, which is most likely related to the antioxidative effects. © 2015 Elsevier Inc. All rights reserved.

1. Introduction An outbreak of nephrolithiasis and kidney damage among children in China has been closely connected to the ingestion of milk contaminated with melamine, and the nephrotoxic effects of melaminetainted foodstuffs were considered an international public health crisis (Yoon et al., 2011). The toxicity of melamine was very low, and more than 90% of the ingested melamine is expelled within 24 h in an animal experiment (Baynes et al., 2008; Brown et al., 2007). However, chronic exposure on the melamine was able to cause cancer or reproductive damage, eye, skin, and respiratory irritant (Mast et al., 1983; Yoon et al., 2011). Recently, our previous studies revealed that the hippocampal pyramidal neurons seemed to be particularly sensitive to melamine (Yang et al., 2010). Although recent literature found that the intravenous administration melamine could be distributed in rats' brain regions, such as cortex, striatum, hippocampus, cerebellum, and brain stem except in the plasma, liver, kidney, spleen, and bladder (Wu et al., 2009), the specific effect and neurotoxic mechanism of melamine on the central nervous system (CNS) still needs to be explored. Chronic melamine exposure was found to induce a decline in cognitive performance, especially spatial learning and memory deficits (An et al., 2011). In mammals, they are largely dependent ⁎ Corresponding author. Tel.: +86 22 23500237. E-mail address: [email protected] (T. Zhang).

http://dx.doi.org/10.1016/j.pbb.2015.03.009 0091-3057/© 2015 Elsevier Inc. All rights reserved.

on the hippocampus (D'Hooge and De Deyn, 2001; Morris et al., 1982), which is a major area of the brain impaired by hypobaric hypoxia at high altitude due to low partial pressure of oxygen (Roach and Hackett, 2001). However, the mechanism underlying the impairment of these cognitive functions is still under investigation (An et al., 2012a; Yang et al., 2011). A substantial body of evidence suggested that oxidative stress and apoptosis were major causative factors for neuronal degeneration and death, which might be involved in most melamine-induced neuropathology (An et al., 2012a; Wang et al., 2011). Despite the severe experimental significance of these cognitive dysfunction (An et al., 2011, 2012a, 2013b; Yang et al., 2011), effective therapeutic approaches have not yet been developed, and exploration of prospective pharmacological agents is imperative. Our previous study found that vitamin C and vitamin E, alone or in combination, were able to protect PC12 cells from the injury induced by melamine through the down-regulation of oxidative stress and prevention of melamine-induced apoptosis, with a predominant effect of vitamin complex (An et al., 2014). Several other investigations showed that a combination of vitamins E and C reduced LPO caused by oxidative stress (Gultekin et al., 2001) and lessened oxidative damage both at biochemical and histological levels (Guney et al., 2007; Ornoy et al., 2009). It was likely that vitamins C and E acted in a synergistic manner, by vitamin E primarily being oxidized to the tocopheroxyl radical and then reduced back to tocopherol by vitamin C (Aydemir et al., 2004). The developing brain, which has only a fraction of the antioxidant

L. An et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 152–159

enzyme activity of the adult brain, is perhaps even more vulnerable to the neurotoxic effects of oxidative stress than the adult brain (Henderson et al., 1999). In addition, certain regions of the CNS, such as the hippocampus and cerebellum, are particularly sensitive to oxidative stress because of their low endogenous levels of biochemical antioxidant, relative to other brain regions (Abel and Hannigan, 1995). Such a depressed defense system may be adequate under normal circumstances. However, in prooxidative conditions, such as during alcohol and nicotine exposure, these low antioxidant defenses could predispose the fetal brain to oxidative damage and induced cognitive impairment (Augustyniak et al., 2005; Hritcu et al., 2009). More importantly, a previous in vivo study from our lab revealed that vitamin complex could enhance synaptic efficacy in melamine-treated rats (An and Zhang, 2014b). Accordingly, a hypothesis was raised that antioxidants could reduce cognitive deficits and oxidative damage induced by melamine. Therefore, in the present study, we tried to apply the combination of lipophilic antioxidant vitamin E and the hydrophilic antioxidant vitamin C in an adolescent rat model treated with melamine and examined melamine-induced free radical production, lipid peroxidation, antioxidative enzymes, and apoptotic related proteins in the hippocampus. In addition, the rat's spatial cognition and histological alterations were evaluated as well. 2. Materials and methods 2.1. Reagents Melamine (purity N 99.5%) was purchased from Yingda Sparseness and Nobel Reagent Chemical Factory, Tianjin, PR China. Before the oral administration, the fresh solution was prepared by diluting stock solution to final dose with sterile endotoxin-free 1% carboxymethylcellulose (CMC) and was administered gavage. The stock solution was stored in 4 °C. Superoxide anion radical, hydroxyl free radical, superoxide dismutase (SOD), and malondialdehyde (MDA) assay kits were purchased from the Nanking Jiancheng Bioengineering Research Institute (Nanking, China). Catalase (CAT), Glutathione peroxidase (GSH-Px), nitric oxide (NO), nitric oxide synthase (NOS), Bcl-2, and caspase-3 ELISA kits were purchased from R&D systems (USA). Other reagents were of A.R. grade. 2.2. Animals and treatment All experiments were performed according to the protocols approved by the Committee for Animal Care at the University of Nankai and in accordance with the practices outlined in the NIH Guide for the Care and Use of Laboratory Animals. Healthy male Wistar rats, 3 weeks old, were obtained from the Laboratory Animal Center, Academy of Military Medical Science of People's Liberation Army, and reared in the animal house of Medical School in the Nankai University. Rats were paired in transparent plastic cages in a temperature-controlled (21 °C) colony room on a 12/12-h light/dark cycle with standard rodent food (Beijing Huafukang Biotechnology Co. Ltd., Beijing, China) and water available ad libitum. Animals were randomly assigned into four experimental groups (n = 8 per group). (1) Melamine-vitamin (MV) group: oral received melamine (30 mg/mL; dissolved in 1% CMC) at a dose of 300 mg/kg/day for 28 consecutive days and then intraperitoneally received vitamins C and E at a dose of 150 and 200 mg/kg, respectively, dissolved in sterile endotoxin-free isotonic saline, once a day for the next 7 consecutive days. (2) Melamine (M) group: oral received melamine solution for 28 consecutive days and then injected saline intraperitoneally for 1 week. (3) Sham (S) group: oral received the same dose and concentration of CMC solution for 28 consecutive days and then injected vitamins intraperitoneally for 1 week. (4) Control (C) group: oral received CMC solution for 28 consecutive days and then injected saline intraperitoneally for 1 week. All the parameters, including the dose, route, and period of melamine administration, were chosen from our previous experiments due

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to its significant effects on the cognitive function (An et al., 2011, 2013a, 2013b) and oxidative stress in the hippocampus (An et al., 2012a, 2012b; An and Zhang, 2014a, 2014b). The dose, route, and period of vitamin combination were referred from our previous study, which showed the reversible effect of vitamins on the impairments of synaptic function in melamine-treated rats (An and Zhang, 2014b), and other findings (Gultekin et al., 2001; Karaoz et al., 2002). Parenteral administration of the vitamins (typically intraperitoneal injection), which was more likely to produce cumulative effect after repetitive administration (Butterfield et al., 2002; Kim et al., 2006), was used to investigate the effect of shortterm vitamin treatment as the previous researches (Barros et al., 2007; Baydas et al., 2003; Celik and Ozkaya, 2002; Ozkan et al., 2005; Tuzcu and Baydas, 2006). 2.3. Morris water maze experiment Twenty-four hours after the last treatment, the spatial learning and memory performance of rats was evaluated using the MWM test. The experimental procedure was carried out as our previous methods (An et al., 2013a; An and Zhang, 2014a; Han et al., 2014). The water maze consisted of a circular tank (150 cm diameter, 50 cm height) that was filled with water (25 ± 1 °C). Black ink was used to render the water opaque. The tank was divided into 4 equal quadrants (I, II, III, and IV) and had four points designed as starting positions. There was a 10-cmdiameter platform submerged 2 cm below the water surface in the center of quadrant III. A camera was located above the center of the maze and a computerized animal tracking system (Ethovision 2.0, Noldus, Wagenigen, Netherlands). The task consisted of two stages, which were initial training (IT) and space exploring test (SET). In the IT stage, animals were subjected to a 4-consecutive days training (two sessions per day and each session consisted of four trials) covering day 36 to day 39. A trial began when the rat was placed in the water from one of the four starting positions randomly, facing the wall. The order of the starting position varied but the same for all animals. Animals were allowed to swim freely and trained to find a hidden platform within 60 s. The time required to find the platform (escape latency) and the swimming speed were recorded. If not succeed, rats was gently guided to the platform and left for 10 s, and escape latency was recorded as 60 s. Rats were dried and returned to their home cages after each trial. Interval between two trials was approximately 5 min, and interval between sessions was approximate 8 h each day (~9 a.m. and ~5 p.m.). In the SET stage, animals were given the probe test at 9 am on day 40. The task consisted of a single trial, with the platform removed. Rats were released individually into water from the starting point of quadrant I and allowed to swim for 60 s. Quadrant dwell time (percentage of time spent in target quadrant) and platform crossings (numbers passing platform area) were recorded. 2.4. Preparation of tissue samples After behavioral test, the rats were decapitated. The brain was longitudinally bisected along the axes, removed, and weighted as previously described (An et al., 2012b; An and Zhang, 2013). The hippocampus of left hemisphere was dissected out and rinsed in 0.1 M phosphate buffer (pH 7.4). After being weighted, it was homogenized with ice-cold saline to be 10% (w/v) homogenates. The mixtures were homogenized using a glass homogenizer for 5 min and centrifuged at 300 ×g at 4 °C for 15 min. The supernatant was collected and stored at − 70 °C for biochemical assay. 2.5. HE staining The hippocampus of right hemisphere was removed and immersed in 4% paraformaldehyde fixed at 4 °C for at least 24 h, and then was dehydrated and embedded in paraffin for tissue sectioning. The coronary slices (5 μm) were obtained and used for hematoxylin/eosin (HE)

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staining in accordance with the standard procedure. They were observed viewed on a Leica microscope (Wetzlar, Germany) and photographed. Three sections per brain were used for analyzing quantitatively. Histologic changes were scored in a masked fashion from 0 to 3 based on the degree of cytoplasmic vacuolization and neuronal necrosis as described by previous researches (Suzuki et al., 1993; Takeda et al., 2003). 2.6. Measurement of oxidative parameters in hippocampus The levels of superoxide anion radical, hydroxyl free radical, MDA, TSOD, CAT, GSH-Px, NO, NOS, Bcl-2, caspase-3, and the protein in hippocampus were determined according to the methods described in the references using commercial kits. The protein levels of samples were measured by the Coomassie brilliant blue G-250 method with bovine serum albumin as standard. 2.7. Statistical analysis All data are presented as mean ± SEM. Two-way repeated measures ANOVA were applied for measuring the differences between four groups during the IT stage, followed by Bonferroni post hoc test. Oneway ANOVAs were performed on the data from the IT and SET stages of the MWM test, body weight gain, HE staining, and biochemical tests, followed by Bonferroni post hoc test. Statistical differences were taken when P b 0.05. The analyses were performed using SPSS 16.0 software. 3. Results 3.1. The effects of melamine and vitamins on body weight One-way ANOVA showed that there were statistical differences of group in the body weight gain after a 4-week melamine exposure (F(3, 28) = 22.81, P b 0.001) and a 1-week vitamin treatment (F(3, 28) = 11.52, P b 0.001). Bonferroni post hoc test showed the body weight gain of the M group was significant lower than the C group (P b 0.05; Table 1), which persisted after 7 days (P b 0.05). Similarly, rats of the MV group gained less weight than those of the C group before vitamin treatment (P b 0.05) and after 1 week vitamin treatment (P b 0.05). There is no significant difference between the M group and the MV group before or after vitamin supplementation. Additionally, no change in the body weight was found between the C group and the S group throughout the experiment. 3.2. MWM experiment results As shown in Fig. 1A, the mean escape latencies were decreased progressively during the four training days. Two-way repeated measures ANOVA showed that there were statistical differences of session (F(7, 224) = 133.18, P b 0.001), session × group interaction Table 1 Measurements of rats' bodyweight in 4 groups. Group

C S M MV

3.3. Histopathological observation As shown in Fig. 2A, the neurons were full and arrange tightly and the nuclei were light-stained in the C group. However, there was no distinctly difference between the S and the C groups (Fig. 2B). It is clearly visible that there are more degenerated neurons in the M group (Fig. 2C). It can be seen that the state in the MV group is that between the C group and the M group (Fig. 2D). One-way ANOVA showed that statistical differences of group in both cytoplasmic vacuolization (F(3, 16) = 18.53, P b 0.001) and (F(3, 16) = 27.05, P b 0.001). Quantitative analysis of brain slices from the M group showed a remarkable neuronal necrosis increase in the number of cytoplasmic vacuolization (P b 0.01; Fig. 2E) and dying neurons (P b 0.01; Fig. 2 F) in the hippocampal CA1 region compared to the C group. Treatment with vitamins significantly attenuated the melamine-induced cytoplasmic vacuolization (P b 0.01) and dying neuronal cells (P b 0.05) compared to the M group. However, significant difference in neuronal necrosis was also found between the MV group and the C group (P b 0.05). There was no statistical difference between the M group and the S group. 3.4. Combination of vitamins C and E reduced hippocampal ROS and MDA

ΔW

Weight (g)

(F(21 , 224) = 8.69, P b 0.001), and group (F(3, 224) = 16.85, P b 0.001). One-way ANOVA showed that there were significant differences on session 3 (F(3, 28) = 7.13, P b 0.01), session 4 (F(3, 28) = 21.61, P b 0.001), session 5 (F(3, 28) = 11.35, P b 0.01), session 6 (F(3, 28) = 25.79, P b 0.001), session 7 (F(3, 28) = 35.05, P b 0.001), and session 8 (F(3, 28) = 27.21, P b 0.001). Bonferroni test revealed that melamine-treated rats spent significantly longer time to find the platform than rats in the C group (session 3 and 5, P b 0.05; session 4, 6, 7, and 8, P b 0.01). Fig. 1B illustrated the swim paths of rats in example trials of the fourth and seventh sessions. Animals tended to explore all four quadrants of the pool in the fourth session. Thereafter, they changed the search strategy. On the seventh session, rats in the C group swam in the direction of the platform, while melamine-treated rats took longer swimming paths. After injecting vitamin complex intraperitoneally, we found that from the fourth to seventh days the escape latency was significantly shortened in the MV group compared to the M group. They were statistically reduced on session 6, 7, and 8 (session 6 and 8, P b 0.05; session 7, P b 0.01), although there were provisionally significant difference between the MV group and the C group on session 4 (P b 0.05). Meanwhile, there was no statistically difference between the S group and the C group during the IT stage. Oneway ANOVA showed that statistical differences of group in both platform crossing (F(3, 28) = 26.34, P b 0.001) and quadrant dwell time (F(3, 28) = 33.52, P b 0.001) during SET stage. Bonferroni test confirmed that platform crossing was significantly reduced in the C group compared to the M group (P b 0.01; Fig. 1C). The animals in the C group spent about twice of the time in the quadrant where the platform was once placed compared to the M group (P b 0.01; Fig. 1D). Moreover, it was found that rats in the MV group took longer time in the target quadrant than the M group (P b 0.05) and also platform crossings increased significantly (P b 0.01). With respect to swimming speed, each group remained constant throughout the test without significant difference among four groups (data not shown).

W(Day 1st)

W(Day 28th)

W(Day 35th)

ΔW1–28

ΔW28–35

54.4 ± 2.9 57.9 ± 3.1 55.6 ± 3.9 57.3 ± 2.7

224.6 ± 9.9 232.3 ± 7.7 198.5 ± 7.9 196.2 ± 7.7

256.0 ± 11.5 268.5 ± 10.3 217.3 ± 9.9 220.7 ± 12.8

168.8 ± 10.7 171.3 ± 9.6 141.0 ± 11.1a 139.7 ± 10.8b

31.6 ± 8.4 34.3 ± 7.6 20.1 ± 7.3a 23.6 ± 8.7b

W(Day 1st), W(Day 28th), and W(Day 35th) individually represent the mean body weight on the first day of melamine treatment, the last day of both melamine treatment and vitamin treatment. ΔW(1–28) and ΔW(28–35) represent the body weight gain from the 1st day to the 28th day and the 28th to the 35th day, respectively. Data are presented as mean ± SEM. a P b 0.05, the M group vs. the C or the S group (n = 8). b P b 0.05, the M group vs the C or the S group (n = 8).

One-way ANOVA showed that statistical differences of group in both superoxide anion radical (F(3, 28) = 47.58, P b 0.001; Table 2) and hydroxyl free radical (F(3, 28) = 54.27, P b 0.001; Table 2). Bonferroni test showed that there was a significant increase in superoxide anion radical (P b 0.01) and hydroxyl free radical (P b 0.01) levels in the M group compared to the C group. It could be seen that the superoxide anion radical (P b 0.01) and hydroxyl free radical (P b 0.01) in the MV group were significantly reduced compared to the M group. Nevertheless, there was no statistical difference between the S group and the C group.

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Fig. 1. Rats' performance in MWM test. (A) Mean escape latency calculated for each session in four groups in IT stage. (B) Representative swim traces of the fourth and seventh sessions in the C, M, and MV groups. (C) Average number of platform area crossings in spatial probe phase. (D) Quadrant dwell time in SET stage. n = 8 for each group. Data are expressed as mean ± SEM. *P b 0.05; **P b 0.01, significant difference as compared between the M group and the C group. #P b 0.05; ##P b 0.01, significant difference as compared between the M group and the MV group. %P b 0.05, significant difference as compared between the MV group and the C group.

One-way ANOVA showed that statistical differences of group in the MDA content (F(3, 28) = 61.80, P b 0.001; Table 2). Bonferroni test showed the MDA level of melamine-treated group was increased considerably compared to the C group (P b 0.01). Such an increase was reversed by treatment of vitamin combination (P b 0.01), while no obvious difference was found between the S group and the C group (P N 0.05).

vitamins resulted in a significantly elevations of enzyme activities of pathological condition, including SOD (P b 0.05), CAT (P b 0.05), and GSH-Px (P b 0.05). It was found that there was a statistical difference between the MV group and the C group in CAT activity (P b 0.05). In addition, vitamin complex treatment had no side effect on the normal rats.

3.6. Effect of combination of vitamins C and E on NO level and NOS activity 3.5. Effect of combination of vitamins C and E on SOD, CAT, and GSH-Px activities One-way ANOVA showed that statistical differences of group in SOD (F(3, 28) = 14.30, P b 0.001; Table 2), CAT (F(3, 28) = 75.52, P b 0.001; Table 2), and GSH-Px (F(3, 28) = 18.18, P b 0.001; Table 2) activities. Bonferroni test revealed the antioxidant effect of vitamin complex in melamine-treated rats. It was found that SOD was considerably reduced after a 4-week melamine treatment period (P b 0.05), the activities of CAT (P b 0.01) and GSH-Px (P b 0.05) were significantly decreased compared to normal physiological condition. However, the combination of

One-way ANOVA showed that statistical differences of group in NO (F(3, 28) = 58.09, P b 0.001; Table 2) and NOS (F(3, 28) = 44.38, P b 0.001; Table 2) levels. Bonferroni test revealed the total NO levels (P b 0.01) and NOS activities (P b 0.01) were dramatically augmented in the M group compared to the C group. Both the NO levels (P b 0.05) and the NOS activities (P b 0.05) in the M group were remarkably reversed by vitamin complex. Nevertheless, there were still statistical differences of both NO level (P b 0.05) and NOS activity (P b 0.05) between the C group and the MV group. Moreover, no effect induced by vitamin complex supplementation was found in the S group.

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Fig. 2. Effect of vitamins C and E combination on the morphology of rat's hippocampal neurons. (A) C group. (B) S group. (C) M group. (D) MV group. Original magnification 100×. Histologic scores for cytoplasmic vacuolization (E) and neuronal necrosis (F). n = 5 for each group. Data are expressed as mean ± SEM. **P b 0.01, significant difference as compared between the M group and the C group. #P b 0.05; ##P b 0.01, significant difference as compared between the M group and the MV group. %P b 0.05, significant difference as compared between the MV group and the C group.

3.7. Effect of combination of vitamins C and E on Bcl-2 and caspase-3 activities One-way ANOVA showed that statistical differences of group in Bcl-2 (F(3, 28) = 59.52, P b 0.001; Table 2) and caspase-3 (F(3, 28) = 65.81, P b 0.001; Table 2) activities. Bonferroni test showed that the Bcl-2 level was significantly lower (P b 0.01), and caspase-3 activity (P b 0.01) was statistically higher in the M group compared to the C group. Following treatment with vitamin complex, Bcl-2 level was significantly elevated (P b 0.05), while the activity of caspase-3 (P b 0.05) were remarkably reduced in the MV group compared to the M group. In addition, it was found that there were significant differences of both Bcl-2 level (P b 0.05) and caspase-3 activity (P b 0.05) between the MV group and the C group. Meanwhile, combination of vitamins had no effect on the hippocampus of normal rats. 4. Discussion Chronic melamine exposure has well been characterized as a common pathological status contributing to neurodegenerative impairment

(An et al., 2011, 2012a; Han et al., 2011; Wang et al., 2011). In vitro, melamine was able to induce acute neuronal death in hippocampus, particularly apoptotic death of CA1 cells 12 hours later (Han et al., 2011). In vivo, 28 days of exposure to melamine produced essentially neuronal loss and necrosis, with a strong impairment of spatial learning and memory (An et al., 2012a). The above approach was normally accepted for establishing a model of melamine-induced neurotoxicity (An et al., 2011; Yang et al., 2011). Previous studies showed that oxidative injury played a key role in the pathogenesis of numerous neurodegenerative diseases, including stroke, Alzheimer's disease, and Parkinson's disease (Henchcliffe and Beal, 2008; Nunomura et al., 2006). Oxygen free radicals and lipid peroxidation might have an etiological role in the development of lesions induced by chronic hippocampal melamine exposure (An et al., 2012a). Therefore, antioxidant therapy may be an important strategy for managing melamine-induced neurotoxicity. Recently, animal models played a critical role in ongoing efforts of melamine to understand the pathology of CNS impairment (An et al., 2011), and laboratorial experiments were directly done to reveal the underlying neurotoxic mechanism (An et al., 2012a; Yang et al., 2011). It was

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Table 2 Biochemical changes in the hippocampus in four groups.

Superoxide anion radical Hydroxyl free radical MDA T-SOD CAT GSH-Px NO NOS Bcl-2 Caspase-3

C group

S group

M group

MV group

0.302 ± 0.044 1.433 ± 0.120 3.052 ± 0.221 68.99 ± 3.27 1.275 ± 0.116 73.76 ± 3.11 3.439 ± 0.226 6.956 ± 0.427 0.364 ± 0.013 0.347 ± 0.012

0.335 ± 0.023 1.412 ± 0.151 2.981 ± 0.217 68.13 ± 3.54 1.321 ± 0.098 70.27 ± 2.98 3.452 ± 0.179 6.702 ± 0.543 0.372 ± 0.025 0.361 ± 0.018

0.443 ± 0.027aa 1.937 ± 0.171aa 4.130 ± 0.269aa 59.61 ± 3.56a 0.635 ± 0.051aa 60.57 ± 3.33a 5.121 ± 0.328aa 8.911 ± 0.662aa 0.236 ± 0.029aa 0.542 ± 0.041aa

0.341 ± 0.027bb 1.565 ± 0.113bb 3.214 ± 0.215bb 70.75 ± 4.00b 0.982 ± 0.071 b c 76.32 ± 2.87b 4.238 ± 0.316b,c 7.939 ± 0.405b,c 0.303 ± 0.020b,c 0.431 ± 0.029b,c

The data are represented as the mean levels of the ROS in the brain regions of cortex, striatum, and amygdala. All data are presented as mean ± SEM. [Superoxide anion radical (nmol/mg protein); Hydroxyl free radical (nmol/mg protein); MDA level (nmol/mg protein); T-SOD activity (U/mg protein); CAT activity (U/mg protein); GSH-Px activity (U/mg protein); NO level (nmol/mg protein); NOS activity (U/mg protein); Bcl-2 level (ng/mg protein); Caspase-3 activity (pmol/mg protein)]. a P b 0.05, the M group vs the C or the S group (n = 8). aa P b 0.01, the M group vs the C or the S group (n = 8). b P b 0.05, the MV group vs the M group (n = 8). bb P b 0.01, the MV group vs the M group (n = 8). c P b 0.05, the MV group vs the C or the S group (n = 8).

becoming increasingly clear that the hippocampus was the main site of neurotoxicity of melamine and cognitive dysfunctions were listed as one of the many complications, along with nephropathy and neuropathy disease (An et al., 2012a). The alternations in specific markers in NF-kappabeta signaling pathway confirmed the involvement of oxidative stress and inflammation in the development of cognitive impairment (Kuhad et al., 2009; Zhao et al., 2013). The current study demonstrated that combination of vitamins C and E could enhance the learning and memory of melamine-treated rats, which was probably a sum of its antioxidative actions. In the present study, the standardized MWM test was utilized to assess rat's spatial learning and memory, as it was shown to be sensitive to decrements in the hippocampal function (D'Hooge and De Deyn, 2001; Morris et al., 1982). It was found that rats displayed learning and memory deficits in the MWM task after chronic melamine exposure, while the impaired functions were significantly restored by vitamin complex supplementation. Our results showed that the escape latency was significantly decreased and the dwell time and platform crossings were statistically increased in the MV group compared to the M group. Furthermore, it could be seen that animals in the MV group selected a better swim path compared to the M group. In addition, morphological findings showed that neuronal loss, shrunken nucleus, and reduced cytoplasm in the MV group were obviously reduced compared to the M group, which could partly explain the improvement in cognition by vitamin complex supplementation. Consequently, combination of vitamins C and E could ameliorate the pathophysiologic and functional alterations resulting from melamine treatment. These findings are consistent with the clinical effects of vitamin complex that have been reported in cognitive impairment diseases (Chao et al., 2012; Ravaglia et al., 2005). Exposure to melamine was considered as an extreme physiological stress state inducing a wide range of deleterious effects at the cellular level (Yang et al., 2010). Oxidative stress is defined as a disturbance in the balance between the production of ROS and antioxidant defense systems (Penugonda et al., 2006). Excessive ROS levels were known to cause damage to major macromolecules in cells, including lipids, proteins, and nucleic acids (Niebroj-Dobosz et al., 2004), culminating in neuronal dysfunction and depression (Mao et al., 2010). The generation and release of damage ROS could be balanced by many antioxidant defense systems consisting of enzymatic and non-enzymatic compounds (Fan et al., 2009). Within the mitochondrial matrix, SOD regarded as the first line of the antioxidant defense system converts superoxide anion to hydrogen peroxide. It can be further metabolized by GSH-Px and CAT, which are considered as a second defensive line of oxidative stress (Li et al., 2008; Paradies et al., 2011). The MDA is another well-known indicator of lipid peroxidation under oxidative stress (Ercan et al., 2010). In pathological

conditions, the burden of ROS productions might damage cellular components and inhibit their normal functions. Therefore, an increase in activities of antioxidant defense systems was considered to be beneficial in the event of oxidative stress. In accordance with the previous reports, our results showed that an increase of oxidative stress and depletion of endogenous antioxidants occurred in the melamine-treated hippocampus. The increase of the antioxidant defense system by vitamin combination finally leads to quenching of free radicals and reduction in ROS and lipid peroxidation. In addition, it was showed that SOD, CAT, and GSHPx activities, and MDA level were significantly restored after treatment of vitamin complex, except for elimination of ROS caused by melamine. The results suggested that the oxidation–antioxidation homeostasis was regulated by vitamins C and E combination via decreasing ROS and lipid peroxidation as well as increasing antioxidant enzyme activities to ameliorate melamine-induced hippocampal oxidative stress. Several pieces of evidence indicated that the mitochondrial production of ROS might be modulated by NO obtained from mitochondrial NOS, which could be converted to various reactive nitrogen species such as nitroxyl anion or the toxic peroxinitrite (Paradies et al., 2011). Moreover, the high levels of NO could react with superoxide anion resulting in peroxinitrite formation, which was able to damage DNA and irreversibly modified proteins such as tyrosine nitration or thiol oxidation (Calabrese et al., 2000; Paradies et al., 2011). Our data suggested that the elevation of NOS activity and NO level triggered by melamine were reversed by treatment of vitamin combination. It was further shown that the reversible effect of vitamin complex against hippocampal injury was related to attenuation of oxidative stress. Oxidative stress could cause serious mitochondrial damage, fully activate caspase, and ultimately lead to apoptosis of neuronal cells (Fukui et al., 2005; Qin et al., 2006). The cognitive deficits caused by melamine exposure were significantly correlated with dysfunction and cellular apoptosis (An et al., 2012a; Yang et al., 2011). Indeed, melamine-induced cognitive impairments were mostly attributed to the dysfunctional hippocampus due to no damage associated with oxidative stress in other brain regions (An et al., 2012a). The protooncogene Bcl-2 is one of the major regulators of the mitochondrial apoptotic pathway (Azad et al., 2008). The stability and expression levels of Bcl-2 protein could be regulated by ROS and NO through various mechanisms, such as post-translational modifications or ubiquitinproteasomal pathway (Azad et al., 2010). In the present study, the chronic administration of melamine led to significant lower Bcl-2 protein expression in the hippocampus, while the reduced Bcl-2 levels were prevented by vitamin combination. Consequently, vitamin complex might increase the expression of Bcl-2 protein under oxidative stress condition by reducing ROS or NO levels. On the other hand, caspase-3 protein is a member of the cysteine-aspartic acid protease

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family, contributing to signaling cascades between death promoting stimuli and the cleavage of protein substrates that contribute to the characteristic apoptotic morphology (Aly and Domenech, 2009; Filiz et al., 2008). In response to death stimuli, the mitochondria might initiate apoptosis through activation of the intrinsic caspase pathway (Graham and Chen, 2001). Some previous studies showed that an increase of Bcl2 prevented the mitochondrial release of cytochrome c, thereby inhibited the activation of caspase cascade and apoptosis (Hwang et al., 2011; Qian et al., 2008). In our current study, caspase-dependent neuronal damage might occur after melamine exposure, and therefore effective therapeutics should target the caspase-dependent pathway. We observed an upregulation in the expression of apoptotic caspase-3 activity after melamine exposure and decreased expression of anti-apoptotic proteins, such as Bcl-2 that ensured progression of neuronal damage. Since vitamin combination was able to maintain significantly these levels by virtue of its anti-apoptotic property (Oral et al., 2006; Tannetta et al., 2008), it could be considered as an important neuroprotective therapeutic agent. However, the detailed mechanism is essential to further elucidate in the following research. Consistent with a recent report (An et al., 2012a), our results suggested that melamine-induced apoptosis in the hippocampus of melamine-treated rats was mediated by Bcl-2 protein and caspase-3 activation, which resulted in activation of the caspase pathway since neuron loss was observed in melamine-treated hippocampal histological experiment 1 week after the behavioral test. The intact cognitive behavior is dependent on intact function of the hippocampus (Morris, 2006), and the neuropathologic lesions of hippocampus can result in the hippocampal dysfunction, as inducing deficits in learning ability. In other respects, correlative studies showed that NO modulated the biological functions of many intracellular signaling proteins and memory-related proteins (Khovryakov et al., 2010; Rivas-Arancibia et al., 2010), including N-methyl-D-aspartate receptor, protein kinase C, activating protein-1, Ras, and caspase-3 (Choi et al., 2000). In agreement with our previous finding, these abnormal changes may significantly regulate the expression of postsynaptic receptor proteins in hippocampus (Yang et al., 2011) and synaptic plasticity (An and Zhang, 2014b) and then further affect the cognitive behavior. The combination of antioxidative vitamins C and E treatment could obviously reduce the neuronal loss, regulate the apoptosis-related proteins, and efficiently rescue spatial cognitive impairment induced by melamine. Since the lasting effects of melamine on physical growth were reported (Dalal and Goldfarb, 2011; Hau et al., 2009), it was most probably that the reduced weight occurred according to food intake, albeit we did not measure the consumption. Similar with our previous finding (An and Zhang, 2014b), we fail to find the beneficial effect of vitamins on the body weight gain, which indicated the curative effect of vitamins was mainly due to its antioxidative effect but not nutrition supplement. One limitation of our study is detecting the T-SOD activity rather than the individual activities of three isoforms, Cu/Zn-SOD, Mn-SOD, and extracellular SOD. Daily treated with vitamin A enhanced Cu/Zn-SOD enzyme activity in frontal cortex and hippocampus (de Oliveira et al., 2011). However, increased Mn-SOD enzyme activity was observed in the striatum and cerebellum regions except for cortex and hippocampus. Interestingly, one investigation on the effect of vitamin E on agerelated changes in difference brain regions reported that vitamin E increased T-SOD in the hippocampus and cerebral cortex of old rats, while only Cu/Zn-SOD increased in the cerebellum of the adults (Jolitha et al., 2006). Therefore, further investigation may need to identify the age-related and region-specific alternations. It was previously reported that vitamin C alone significantly improved cognitive function in control rats (Parle and Dhingra, 2003). Combination of vitamin C and E has been also reported to improve passive avoidance learning and memory (Hasanein and Shahidi, 2010). However, under the conditions of this study, the vitamin treatment did not significantly improve memory in control rats. Further investigation to estimate the contents of vitamins may need.

In summary, it was evidently demonstrated that the cognitive deficits, induced by melamine, were significantly improved by combination of vitamins C and E supplementation. It considerably reduced oxidative stress, augmented antioxidant mechanisms, and acted as an antiapoptotic agent, and consequently increased neuronal survivability in melamine-treated condition. All these findings provide a support for further testing of vitamins C and E complex as a therapeutic agent in higher mammals for preventing cognitive deficits after melamine exposure. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (31171053, 11232005), and 111 Project (B08011). References Abel EL, Hannigan JH. Maternal risk factors in fetal alcohol syndrome: provocative and permissive influences. Neurotoxicol Teratol 1995;17:445–62. Aly HA, Domenech O. Cytotoxicity and mitochondrial dysfunction of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in isolated rat hepatocytes. Toxicol Lett 2009;191:79–87. An L, Zhang T. Spatial cognition and sexually dimorphic synaptic plasticity balance impairment in rats with chronic prenatal ethanol exposure. Behav Brain Res 2013; 256:564–74. An L, Zhang T. Prenatal melamine exposure induces impairments of spatial cognition and hippocampal synaptic plasticity in male adolescent rats. Reprod Toxicol 2014a;49: 78–85. An L, Zhang T. Vitamins C and E reverse melamine-induced deficits in spatial cognition and hippocampal synaptic plasticity in rats. Neurotoxicology 2014b;44:132–9. An L, Li Z, Yang Z, Zhang T. Cognitive deficits induced by melamine in rats. Toxicol Lett 2011;206:276–80. An L, Li Z, Yang Z, Zhang T. Melamine induced cognitive impairment associated with oxidative damage in rat's hippocampus. Pharmacol Biochem Behav 2012a;102: 196–202. An L, Liu S, Yang Z, Zhang T. Cognitive impairment in rats induced by nano-CuO and its possible mechanisms. Toxicol Lett 2012b;213:220–7. An L, Yang Z, Zhang T. Imbalanced synaptic plasticity induced spatial cognition impairment in male offspring rats treated with chronic prenatal ethanol exposure. Alcohol Clin Exp Res 2013a;37:763–70. An L, Yang Z, Zhang T. Melamine induced spatial cognitive deficits associated with impairments of hippocampal long-term depression and cholinergic system in Wistar rats. Neurobiol Learn Mem 2013b;100:18–24. An L, Li Z, Zhang T. Reversible effects of vitamins C and E combination on oxidative stressinduced apoptosis in melamine-treated PC12 cells. Free Radic Res 2014;48:239–50. Augustyniak A, Michalak K, Skrzydlewska E. The action of oxidative stress induced by ethanol on the central nervous system (CNS). Postepy Hig Med Dosw (Online) 2005;59:464. Aydemir O, Naziroglu M, Celebi S, Yilmaz T, Kukner AS. Antioxidant effects of alpha-, gammaand succinate-tocopherols in guinea pig retina during ischemia-reperfusion injury. Pathophysiology 2004;11:167–71. Azad N, Iyer AKV, Manosroi A, Wang L, Rojanasakul Y. Superoxide-mediated proteasomal degradation of Bcl-2 determines cell susceptibility to Cr (VI)-induced apoptosis. Carcinogenesis 2008;29:1538–45. Azad N, Iyer A, Vallyathan V, Wang L, Castranova V, Stehlik C, et al. Role of oxidative/ nitrosative stress‐mediated Bcl‐2 regulation in apoptosis and malignant transformation. Ann N Y Acad Sci 2010;1203:1–6. Barros DO, Xavier SM, Barbosa CO, Silva RF, Freitas RL, Maia FD, et al. Effects of the vitamin E in catalase activities in hippocampus after status epilepticus induced by pilocarpine in Wistar rats. Neurosci Lett 2007;416:227–30. Baydas G, Nedzvetskii VS, Tuzcu M, Yasar A, Kirichenko SV. Increase of glial fibrillary acidic protein and S-100B in hippocampus and cortex of diabetic rats: effects of vitamin E. Eur J Pharmacol 2003;462:67–71. Baynes RE, Smith G, Mason SE, Barrett E, Barlow BM, Riviere JE. Pharmacokinetics of melamine in pigs following intravenous administration. Food Chem Toxicol 2008; 46:1196–200. Brown CA, Jeong KS, Poppenga RH, Puschner B, Miller DM, Ellis AE, et al. Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007. J Vet Diagn Invest 2007;19:525–31. Butterfield DA, Castegna A, Drake J, Scapagnini G, Calabrese V. Vitamin E and neurodegenerative disorders associated with oxidative stress. Nutr Neurosci 2002;5:229–39. Calabrese V, Bates TE, Stella AM. NO synthase and NO-dependent signal pathways in brain aging and neurodegenerative disorders: the role of oxidant/antioxidant balance. Neurochem Res 2000;25:1315–41. Celik S, Ozkaya A. Effects of intraperitoneally administered lipoic acid, vitamin E, and linalool on the level of total lipid and fatty acids in guinea pig brain with oxidative stress induced by H2O2. J Biochem Mol Biol 2002;35:547–52. Chao J, Leung Y, Wang M, Chang RC. Nutraceuticals and their preventive or potential therapeutic value in Parkinson's disease. Nutr Rev 2012;70:373–86. Choi YB, Tenneti L, Le DA, Ortiz J, Bai G, Chen HS, et al. Molecular basis of NMDA receptorcoupled ion channel modulation by S-nitrosylation. Nat Neurosci 2000;3:15–21.

L. An et al. / Pharmacology, Biochemistry and Behavior 132 (2015) 152–159 Dalal RP, Goldfarb DS. Melamine-related kidney stones and renal toxicity. Nat Rev Nephrol 2011;7:267–74. de Oliveira MR, Lorenzi R, Schnorr CE, Morrone M, Moreira JC. Increased 3-nitrotyrosine levels in mitochondrial membranes and impaired respiratory chain activity in brain regions of adult female rats submitted to daily vitamin A supplementation for 2 months. Brain Res Bull 2011;86:246–53. D'Hooge R, De Deyn PP. Applications of the Morris water maze in the study of learning and memory. Brain Res Rev 2001;36:60–90. Ercan S, Ozturk N, Celik-Ozenci C, Gungor NE, Yargicoglu P. Sodium metabisulfite induces lipid peroxidation and apoptosis in rat gastric tissue. Toxicol Ind Health 2010;26: 425–31. Fan SH, Zhang ZF, Zheng YL, Lu J, Wu DM, Shan Q, et al. Troxerutin protects the mouse kidney from d-galactose-caused injury through anti-inflammation and anti-oxidation. Int Immunopharmacol 2009;9:91–6. Filiz G, Caragounis A, Bica L, Du T, Masters CL, Crouch PJ, et al. Clioquinol inhibits peroxide-mediated toxicity through up-regulation of phosphoinositol-3-kinase and inhibition of p53 activity. Int J Biochem Cell Biol 2008;40:1030–42. Fukui K, Takatsu H, Shinkai T, Suzuki S, Abe K, Urano S. Appearance of amyloid beta-like substances and delayed-type apoptosis in rat hippocampus CA1 region through aging and oxidative stress. J Alzheimers Dis 2005;8:299–309. Graham SH, Chen J. Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab 2001;21:99–109. Gultekin F, Delibas N, Yasar S, Kilinc I. In vivo changes in antioxidant systems and protective role of melatonin and a combination of vitamin C and vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats. Arch Toxicol 2001;75:88–96. Guney M, Ozguner F, Oral B, Karahan N, Mungan T. 900 MHz radiofrequency-induced histopathologic changes and oxidative stress in rat endometrium: protection by vitamins E and C. Toxicol Ind Health 2007;23:411–20. Han YG, Liu SC, Zhang T, Yang Z. Induction of apoptosis by melamine in differentiated PC12 cells. Cell Mol Neurobiol 2011;31:65–71. Han G, An L, Yang B, Si L, Zhang T. Nicotine-induced impairments of spatial cognition and long-term potentiation in adolescent male rats. Hum Exp Toxicol 2014;33:203–13. Hasanein P, Shahidi S. Effects of combined treatment with vitamins C and E on passive avoidance learning and memory in diabetic rats. Neurobiol Learn Mem 2010;93:472–8. Hau AK, Kwan TH, Li PK. Melamine toxicity and the kidney. J Am Soc Nephrol 2009;20: 245–50. Henchcliffe C, Beal MF. Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis. Nat Clin Pract Neurol 2008;4:600–9. Henderson GI, Chen JJ, Schenker S. Ethanol, oxidative stress, reactive aldehydes, and the fetus. Front Biosci 1999;4:D541–50. Hritcu L, Ciobica A, Gorgan L. Nicotine-induced memory impairment by increasing brain oxidative stress. Cent Eur J Biol 2009;4:335–42. Hwang DS, Kim HG, Kwon HJ, Cho JH, Lee CH, Lee JM, et al. Dangguijakyak-san, a medicinal herbal formula, protects dopaminergic neurons from 6-hydroxydopamine-induced neurotoxicity. J Ethnopharmacol 2011;133:934–9. Jolitha AB, Subramanyam MV, Asha Devi S. Modification by vitamin E and exercise of oxidative stress in regions of aging rat brain: studies on superoxide dismutase isoenzymes and protein oxidation status. Exp Gerontol 2006;41:753–63. Karaoz E, Gultekin F, Akdogan M, Oncu M, Gokcimen A. Protective role of melatonin and a combination of vitamin C and vitamin E on lung toxicity induced by chlorpyrifos-ethyl in rats. Exp Toxicol Pathol 2002;54:97–108. Khovryakov A, Podrezova E, Kruglyakov P, Shikhanov N, Balykova M, Semibratova N, et al. Involvement of the NO synthase system in stress-mediated brain reactions. Neurosci Behav Physiol 2010;40:333–7. Kim HK, Kim JH, Gao X, Zhou JL, Lee I, Chung K, et al. Analgesic effect of vitamin E is mediated by reducing central sensitization in neuropathic pain. Pain 2006;122: 53–62. Kuhad A, Bishnoi M, Tiwari V, Chopra K. Suppression of NF-kappabeta signaling pathway by tocotrienol can prevent diabetes associated cognitive deficits. Pharmacol Biochem Behav 2009;92:251–9. Li Y, Bao Y, Jiang B, Wang Z, Liu Y, Zhang C, et al. Catalpol protects primary cultured astrocytes from in vitro ischemia-induced damage. Int J Dev Neurosci 2008;26:309–17. Mao QQ, Zhong XM, Feng CR, Pan AJ, Li ZY, Huang Z. Protective effects of paeoniflorin against glutamate-induced neurotoxicity in PC12 cells via antioxidant mechanisms and Ca(2+) antagonism. Cell Mol Neurobiol 2010;30:1059–66. Mast RW, Jeffcoat AR, Sadler BM, Kraska RC, Friedman MA. Metabolism, disposition and excretion of [14C]melamine in male Fischer 344 rats. Food Chem Toxicol 1983;21: 807–10.

159

Morris RG. Elements of a neurobiological theory of hippocampal function: the role of synaptic plasticity, synaptic tagging and schemas. Eur J Neurosci 2006;23:2829–46. Morris RG, Garrud P, Rawlins JN, O'Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature 1982;297:681–3. Niebroj-Dobosz I, Dziewulska D, Kwiecinski H. Oxidative damage to proteins in the spinal cord in amyotrophic lateral sclerosis (ALS). Folia Neuropathol 2004;42:151–6. Nunomura A, Castellani RJ, Zhu X, Moreira PI, Perry G, Smith MA. Involvement of oxidative stress in Alzheimer disease. J Neuropathol Exp Neurol 2006;65:631–41. Oral B, Guney M, Demirin H, Ozguner M, Giray SG, Take G, et al. Endometrial damage and apoptosis in rats induced by dichlorvos and ameliorating effect of antioxidant vitamins E and C. Reprod Toxicol 2006;22:783–90. Ornoy A, Tsadok MA, Yaffe P, Zangen SW. The Cohen diabetic rat as a model for fetal growth restriction: vitamins C and E reduce fetal oxidative stress but do not restore normal growth. Reprod Toxicol 2009;28:521–9. Ozkan Y, Yilmaz O, Ozturk AI, Ersan Y. Effects of triple antioxidant combination (vitamin E, vitamin C and alpha-lipoic acid) with insulin on lipid and cholesterol levels and fatty acid composition of brain tissue in experimental diabetic and non-diabetic rats. Cell Biol Int 2005;29:754–60. Paradies G, Petrosillo G, Paradies V, Ruggiero FM. Mitochondrial dysfunction in brain aging: role of oxidative stress and cardiolipin. Neurochem Int 2011;58:447–57. Parle M, Dhingra D. Ascorbic Acid: a promising memory-enhancer in mice. J Pharmacol Sci 2003;93:129–35. Penugonda S, Mare S, Lutz P, Banks WA, Ercal N. Potentiation of lead-induced cell death in PC12 cells by glutamate: protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant. Toxicol Appl Pharmacol 2006;216:197–205. Qian Y, Wang H, Yao W, Gao X. Aqueous extract of the Chinese medicine, DangguiShaoyao-San, inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade. Cell Biol Int 2008;32:304–11. Qin F, Yan C, Patel R, Liu W, Dong E. Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction. Free Radic Biol Med 2006;40:1827–42. Ravaglia G, Forti P, Maioli F, Martelli M, Servadei L, Brunetti N, et al. Homocysteine and folate as risk factors for dementia and Alzheimer disease. Am J Clin Nutr 2005;82:636–43. Rivas-Arancibia S, Guevara-Guzman R, Lopez-Vidal Y, Rodriguez-Martinez E, ZanardoGomes M, Angoa-Perez M, et al. Oxidative stress caused by ozone exposure induces loss of brain repair in the hippocampus of adult rats. Toxicol Sci 2010;113:187–97. Roach RC, Hackett PH. Frontiers of hypoxia research: acute mountain sickness. J Exp Biol 2001;204:3161–70. Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulating effects of FK506 and cyclosporine. Transplantation 1993;55:1265–72. Takeda K, Jin MB, Fujita M, Fukai M, Sakurai T, Nakayama M, et al. A novel inhibitor of Rho-associated protein kinase, Y-27632, ameliorates hepatic ischemia and reperfusion injury in rats. Surgery 2003;133:197–206. Tannetta DS, Sargent IL, Linton EA, Redman CW. Vitamins C and E inhibit apoptosis of cultured human term placenta trophoblast. Placenta 2008;29:680–90. Tuzcu M, Baydas G. Effect of melatonin and vitamin E on diabetes-induced learning and memory impairment in rats. Eur J Pharmacol 2006;537:106–10. Wang Y, Liu F, Wei Y, Liu D. The effect of exogenous melamine on rat hippocampal neurons. Toxicol Ind Health 2011;27:571–6. Wu YT, Huang CM, Lin CC, Ho WA, Lin LC, Chiu TF, et al. Determination of melamine in rat plasma, liver, kidney, spleen, bladder and brain by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2009;1216:7595–601. Yang JJ, Yang Z, Zhang T. Action potential changes associated with impairment of functional properties of sodium channels in hippocampal neurons induced by melamine. Toxicol Lett 2010;198:171–6. Yang J, An L, Yao Y, Yang Z, Zhang T. Melamine impairs spatial cognition and hippocampal synaptic plasticity by presynaptic inhibition of glutamatergic transmission in infant rats. Toxicology 2011;289:167–74. Yoon YS, Kim DH, Kim SK, Song SB, Uh Y, Jin D, et al. The melamine excretion effect of the electrolyzed reduced water in melamine-fed mice. Food Chem Toxicol 2011;49: 1814–9. Zhao Z, Huang G, Wang B, Zhong Y. Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate partially attenuates hippocampal MMP-9 activation and improves cognitive deficits in streptozotocin-induced diabetic rats. Behav Brain Res 2013; 238:44–7.