Astragaloside Ⅳ Protects Against Aβ1-42-induced Oxidative Stress, Neuroinflammation and Cognitive Impairment in Rats

Yanfang Pan, Xiaotao Jia, Erfei Song, Xiaozhong Peng

1Department of Pathology, Shaanxi University of Chinese Medicine, Xianyang,Shaanxi 712046, China

2Department of Neurology, The Affiliated Xi’an Central Hospital of Xi’an Jiaotong University College of Medicine, Xi’an 710003, China

3Department of Biology, York University, Toronto, ON M3J 1P3, Canada

4State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China

ALZHEIMER’S disease (AD), characterized by cognition impairments, personality alterations and visual skills deficits, is an age-related and irreversible neurodegenerative disorder.1-2Amyloid beta 1-42 protein (Aβ) deposition plays an essential role in the pathogenesis development of AD.3-4Although the mechanisms through which Aβ exerts its toxicity have not yet been completely understood, recent evidence suggests that oxidative stress and neuroinflammation induced by Aβ1-42 play a pivotal role in the pathogenesis of AD and cognitive impairment.5-6Besides, aggregated Aβ in the brain elicits the activation of microglia cells and astrocytes,leading to the production of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), triggering the damage and loss of neurons.7-8The interactions between reactive oxygen species and proinflammatory factors aggravates cognitive dysfunction.9-10Therefore, it is urgent to discover new compounds with antioxidant and anti-inflammatory properties for the treatment of cognitive decline during AD.

Although several therapeutic interventions have been applied for reducing amyloid formation by using cholinesterase inhibitors to restore cholinergic deficits and using COX-2 inhibitors to regulate neuroinflammation,11-12these drugs are not well accepted because of their severe side effects. In light of these considerations,natural herbal sources may provide greater therapeutic benefit by reducing or prevention of oxidative stress and neuroinflammation in the treatment of AD.

AstragalosideⅣ (AS-Ⅳ), a small molecular saponin purified from Astragalus membranaceus, is a primary active constituent in Huangqi (Astragali Radix).AS-Ⅳhas been applied for the treatment of cardiovascular diseases, hepatic and renal disorders.13Pharmaceutical studies have shown the prominent antioxidant effect of AS-Ⅳ. The antioxidant mechanisms of AS-Ⅳinclude free radical scavenging activity, reducing lipid peroxidation, and increasing antioxidant enzymes.14-16A recent study reported that AS-Ⅳ protects from the ischemic brain injury mainly via suppressing oxidative damage after chronic cerebral hypoperfusion.17-19However, it is unknown that whether AS-Ⅳ can ameliorate Aβ1-42-induced oxidative stress, neuroinflammation and cognitive dysfunction. Therefore, the present work was designed to investigate the neuroprotective effects of AS-Ⅳ against Aβ1-42-induced oxidative stress, neuroinflammation and memory deficit in an in vivo amnesia-like rat model.

MATERIALS AND METHODS

Rats

Male Sprague-Dawley (SD) rats supplied by the Research Animal Center of Xi’an Jiaotong University with a body weight between 230 and 250 g were used. The rats were housed at 23°C with a 12-hour light-dark cycle. This study was conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and with the approval of the Shaanxi Animal Research Ethics Committee.

Drugs

To induce peptide aggregation, the Aβ1-42 (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% saline (5 mg/ml) and incubated at 37°C for 4 days before surgery.20-21The AS-Ⅳ was purchased from Biopurify Phytochemicals (Chengdu, China).

Grouping and drug injections

Sixty-eight rats were divided into six groups randomly,control group, Aβ1-42 group, AS-Ⅳ group, Aβ1-42 plus 5 mg/kg·d AS-Ⅳ group, Aβ1-42 plus 25 mg/kg·d AS-Ⅳgroup, and Aβ1-42 plus 50 mg/kg·d AS-Ⅳ group.

The surgery was performed according to our previous report with minor modifications.22Briefly, rats were put in a stereotaxic apparatus under the anesthesia with an intraperitoneal injection of chloral hydrate(0.3 g/kg). Then, the rat skulls were opened and the burr holes at the corresponding position were drilled for Aβ1-42 intracerebroventricular injection. The specific area for injection was: anteroposterior: -0.8 mm from Bregma, medial/lateral: ±1.3 mm from midline,and dorsal/ventral: -4.0 mm from dura. The experimental procedures for chronic AS-Ⅳ treatment are shown in Fig. 1. Seven days after Aβ1-42 injection, all rats were tested in Morris water maze.

Morris water maze task

As described previously,22spatial memory testing was carried out using Morris water maze (MWM). A large circular black-painted pool (150 cm in diameter; 50 cm in height) was divided into four quadrants at the equal distance from the rim. A small escape platform(14 cm in diameter; 29 cm in height) was fixed at the center of a quadrant and submerged approximately 1.0 cm below water surface (maintained at 23°C ± 2°C).In the room for water maze, several landmarks were fixed on the walls. After Aβ1-42 injection for 7 days,memory training was performed. For hidden platform tests, the treated rats were released to swim freely in the maze to search the underwater platform. We performed the training four times a day for five consecutive days. In each trial, the rats were placed into the water facing the pool wall at one of the four equal quadrants (Zone 1, 2, 3, and 4) designated by computer software. Rats were allowed to swim until they found the platform or until 120 seconds elapsed. In probe trials on the sixth day, the rats were allowed to swim for 120 seconds after removing the platform. The swim escape latency (s), average swiming speed (cm/s),and time spent in the target quadrant (%) were measured. Then, the visual platform test was performed.The platform was attached to a highly visible cover and elevated to approximately 2 cm above the water surface. The swimming speed and time needed to reach the platform were recorded and analyzed by EthoVision 3.0 (Noldus Information Technology, Wageningen, the Netherlands).

Assay for antioxidant enzymes activity

After behavioral tests, the animals were sacrificed immediately by decapitation. The bilateral hippocampus was immediately removed, then weighed. Hippocampus homogenates with 5% tissue (w/v) in 0.9% saline solution were prepared. Supernatants collected after the homogenization were centrifuged at 3000 ×g for 15 minutes at 4°C. Then the enzyme activities of superoxide dismutase (SOD), glutathione peroxidase(GSH-px) and catalase (CAT) were measured by following the manufacturer’s instructions. The activity of CAT was expressed as nmol/mg protein, and the GSH-px and SOD activity were expressed as U/mg protein.Assay kits for SOD, GSH-px and CAT were purchased from Nanjing Jian-cheng Bioengineering Institute(Nanjing, China).

Assay for pro-inflammatory cytokines

Hippocampal tissue collected was homogenized with RIPA lysis buffer (150 mmol/L NaCl, 0.5% sodium deoxycholate, 5 mmol/L EDTA, 0.5% NP-40, 50 mmol/L Tris-HCl, pH 6.8) supplemented with a commercial protease and phosphatase cocktail (Applygen, Beijing,China). The tissue homogenates of the samples were centrifuged at full speed for 15 minutes at 4°C. BCA Protein Assay (Thermo Pierce, Rock-ford, IL, USA)were performed to determine protein concentrations.Then the levels of IL-1β and TNF-α in tissue lysates were checked by the commercial ELISA kits (R&D systems, Minneapo-lis, MN, USA) following the manufacturer’s instructions. The results were expressed as pg/mg protein.

Figure 1. Schematic diagram of drug treatment and behavioral tests. Aβ1-42 was injected into the intracerebroventricular of rats. After a recovery period for 7 days, AS-Ⅳ was intraperitoneally administrated at the doses of 5, 25 and 50 mg/kg·d respectively for 5 consecutive days. Aβ1-42: amyloid-beta 1-42; AS-Ⅳ: astragalosideⅣ.

Statistical analysis

All data were represented as the mean values ± standard error (SE). In the MWM test, a two-way analysis of variance (ANOVA) with repeated measures was used in the analysis of spatial learning task. Oneway ANOVA followed by Dunnett’s post-hoc test were performed in intergroup comparisons to determine the significant differences. All statistical analyses were performed by SPSS 17.0. Statistical significance was accepted at P＜0.05.

RESULTS

AS-Ⅳ treatment prevented against Aβ1-42-induced spatial learning and memory impairment in a dosedependent manner

To explore the neuroprotective effect of AS-Ⅳ against Aβ1-42-induced cognitive impairment, we tested reference memory in the MWM. As expected, Aβ1-42 significantly impaired the rat performance in both hidden platform tests and probe trials (F1.39=9.078, P＜0.05).When the rats were treated with AS-Ⅳ alone, their average latencies to find the hidden platform did not differ from those of the control group (Fig. 2A). However,AS-Ⅳ (25 and 50 mg/kg·d) significantly decreased the animal escape latency induced by Aβ1-42 (F1.39=5.041,P＜0.05; F1.39=6.243, P＜0.05).

To further evaluate the animal spatial memory ability in MWM, we performed probe trials on the 6th day. The swimming time spent and distance swum in target quadrant was compared among the groups. As shown in Fig. 2B, AS-Ⅳ (50 mg/kg·d) alone did not show an influence on memory behavior compared to the control group (F1.39=1.289, P＞0.05). Interestingly, chronic AS-Ⅳ (5, 25 and 50 mg/kg·d) treatment prevented the Aβ1-42-induced memory deficit in a dose-dependent manner. As depicted in Fig. 2B, the time percentages in the target quadrant in both AS-Ⅳ(25 mg/kg·d) and AS-Ⅳ (50 mg/kg·d) plus Aβ1-42 treated rats were significantly higher than those in the Aβ1-42 alone group (F1.39=5.279, P＜0.05; F1.39=7.023, P＜0.05). After the probe trials, rats escape latencies were conducted with visible platform. The differences in escape time and swimming speed were not significant between all groups (Fig. 2C, 2D). The results suggested that the vision and ability of motor were not affected in all the rats.

AS-Ⅳ effectively reversed the Aβ1-42-induced decrease of SOD, GSH-Px and CAT activities in the hippocampus of rats

To further elucidate the probable biochemical mechanisms of the neuroprotective effect of AS-Ⅳ in Aβ1-42-mediated impairment in spatial memory, we subsequently tested whether AS-Ⅳ influenced antioxidant activity in the hippocampus of amnesia-like rat brain. We first tested the enzyme activities of SOD,GSH-Px and CAT. As shown in Fig. 3, the activities of SOD, GSH-Px and CAT were significantly decreased by Aβ1-42 treatment when compared to the control rats. However, supplementation of AS-Ⅳ (25 and 50 mg/kg·d) significantly increased SOD (Fig. 3A,F1.23=5.042, P＜0.05; F1.23=5.986, P＜0.05), GSH-Px(Fig. 3B, F1.23=5.124, P＜0.05; F1.23=6.028, P＜0.05),and CAT (Fig. 3C, F1.23=5.369, P＜0.05; F1.23=6.272,P＜0.05) activities when compared with the Aβ1-42-treated rats. AS-Ⅳ (50 mg/kg·d) alone supplementation did not show any significant changes in the activities of SOD, GSH-Px and CAT when compared to the control group rats (F1.23=1.056, all P＞0.05). Taken together, the above results suggest neuroprotective effects of AS-Ⅳ on Aβ1-42-induced cognitive deficits might be mediated through its antioxidative effect.

AS-Ⅳ markedly suppressed pro-inflammatory cytokines accumulation induced by Aβ1-42 in rat hippocampus

In this study, the levels of two pro-inflammatory cytokines(IL-1β and TNF-α) in hippocampus were measured. ELISA results showed that IL-1β and TNF-α levels of Aβ1-42 treated rats were significantly increased compared to the control group as shown in Fig. 4 (F1.23=5.869,P＜0.05). Meanwhile, the treatment of AS-Ⅳ (25 and 50 mg/kg·d) significantly attenuated the Aβ1-42-induced up-regulation of these two pro-inflammatory cytokines in the hippocampal region of the rat brain (F1.23=5.859, P＜0.05; F1.23=6.564, P＜0.05). The results suggest that AS-Ⅳ prevented the pro-inflammatory cytokines accumulation induced by Aβ1-42 in rat hippocampus.

Figure 2. AS-Ⅳ reatments attenuated Aβ1-42-induced spatial learning and memory impairment in rats. A. Rats with different treatments as labeled in the figure were trained for five consecutive days and the average escape latencies of rats were checked by the Morris water maze. B. The probe testing in various groups were performed four times per day and the percentages of total time in the target quadrant were calculated. C. Visible platform test was performed in rats with different treatments. D. The swimming speed (cm/s) in the various groups were evaluated. The data were represented as mean±SE.(n=10). *P＜0.05 compared with the control group. #P＜0.05, ##P＜0.01 compared with the Aβ1-42 alone group.

DISCUSSION

AD is a progressive neurodegenerative disorder with a complex pathogenesis.23The current hypothesis suggests that oxidative stress and neuroinflammation have a vital role in the progress of AD.24-25The impairment of cognitive in patients with AD is related with elevated Aβ levels in brain. The injection of Aβ to rats has been shown to cause behavioral and pathological symptoms of AD, such as learning and memory deficit,synaptotoxicity, oxidative stress, inflammation, neuronal injury and death.26-29In the present study, the Aβ1-42-induced AD model was used to clarify the neuroprotective potential of the AS-Ⅳ.

The most commonly method used to assess the hippocampal-dependent spatial learning and memory ability is MWM test.30As previously reported,31our present results confirmed that Aβ1-42 treatment significantly impaired the spatial learning and memory in MWM performance. Interestingly, AS-Ⅳ alone did not affect the learning and memory capacity in rats. AS-Ⅳadministration (25, 50 mg/kg) can dose-dependently reverse the cognitive decline in rats induced by Aβ1-42 in hidden platform test and the MWM probe trials.In addition, the performance of rats in visible platform test indicated that AS-Ⅳ and Aβ1-42 did not affect rat’s vision and motor ability.

Figure 3. AS-Ⅳ attenuated the oxidative stress in the hippocampus of rats treated with Aβ1-42. Rats were received different treatments as labeled in the figure for five days and the activities of superoxide dismutase (SOD, A), glutathione peroxidase (GSH-px, B) and catalase (CAT, C) in hippocampus were checked. Values were expressed as mean ± SE (n=8).**P＜0.01 compared with the control group; #P＜0.05, ##P＜0.01 compared with the Aβ1-42 group.

Figure 4. AS-Ⅳ prevented the increase of IL-1β and TNF-α induced by Aβ1-42. ELISA for interleukin-1 beta (IL-1β, A) and tumor necrosis factor-alpha (TNF-α, B) were performed in the hippocampus tissue of rats with various treatments. Values are expressed as mean ± SE (n=8). **P＜0.01 compared with the control group; #P＜0.05, ##P＜0.01 compared with the Aβ1-42 group.

Previous studies involving in vivo and in vitro experiments have shown that Aβ induces oxidative damage.32Consistent with other prior reports,33rats treated with Aβ1-42 in this study exhibited a significant increase in oxidative stress compared with the control group. Endogenous enzymes like SOD, GSH-Px and CAT maintain the redox homeostasis and low oxidant level in tissue.34In the present study, the SOD activity was markedly lower in Aβ1-42-treated rats than that in the control rats. The administration of AS-Ⅳ significantly restored the SOD activity in Aβ1-42-treated rats. GSH-Px exerts an important role in scavenging free radical. Our present data showed that Aβ1-42 decreased the level of GSH-Px compared to the control rats. Administration of AS-Ⅳ significantly restores GSH-Px levels decreased by Aβ1-42. CAT is an enzyme that is responsible for catalyzing the decomposition of H2O2. The maintenance of reactive oxygen species is important for proper cell function.35In the present study, CAT activity was significantly compromised by Aβ1-42-injection in rats, while the administration of AS-Ⅳ (25, 50 mg/kg) markedly ameliorates these abnormalities. Overall, the results in our study suggested that AS-Ⅳ injection was effective in restoring the activities of SOD, GSH-Px and CAT, thus clearing the free radicals and protecting from oxidative damage induced by Aβ1-42.

In the pathogenesis of AD, the glial cell activation and pro-inflammatory mediators release play critical roles. IL-1β and TNF-α are basic indicators of the inflammation.36Previous studies have shown that Aβ1-42 caused cognitive injury by inducing the activation of microglia cells and over-producing pro-inflammatory cytokines in hippocampus.37It has been reported that IL-1β can increase the production and accumulation of Aβ.38In the present study, we demonstrated that intracerebroventricular injection Aβ1-42 significantly increased the IL-1β and TNF-α level in the hippocampus. However, administration of AS-Ⅳ (25, 50 mg/kg) significantly and dose-dependently suppressed the inflammatory responses in Aβ1-42-treated rats, suggesting the anti-neuroinflammation role of AS-Ⅳ in neuroprotection.

In summary, our results indicated that AS-Ⅳdose-dependently ameliorates Aβ1-42-induced spatial learning and memory impairments in rats. Restored activities of antioxidant enzymes and declined pro-inflammatory cytokines release are accountable for the neuroprotective effects of AS-Ⅳ against Aβ1-42 induced injury in the brain. It is necessary to further clarify the detailed mechanism of AS-Ⅳ in the treatment of AD both in vitro and in vivo. Therefore, AS-Ⅳmay be an effective therapeutic agent in improving the cognitive functions in patients of AD.

Conflicts of interest statement

The authors have no conflicts of interest to disclose.

1. Goedert M, Spillantini MG. A century of Alzheimer’s disease. Science 2006; 314(5800):777-81. doi:10.1126/science.1132814.

2. Anand R, Gill KD, Mahdi AA. Therapeutics of Alzheimer’s disease: past, present and future. Neuropharmacology 2014; 76 PtA:27-50. doi: 10.1016/j.neuropharm.2013.07.004.

3. Esparza TJ, Zhao H, Cirrito JR, et al. Amyloid-beta oligomerization in Alzheimer dementia versus high-pathology controls. Ann Neurol 2013;73(1):104-19. doi: 10.1002/ana.23748.

4. Zetterberg H, Blennow K, Hanse E. Amyloid beta and APP as biomarkers for Alzheimer’s disease.Exp Gerontol 2010; 45(1):23-9. doi: 10.1016/j.exger.2009.08.002.

5. Yatin SM, Yatin M, Aulick T, et al. Alzheimer’s amyloid beta-peptide associated free radicals increase rat embryonic neuronal polyamine uptake and ornithine decarboxylase activity: protective effect of vitamin E. NeurosciLett 1999; 263(1):17-20. doi: 10.1016/S0304-3940(99)00101-96.

6. Zotova E, Nicoll JA, Kalaria R, et al. Inflammation in Alzheimer’s disease: relevance to pathogenesis and therapy. Alzheimers Res Ther 2010; 2(1):1. doi:10.1186/alzrt24.

7. Hickman SE, Allison EK, El KJ. Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer’s disease mice. J Neurosci 2008; 28(33):8354-60. doi: 10.1523/JNEUROSCI.0616-08.2008.

8. Johnston H, Boutin H, Allan SM. Assessing the contribution of inflammation in models of Alzheimer’s disease. Biochem Soc Trans 2011; 39(4):886-90. doi:10.1042/BST0390886.

9. Godbout JP, Chen J, Abraham J, et al. Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system. FASEB J 2005; 19(10):1329-31. doi:10.1096/fj.05-3776fj.

10. Ullah F, Ali T, Ullah N, et al. Caffeine prevents d-galactose-induced cognitive deficits, oxidative stress,neuroinflammation and neurodegeneration in the adult rat brain. Neurochem Int 2015; 90:114-24. doi:10.1016/j.neuint.2015.07.001.

11. Tan CC, Yu JT, Wang HF, et al. Efficacy and safety of donepezil, galantamine, rivastigmine, and memantine for the treatment of Alzheimer’s disease: a systematic review and meta-analysis. J Alzheimers Dis 2014;41(2):615-31. doi: 10.3233/JAD-132690.

12. Saini SS, Gesselllee DL, Peterson JW. The cox-2-specific inhibitor celecoxib inhibits adenylyl cyclase.Inflammation 2003; 27(2):79-88. doi: 10.1023/A:1023226616526.

13. Fu J, Wang Z, Huang L, et al. Review of the botanical characteristics, phytochemistry, and pharmacology of Astragalus membranaceus (Huangqi). Phytother Res 2014; 28(9):1275-83. doi: 10.1002/ptr.5188.

14. Zhang ZG, Wu L, Wang JL, et al. AstragalosideⅣprevents MPP(+)-induced SH-SY5Y cell death via the inhibition of Bax-mediated pathways and ROS production. Mol Cell Biochem 2012; 364(1-2):209-16. doi:10.1007/s11010-011-1219-1.

15. Li X, Wang X, Han C, et al. Astragaloside Ⅳ suppresses collagen production of activated hepatic stellate cells via oxidative stress-mediated p38 MAPK pathway. Free Radic Biol Med 2013; 60:168-76. doi:10.1016/j.freeradbiomed.

16. Hu JY, Han J, Chu ZG, et al. AstragalosideⅣattenuates hypoxia-induced cardiomyocyte damage in rats by upregulating superoxide dismutase-1 levels.Clin Exp Pharmacol Physiol 2009; 36(4):351-7. doi:10.1111/j.1440-1681.2008.05059.x.

17. Li M, Qu YZ, Zhao ZW, et al. AstragalosideⅣprotects against focal cerebral ischemia/reperfusion injury correlating to suppression of neutrophils adhesion-related molecules. Neurochem Int 2012; 60(5):458-65.doi: 10.1016/j.neuint.2012.01.026.

18. Liu G, Song J, Guo Y, et al. Astragalus injection protects cerebral ischemic injury by inhibiting neuronal apoptosis and the expression of JNK3 after cerebral ischemia reperfusion in rats. Behav Brain Funct 2013;9:36. doi: 10.1186/1744-9081-9-36.

19. Kim S, Kang IH, Nam JB, et al. Ameliorating the effect of astragalosideⅣon learning and memory deficit after chronic cerebral hypoperfusion in rats. Molecules 2015; 20(2):1904-21. doi: 10.3390/molecules 20021904.

20. Paranjape GS, Terrill SE, Gouwens LK, et al. Amyloid-β(1-42) protofibrils formed in modified artificial cerebrospinal fluid bind and activate microglia. J Neuroimmune Pharmacol 2013; 8(1):312-22. doi:10.1007/s11481-012-9424-6.

21. Nakamura S, Murayama N, Noshita T, et al. Progressive brain dysfunction following intracerebroventricular infusion of beta (1-42)-amyloid peptide. Brain Res 2001; 912(2):128-36. doi: 10.1016/S0006-8993(01)02704-4.

22. Pan YF, Chen XR, Wu MN, et al. Arginine vasopressin prevents against Aβ25-35-induced impairment of spatial learning and memory in rats. Horm Behav 2010;57(4-5):448-54. doi: 10.1016/j.yhbeh.2010.01.015.

23. Butterfield DA, Boyd-Kimball D. Amyloid β-peptide(1-42) contributes to the oxidative stress and neurodegeneration found in Alzheimer disease brain.Brain Pathol 2004; 14(4):426-32. doi: 10.1111/j.1750-3639.2004.tb00087.x.

24. Praticò D. Oxidative stress hypothesis in Alzheimer’s disease: a reappraisal. Trends Pharmacol Sci 2008;29(12):609-15. doi: 10.1016/j.tips.2008.09.001.

25. Kamer A , Craig RG, Dasanayake AP, et al. Inflammation and Alzheimer’s disease: possible role of periodontal diseases. Alzheimers Dement 2008; 4(4):242-50. doi: 10.1016/j.jalz.2007.08.004.

26. Cai HY, Holscher C, Yue XH, et al. Lixisenatide rescues spatial memory and synaptic plasticity from amyloid β protein-induced impairments in rats. Neuroscience 2014; 277:6-13. doi: 10.1016/j.neuroscience.2014.02.022.

27. Klein WL, Jr SW, Teplow DB. Small assemblies of unmodified amyloid beta-protein are the proximate neurotoxin in Alzheimer’s disease. Neurobiol Aging 2004; 25(5):569-80. doi: 10.1016/j.neurobiolaging.2004.02.010.

28. Nillert N, Pannangrong W, Welbat JU, et al. Neuroprotective effects of aged garlic extract on cognitive dysfunction and neuroinflammation induced by β-amyloid in rats. Nutrients 2017; 9(1). pii: E24. doi: 10.3390/nu9010024.

29. Wang J, Ho L, Zhao W, et al. Grape-derived polyphenolics prevent Aβ oligomerization and attenuate cognitive deterioration in a mouse model of Alzheimer’s disease. J Neurosci 2008; 28(25):6388-92. doi:10.1523/JNEUROSCI.0364-08.2008.

30. Dong Z, Bai Y, Wu X, et al. Hippocampal long-term depression mediates spatial reversal learning in the Morris water maze. Neuropharmacology 2013; 64:65-73. doi: 10.1016/j.neuropharm.2012.06.027.

31. Jia XT, Ye-Tian, Yuan-Li, et al. Exendin-4, a glucagon-like peptide 1 receptor agonist, protects against amyloid-β peptide-induced impairment of spatial learning and memory in rats. Physiol Behav 2016; 159:72-9.doi: 10.1016/j.physbeh. 2016.03.016.

32. Wan L, Nie G, Zhang J, et al. β-Amyloid peptide increases levels of iron content and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease. Free Radic Biol Med 2011; 50(1):122-9. doi: 10.1016/j.freeradbiomed.2010.10.707.

33. Abdul HM, Sultana R, St Clair DK, et al. Oxidative damage in brain from human mutant APP/PS-1 double knock-in mice as a function of age. Free Radic Biol Med 2008; 45(10):1420-5. doi: 10.1016/j.freerad biomed.2008.08.012.

34. Chen H, Yoshioka H, Kim GS, et al. Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection.Antioxid Redox Signal 2011; 14(8):1505-17. doi:10.1089/ars.2010.3576.

35. Pong K, Rong Y, Doctrow SR, et al. Attenuation of zinc-induced intracellular dysfunction and neurotoxicity by a synthetic superoxide dismutase/catalase mimetic, in cultured cortical neurons. Brain Res 2002; 950(1-2):218-30. doi:10.1016/S0006-8993(02)03040-8.

36. Hofmann U, Heuer S, Meder K, et al. The proinflam-matory cytokines TNF-alpha and IL-1 beta impair economy of contraction in human myocardium. Cytokine 2007; 39(3):157-62. doi: 10.1016/j.cyto.2007.07.185.

37. Detloff MR, Fisher LC, Mcgaughy V, et al. Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats. Exp Neurol 2008; 212(2):337-47. doi: 10.1016/j.expneurol.2008.04.009.

38. Rogers JT, Leiter LM, Mcphee J, et al. Translation of the alzheimer amyloid precursor protein mRNA is up-regulated by interleukin-1 through 5’-untranslated region sequences. J Biol Chem 1999; 274(10):6421-31. doi: 10.1074/jbc.274.10.6421.