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Neuro-protective Mechanism of Isoflavones on Senescence-accelerated Mice
Chinese Journal of Natural Medicines 2010, 8(4): 02800284
Chinese Journal of Natural Medicines
Neuro-protective Mechanism of Isoflavones on Senescence-accelerated Mice YANG Hong1, 2, JIN Gui-Fang2, REN Dong-Dong2, LUO Si-Jing2, ZHOU Tian-Hong1* 1
College of Life Science and TechnologyēJinan University, Guangzhou 510632; College of Basic Courses, Guangzhou Higher Education Mega Center, Guangdong Pharmaceutical University, Guangzhou 510006, China 2
Available online 20 July 2010
[ABSTRACT] AIM: To investigate the neuro-protective mechanism of isoflavones, using senescence-accelerated SAM-P/8 mice. METHODS: Various dosages of isoflavones were intragastrically administered to SAM-P/8 senescence-accelerated mice. The cortex acetylcholinesterase (AchE) activity, serum superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were examined. In addition, the concentration of malondialdehyde (MDA) was examined. ȕ-secretase activity in the hippocampus tissue was determined by fluorometry. Changes of the hippocampal neurons were observed under a light microscope. RESULTS: Mice treated with isoflavones performed significantly better in the cognitive test than the no-treatment control group (P < 0.05). The cortex AchE activity, serum SOD and GSH-Px activities were notably higher than those of the untreated mouse (P < 0.05). MDA concentration and the ȕ-secretase activity in the hippocampal tissue were both lower than those in no-treatment control (P < 0.05). The numbers of hippocampal neurons of isoflavones treatment mice were increased and the cellular morphology was significantly improved. CONCLUSION: Isoflavones can indirectly increase the concentration of the cholinergic neural transmitter Ach through regulation of AchE. Through reduction of hippocampal ȕ-secretase activity, the strong anti-oxidative activity of isoflavones can decrease the formation and deposition of insoluable ȕ-amyloid (Aȕ) debris, relieve the resulting toxicity and damage to neurons, and thereby effectively protect the nervous system. [KEY WORDS] Isoflavones; Senescence-accelerated mice; Acetylcholinesterase; ȕ-Secretase [CLC Number] R965
1
[Document code] A
[Article ID] 1672-3651(2010)04-0280-05
Introduction
Alzheimer’s disease (AD) is a chronic nervous system degenerative disease with complicated etiology. It is characterized by progressive decline of cognitivity and memory. Studies showed that ȕ-Amyloid (AE) deposition, cholinergic nerve damage and oxidative stress damage were the main causation factors in the pathological process of AD. The protective role of estrogen on brain has become a hot research topic worldwide in recent years. Estrogen can prevent or relieve Alzheimer’s disease through multiple protective [Received on] 30-Mar-2010 [Research Funding] This project was supported by Guangdong Technology Bureau (Nos. 2004B30101001 and 2009B030801343) and by research and development fund from Guangdong Chinese Medicine Administration (No.103079). [ Corresponding author] ZHOU Tian-Hong: Prof., Tel: 86-13392655005, E-mail: [email protected] Copyright © 2010, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.
mechanisms. However, severe side-effects after long time use of estrogen restricted its clinical application [1]. Data suggested that plant estrogen analogues have estrogen-like positive functions but no carcinogenesis side-effect[2]. Isoflavones are a natural plant estrogen extracted from soy beans. Previous data from our laboratory demonstrated that isoflavones had remarkable neural protective effects both in vivo and in vitro[3-4]. To further explore the mechanism of isoflavones, we utilized senescence-accelerated SAM-P/8 mice model in this study. Through the examination of the effects of isoflavones on the protection of cholinergic neural functions, the inhibition of AE deposition, and the anti-oxidative stress role, we discussed the possible mechanism of isoflavones, providing scientific basis for clinical usage of isoflavones in AD treatment.
2
Materials
2.1 Drugs Isoflavones extract was prepared from soy beans in our laboratory, with 70.98% of the content being genistein
YANG Hong, et al. /Chinese Journal of Natural Medicines 2010, 8(4): 280284
46.59%, daidzin 21.215% and glycitein 3.175%, determined by HPLC; 17-ȕestradiol was purchased from Sigma Inc. (St. Louis, USA); SOD, MDA and GSH-Px assay kits, BCA protein quantificatioin kit were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); ȕ-secretase assay kit was bought from R&D Systems Co. Ltd (Minneapolis, USA). 2.2 Experimental animal Specific pathogen free 6-month old SAM-P/8 mice were purchased from Tianjin University of Traditional Chinese Medicine (Tianjin, China). All experiments were conducted according to the guidelines issued by the institutional Animal Care Committee and in compliance with the regulations formulated by the Chinese government.
3 3.1
Methods
Animal treatment groups and drug administration Fifty male SAM-P/8 mice were randomly divided into 5 groups: no treatment control group, low, middle, high dose isoflavones group and estradiol treatment positive control group. The no treatment control group was given saline intragastrically while the positive control group was administered with 0.298 mg·kg-1 estradiol intragastrically. The three drug treatment groups were intragastrically administered daily with 50, 100 and 200 mg·kg-1 isoflavones respectively for 25 consecutive days. 3.2 Determination of AchE activity Cortex tissue of the brain was extracted, weighted and added with pre-chilled saline (10 g·L-1) to submerge the tissue. The brain tissue was then homogenized in an ice-bath. The mixture was centrifuged at 4 000 r·min-1, 4 qC for 20 min. The supernatant was transferred to clean tubes and stored at 70 qC before use [5]. Determination of AchE activity was conducted by a Model 721 UV-Visor spectrophotometer according to the instructions of the kit manual. 3.3 Determination of serum SOD, GSH-Px activities and MDA level Blood was drawn from the eye balls of mice, which were then killed. After 4 h settlement, the blood was centrifuged to obtain serum. Serum SOD, GSH-Px and MDA levels were then measured according to manufacture’s instructions[6]. 3.4 Determination of hippocampal E-secretase activity E-secretase activity was determined following Tyler et [7] al. and kit protocol. Mice brain tissue was extracted and washed in pre-chilled saline before both sides of hippocampuses were immediately excised on the ice plate. The hippocampuses were then weighed to accuracy, cut to small pieces and transferred to a 2mL glass homogenizer. Lysis buffer (4 mL·g1) was added to the homogenizer and the tissue was thoroughly ground over an ice-bath. The mixture was then transferred to a sterile EP tube and incubated on ice for 30 min to allow the complete reaction of lysis buffer and homogenate. Later, the mixture was centrifuged at 12 000 r·min1 for 30 min at 4qC and the supernatant was made into
200 L aliquots before it was stored at 70 qC. Protein concentrations were determined by the BCA method. Final concentration of each protein sample was adjusted to 1.5 mg·mL1 using the lysis buffer. In the darkroom, the protein extract (50 L), 2x reaction buffer (50 L) and substrate (5 L) were sequentially added to a black 96-well plate. Each sample was assayed in duplicate. Substrate control and sample control (mixture of multiple protein extracts) were also prepared. The plate was incubated at 37 qC for 1 h before the fluorescence units were determined with a fluorescent plate reader. The excitation wavelength was set to 355 nm and the emission wavelength to 510 nm[8]. 3.5 Morphological observation of AD mice hippocampal structure Hippocampal tissues of the brain were dehydrated and embedded in paraffin before they were sliced and stained with Hematoxylin and Eosin (HE) dye. Slices were observed under a light microscope with images taken. 3.6 Statistical analysis Data were analyzed using ANOVA for significance test. All results were expressed as x ±s.
4
Results and Discussion
4.1 Effect of isoflavones on cortex AchE activity of AD mice As listed in Table 1, all three isoflavones treatment groups showed significantly decreased AchE activity (P < 0.05) in a dose-dependent manner. In addition, the effect of the high-dose treatment group was better than the effect of estradiol positive control group. Table 1 Comparison of AchE activity in cortex of AD mice ( x ± s, n = 10) Group
Dose (mg·kg1) AchE activity (U·mg1)
No treatment control
0.690 1 ± 0.008 2
Low isoflavones dose
50
0.657 8 ± 0.026 0*
Middle isoflavones dose
100
0.643 7 ± 0.033 2*
High isoflavones dose
200
0.637 7 ± 0.015 02*
Estradiol positive control
0.298
0.645 7 ± 0.013 64*
*P < 0.05 vs control group
4.2 Effects of isoflavones on mouse serum SOD, GSH-Px and MDA levels All three isoflavones treatment groups showed significantly increased SOD and GSH-Px activities than the notreatment control group (P < 0.05), while the MDA levels were significantly lower than the no-treatment control (P < 0.05). Effect of the high-dose treatment group was close to that of the estradiol positive control. 4.3 Effect of isoflavones on the mouse hippocampal -secretase activity As shown in Table 3, all three isoflavones treatment groups had significantly decreased-secretase activity in the m o u s e h i p p o c a m p u s ( P < 0 . 0 5 ). T h e h i g h - d o s e
YANG Hong, et al. /Chinese Journal of Natural Medicines 2010, 8(4): 280284 Table 2 Comparisons of SOD activity, GSH-Px activity and MDA levels in AD mice ( x ± s, n = 10) Group
Dose (mg·kgí1)
SOD (U·mL1) 88.20 ± 12.11
199.9 ± 16.45
21.78 ± 5.074
50
105.0 ± 10.34*
220.4 ± 10.70*
14.65 ± 2.080*
No treatment control Low isoflavones dose
GSH-Px (U·L1)
MDA (ȝmol·L1)
Middle isoflavones dose
100
106.4 ± 5.021*
226.9 ± 14.89*
13.12 ± 2.089*
High isoflavones dose
200
114.6 ± 11.85*
245.5 ± 29.32*
13.00 ± 1.698*
115.0 ± 13.25*
235.5 ± 10.88*
13.63 ± 2.508*
Estradiol positive control
0.298
*P < 0.05 vs control group
Table 3
Comparison of the E-secretase activity (fluores-
cence units) in hippocampus of AD mice ( x ± s, n = 10) Group
Dose (mg·kgí1)
No treatment control Low isoflavones dose Middle isoflavones dose High isoflavones dose Positive control Substrate control Sample control
Fluorescence unit 22 729 ± 125.1
50 100 200 0.298
22 577 ± 50.62* 22 563 ± 26.79* 22 526 ± 44.08* 22 532 ± 119.8589* 758.7 ± 27.16 477.7 ± 22.23
*P < 0.05 vs control group
isoflavones treatment group was more effective than the estradiol positive control group. 4.4 Effect of isoflavones on the AD mouse hippocampal structure As shown in Fig.1, the no treatment control group possessed reduced hippocampal neuron numbers with many of the cells showing decreased volume and increased nuclear condensation. However hippocampal neurons from the isoflavones treated mice showed distinct cell membrane, highly-ordered arrangement, complete composition and a much higher cell number than the no treatment control group.
Low isoflavones dose (50 mg·kg1)
No treatment control
Meddle isoflavones dose (100mg·kg1)
High isoflavones dose (200 mg·kg1)
Estradiol positive control
Fig. 1. Histological section of hippocampus from AD mice treated with various amounts of isoflavones (HE, 40×)
YANG Hong, et al. /Chinese Journal of Natural Medicines 2010, 8(4): 280284
The treatment groups also showed similar structures and cell numbers to the estradiol positive control group.
5
Discussion
Soybean isoflavones have multiple phenolic hydroxyl groups, therefore its anti-oxidative activity is far superior than that of the glycosides. In this research, we significantly improved the percentage of aglycones in soybean isoflavones through optimized extraction process, making its biological activity more potent in vivo. Researches in recent years demonstrated that soybean isoflavones had good treatment outcomes for improving cognition and memory. These researches were mostly based on cholinergic neural damage models induced by hippocampal injection of D-galactose or Aȕ. However, these models could only reflect a single aspect of pathological symptoms and the corresponding detection methods were also limited, thereby unable to fully reveal the anti-AD mechanism of isoflavones. In this study, we selected a currently more-ideal model that can better reflect the pathological characteristics of AD patients, the SAM-P/8 senescence-accelerated mice model. These mice showed large amount of amyloidҏ (A) deposition in their brains [9], alteration of neural transmitters related to learning and memory (such as acetyl choline in cortex and hippocampus of the brain) [10-11], increased lipid peroxide levels in the brain compared to SAMR1 mice of the same age, disturbed anti-oxidation system, detectable oxidative stress damage [12-13] , and aging-related learning-memory function degeneration. Through systematical studies, this model can provide direct back-up evidence for the anti-AD mechanism of isoflavones. It has been reported that a large number of cholinergic neurons in AD patients’ basal forebrains were damaged or dead, causing significantly decreased ability to acquire choline. Interestingly, the extent of these effects was positively correlated with damage of patients’ cognitive functions [14-15]. Acetylcholinesterase (AchE) is an Ach specific hydrolase. Increased AChE activity may lead to expedited hydrolysis of the hippocampal cholinergic neural transmitter, Ach. When the Ach content was reduced, the neural transmitter’s function also dramatically decreased, therefore causing decreased learning and memory performance. Results from this study showed that isoflavones-treated AD mice could significantly reduce the cortex AchE activity. The results implicated that isoflavones could regulate AchE activity in the brain and indirectly increase the content of cholinergic neural transmitter Ach, which in turn improved the central cholinergic function and increased mouse learning and memory ability. Researches in recent years discovered that oxidative damage played an important role in neuron degeneration and cell apoptosis. The free radical theory of AD pointed out that imbalance of oxidation-reduction in the body could increase the production of lipid peroxide, cause alteration of numerous cellular enzyme activities, and finally speed up the neural
degeneration process [16-17]. In this study, we examined serum levels of three biochemical anti-oxidation indicators, MDA, SOD and GSH-Px. Results demonstrated that isoflavones could increase the activities of SOD and GSH-Px, while decreasing the level of MDA. In other words, isoflavones strengthened the anti-oxidation ability of the body, reduced neural damages from free radicals, and effectively postponed the degeneration and apoptosis of neurons. Among the many causation factors of AD, the most commonly recognized initiator is deposition of AESenile plaques found in the brain of AD patients are considered as a consistent characteristic of Alzheimer’s disease, and likely the causation of AD [18-19]. Senile plaques are mainly made up of AE. Results in this study indicated that the isoflavones could directly inhibit E-secretase activity, effectively decrease the formation and deposition of AE, reduce the AE mediated neural toxicity and apoptosis, improve the neuronal cell number and morphology, thereby protecting the important tissue structure of hippocampus. Among the many proposed etiologic mechanisms of AD, the most convincing hypothesis is the amyloid waterfall theory. According to this theory, genetic or environmental factors cause abnormal accumulation of Aȕ ҏin cells, forming amyloid plaques; meanwhile, these factors activate neuroglia cells and produce many inflammatory mediators; Through oxidative reaction, apoptosis and other pathways, some mediators directly damage neurons and synapses, while others further promote the synthesis of Aȕ, leading to neuron degeneration and chronic pathogenesis of AD. Combined data in this study suggested that isoflavones could target several factors of AD, such as the cholinergic neural function, anti-oxidation ability and Aȕ formation. Therefore, isoflavones has neural protective functions from multiple etiological angles of AD, improving the chronic degeneration of the nervous system. This study provides a thorough theoretical basis for the anti-AD effect of isoflavones, and suggests a bright future for isoflavones to replace estrogen in AD treatment.
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Rossouw JE, Anderson GL, Prentice RL, et a1. Risksand benefits of estrogen plus progestin in healthypostmenopausal women: principal results from the women health initiative randomized controlled tria1[J].JAMA,2002, 288(3): 321-333. Bang OH, Hong D, Kim JB, et al.Neuroprotective effect of genistein against beta amyloid-induced neurotoxicity [J].Neurobiol Dis, 2004,16(1): 21-28. Yang H, Chen WQ, Zhao YH, et al. Effect of soybean i soflavones active-extracts on learning and memory in AD mice.[J].Chin Tradit Herb Drug, 2005, 36(6):885-886. Yang H, Chen WQ, Zhao YH, et al. The protective effect of components of isoflavones on the PC12 cells damage induced by Aȕ1-42. [J]. Chin Pharmacol Bull, 2005,21(9):1089-1091. Yang WM, Yang JF, Chen B, et al. Effects of Zhinao cap-
YANG Hong, et al. /Chinese Journal of Natural Medicines 2010, 8(4): 280284 sule on learning, memory and cerebral cholinergic neurotransmitter system in rat models of Alzheimer disease [J]. Chin Clin Rehabilit, 2004, 8(28): 6262-6264. [6] Li QM, Meng RS, Wei CX, et al. Effects of Naohuandan Recipe on learning and memory abilities of SAM-P/8 mice and its role in anti-oxidation and anti-apoptosis [J]. Chin Integr Med, 2006, 4(1): 48-51. [7] Tyler SJ, Dawbarn D, Wilcock GK, et al. Alpha- and beta-secretase: profound changes in Alzheimer’s disease [J]. Biochem Biophys Res Commun, 2002, 299: 373-376. [8] Zhang J, Yan FL, Guan XN. Effect of Egb761 on Activity of ȕ-secterase in hippocampus of old rats after chronic cerebral hypoperfusion [J]. Chin Clin Neu Sci, 2006, 14(5): 454-457. [9] Petursdottir AL, Farr SA, Morley JE, et al. Lipid peroxidation in brain during aging in the senescence-accelerated mouse (SAM) [J]. Neurobiol Aging, 2007, 28 (8): 1170-1178. [10] Chan YC, Hosoda K, Tsai CJ, et al. Favorable effects of tea on reducing the cognitive deficits and brain morphological changes in senescence-accelerated mice [J]. J Nutr Sci Vitaminol (Tokyo), 2006, 52(4): 266-273. [11] Liao JW, Hsu CK, Wang MF, et al. Beneficial effect of Toona sinensis Roemor on improving cognitive performance and brain degeneration in senescence-accelerated mice [J]. Br J Nutr, 2006, 96 (2): 400-407. [12] Flood JF, Farr SA, Uezu K, et al. Age-related changes in
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septal serotonergic, GABAergic and glutamatergic facilitation of retention in SAMP 8 mice [J]. Mech Ageing Dev, 1998, 105(122): 173-188. Zhao WH, Yu JC, Ji XC, et al. Learning and memory deficits in SAMP8 and their biochemical mechanisms[J]. J Xi’an Jiaotong Univ (Med Sci), 2008, 29(3): 289-293. Talesa VN. Acetylcholinesterase in Alzheimer’s disease [J].Mech Ageing Dev, 2001, 122(16): 1961-1969. Ma DL, Hu HT, Yang PB, et al. Protective effect of rAAV vector mediated hBDNF on rat basal forebrain cholinergic neurons damaged by amyloid ȕ-peptide [J]. J Xi’an Jiaotong Univ (Med Sci), 2004, 25 (1): 27-29. Yatin S, Aksenov M, Butterfield DA. The antioxidant vitamin E modulates amyloid beta 2 peptide-induced creatine kinase activity inhibition and increased protein oxidation: implications for the free radical hypothesis of Alzheimer’s disease [J]. Neurochem Res, 1999, 24 (3): 427-435. Shi Q, Gibson GE. Oxidative stress and transcriptional regulation in Alzheimer’s disease [J]. Alzheimer Dis Assoc Disord, 2007, 21(4): 276-291. Frost D, Gorman PM, Yip CM. Co-incorporation of Abeta 40 and A beta42 to form mixed pre-fibrillar aggregates [J]. Eur J Biochem, 2003, 270(4): 654-663. Townsend KP, Obregon D, Quadtros A, et al. Proinflammatory and vasoactive effects of abeta in the cerebrovasculature [J].Ann NY Acad Sci, 2002, 977(1):65-76.
䅍㵋䢶䊋ⰵ㌍㯺㎰⿐㾂㭔㪒㈎⡄⿅⭥〛䐧䁱㈠ ཷ
܃1, 2, ࠡ ֹک2, ఉՋՋ 2, ࡇ 2, ᄻඟ ۿ1*
1
᰽Ӗ༰ಓࢳ༰ޏ೬༰ၝ, ڜᄼ 510632
2
ڜՊྐ༰ၝ҄ݮ༰ၝ, ڜᄼ 510006
ᨬ 㽕Ⳃⱘ˖൰ඉ࿓ܻර፣၍ճࣙമच໌ܤ೪ಊ࠼ͬܙԅᆴဈݯᄥdᮍ⊩˖ྻϢලޔ२࿓ܻර፣၍ڙวᄭ९ SAM-P/8 ࣙമच໌ܤ೪; Љ໌೪ଣЊྐྵᱟө߁ᱡਝ(AchE)ݣ; Љ໌೪༳ூбཾܤ୦ܤਝ(SOD)cځᣧإᣔܤཾڶਝ(GSH-Px)ݣ ॏۤτ֝ (MDA) ૉէ; ֥ڛЉՇں৴ᆦᄎ ȕ-דਿਝݣॏ; ມฑࡄڔГں৴ᆦᄎಊ࠼၍ͦԅέܤd㒧ᵰ˖ᄭ९ᆦ໌೪ଣЊ AchE ݣc༳ூ SOD ۤ GSH-Px ԅݣॏੜມغဟ੦ᆦ(P < 0.05); MDA ഃ଼cں৴ᆦᄎ ȕ-דਿਝݣॏ࢈Ԏဟ੦ᆦ(P < 0.05), ں৴ಊ࠼၍२ۤͦൟੜມ؟d㒧䆎˖࿓ܻර፣၍ඹڶԸࠋ AchE ݣ, ࠄඔغө߁ટಊ࠼Ԟᄩ ACh ۃ२, ؟ᄯ ө߁ટٟટ, ႙ஜ༰ޚ࿋ટॏ; யပஜࢦཾݣܤ, ωඹߦڶԎں৴ ȕ-דਿਝݣ, ߈Ϣగ Aȕ ാଫԅСಓۤцݲ, ߈ ாୣӽᄡԅಊ࠼՝ۤಊ࠼၍ͦԅ, ပ໒݈֟ಊ࠼ͬܙᆴဈd ݇䬂䆡 ࿓ܻර፣၍; ࣙമच໌ܤ೪; ྐྵᱟө߁ᱡਝ; ȕ-דਿਝ
䞥乍Ⳃ
ڜՊಘࢳޏද(ંົܣޙNos. 2004B30101001, 2009B030801343)ĢڜՊಘᄯྤྐڕसࡥࢳཙົં(No. 103079)
Chinese Journal of Natural Medicines
Neuro-protective Mechanism of Isoflavones on Senescence-accelerated Mice YANG Hong1, 2, JIN Gui-Fang2, REN Dong-Dong2, LUO Si-Jing2, ZHOU Tian-Hong1* 1
College of Life Science and TechnologyēJinan University, Guangzhou 510632; College of Basic Courses, Guangzhou Higher Education Mega Center, Guangdong Pharmaceutical University, Guangzhou 510006, China 2
Available online 20 July 2010
[ABSTRACT] AIM: To investigate the neuro-protective mechanism of isoflavones, using senescence-accelerated SAM-P/8 mice. METHODS: Various dosages of isoflavones were intragastrically administered to SAM-P/8 senescence-accelerated mice. The cortex acetylcholinesterase (AchE) activity, serum superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were examined. In addition, the concentration of malondialdehyde (MDA) was examined. ȕ-secretase activity in the hippocampus tissue was determined by fluorometry. Changes of the hippocampal neurons were observed under a light microscope. RESULTS: Mice treated with isoflavones performed significantly better in the cognitive test than the no-treatment control group (P < 0.05). The cortex AchE activity, serum SOD and GSH-Px activities were notably higher than those of the untreated mouse (P < 0.05). MDA concentration and the ȕ-secretase activity in the hippocampal tissue were both lower than those in no-treatment control (P < 0.05). The numbers of hippocampal neurons of isoflavones treatment mice were increased and the cellular morphology was significantly improved. CONCLUSION: Isoflavones can indirectly increase the concentration of the cholinergic neural transmitter Ach through regulation of AchE. Through reduction of hippocampal ȕ-secretase activity, the strong anti-oxidative activity of isoflavones can decrease the formation and deposition of insoluable ȕ-amyloid (Aȕ) debris, relieve the resulting toxicity and damage to neurons, and thereby effectively protect the nervous system. [KEY WORDS] Isoflavones; Senescence-accelerated mice; Acetylcholinesterase; ȕ-Secretase [CLC Number] R965
1
[Document code] A
[Article ID] 1672-3651(2010)04-0280-05
Introduction
Alzheimer’s disease (AD) is a chronic nervous system degenerative disease with complicated etiology. It is characterized by progressive decline of cognitivity and memory. Studies showed that ȕ-Amyloid (AE) deposition, cholinergic nerve damage and oxidative stress damage were the main causation factors in the pathological process of AD. The protective role of estrogen on brain has become a hot research topic worldwide in recent years. Estrogen can prevent or relieve Alzheimer’s disease through multiple protective [Received on] 30-Mar-2010 [Research Funding] This project was supported by Guangdong Technology Bureau (Nos. 2004B30101001 and 2009B030801343) and by research and development fund from Guangdong Chinese Medicine Administration (No.103079). [ Corresponding author] ZHOU Tian-Hong: Prof., Tel: 86-13392655005, E-mail: [email protected] Copyright © 2010, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.
mechanisms. However, severe side-effects after long time use of estrogen restricted its clinical application [1]. Data suggested that plant estrogen analogues have estrogen-like positive functions but no carcinogenesis side-effect[2]. Isoflavones are a natural plant estrogen extracted from soy beans. Previous data from our laboratory demonstrated that isoflavones had remarkable neural protective effects both in vivo and in vitro[3-4]. To further explore the mechanism of isoflavones, we utilized senescence-accelerated SAM-P/8 mice model in this study. Through the examination of the effects of isoflavones on the protection of cholinergic neural functions, the inhibition of AE deposition, and the anti-oxidative stress role, we discussed the possible mechanism of isoflavones, providing scientific basis for clinical usage of isoflavones in AD treatment.
2
Materials
2.1 Drugs Isoflavones extract was prepared from soy beans in our laboratory, with 70.98% of the content being genistein
YANG Hong, et al. /Chinese Journal of Natural Medicines 2010, 8(4): 280284
46.59%, daidzin 21.215% and glycitein 3.175%, determined by HPLC; 17-ȕestradiol was purchased from Sigma Inc. (St. Louis, USA); SOD, MDA and GSH-Px assay kits, BCA protein quantificatioin kit were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); ȕ-secretase assay kit was bought from R&D Systems Co. Ltd (Minneapolis, USA). 2.2 Experimental animal Specific pathogen free 6-month old SAM-P/8 mice were purchased from Tianjin University of Traditional Chinese Medicine (Tianjin, China). All experiments were conducted according to the guidelines issued by the institutional Animal Care Committee and in compliance with the regulations formulated by the Chinese government.
3 3.1
Methods
Animal treatment groups and drug administration Fifty male SAM-P/8 mice were randomly divided into 5 groups: no treatment control group, low, middle, high dose isoflavones group and estradiol treatment positive control group. The no treatment control group was given saline intragastrically while the positive control group was administered with 0.298 mg·kg-1 estradiol intragastrically. The three drug treatment groups were intragastrically administered daily with 50, 100 and 200 mg·kg-1 isoflavones respectively for 25 consecutive days. 3.2 Determination of AchE activity Cortex tissue of the brain was extracted, weighted and added with pre-chilled saline (10 g·L-1) to submerge the tissue. The brain tissue was then homogenized in an ice-bath. The mixture was centrifuged at 4 000 r·min-1, 4 qC for 20 min. The supernatant was transferred to clean tubes and stored at 70 qC before use [5]. Determination of AchE activity was conducted by a Model 721 UV-Visor spectrophotometer according to the instructions of the kit manual. 3.3 Determination of serum SOD, GSH-Px activities and MDA level Blood was drawn from the eye balls of mice, which were then killed. After 4 h settlement, the blood was centrifuged to obtain serum. Serum SOD, GSH-Px and MDA levels were then measured according to manufacture’s instructions[6]. 3.4 Determination of hippocampal E-secretase activity E-secretase activity was determined following Tyler et [7] al. and kit protocol. Mice brain tissue was extracted and washed in pre-chilled saline before both sides of hippocampuses were immediately excised on the ice plate. The hippocampuses were then weighed to accuracy, cut to small pieces and transferred to a 2mL glass homogenizer. Lysis buffer (4 mL·g1) was added to the homogenizer and the tissue was thoroughly ground over an ice-bath. The mixture was then transferred to a sterile EP tube and incubated on ice for 30 min to allow the complete reaction of lysis buffer and homogenate. Later, the mixture was centrifuged at 12 000 r·min1 for 30 min at 4qC and the supernatant was made into
200 L aliquots before it was stored at 70 qC. Protein concentrations were determined by the BCA method. Final concentration of each protein sample was adjusted to 1.5 mg·mL1 using the lysis buffer. In the darkroom, the protein extract (50 L), 2x reaction buffer (50 L) and substrate (5 L) were sequentially added to a black 96-well plate. Each sample was assayed in duplicate. Substrate control and sample control (mixture of multiple protein extracts) were also prepared. The plate was incubated at 37 qC for 1 h before the fluorescence units were determined with a fluorescent plate reader. The excitation wavelength was set to 355 nm and the emission wavelength to 510 nm[8]. 3.5 Morphological observation of AD mice hippocampal structure Hippocampal tissues of the brain were dehydrated and embedded in paraffin before they were sliced and stained with Hematoxylin and Eosin (HE) dye. Slices were observed under a light microscope with images taken. 3.6 Statistical analysis Data were analyzed using ANOVA for significance test. All results were expressed as x ±s.
4
Results and Discussion
4.1 Effect of isoflavones on cortex AchE activity of AD mice As listed in Table 1, all three isoflavones treatment groups showed significantly decreased AchE activity (P < 0.05) in a dose-dependent manner. In addition, the effect of the high-dose treatment group was better than the effect of estradiol positive control group. Table 1 Comparison of AchE activity in cortex of AD mice ( x ± s, n = 10) Group
Dose (mg·kg1) AchE activity (U·mg1)
No treatment control
0.690 1 ± 0.008 2
Low isoflavones dose
50
0.657 8 ± 0.026 0*
Middle isoflavones dose
100
0.643 7 ± 0.033 2*
High isoflavones dose
200
0.637 7 ± 0.015 02*
Estradiol positive control
0.298
0.645 7 ± 0.013 64*
*P < 0.05 vs control group
4.2 Effects of isoflavones on mouse serum SOD, GSH-Px and MDA levels All three isoflavones treatment groups showed significantly increased SOD and GSH-Px activities than the notreatment control group (P < 0.05), while the MDA levels were significantly lower than the no-treatment control (P < 0.05). Effect of the high-dose treatment group was close to that of the estradiol positive control. 4.3 Effect of isoflavones on the mouse hippocampal -secretase activity As shown in Table 3, all three isoflavones treatment groups had significantly decreased-secretase activity in the m o u s e h i p p o c a m p u s ( P < 0 . 0 5 ). T h e h i g h - d o s e
YANG Hong, et al. /Chinese Journal of Natural Medicines 2010, 8(4): 280284 Table 2 Comparisons of SOD activity, GSH-Px activity and MDA levels in AD mice ( x ± s, n = 10) Group
Dose (mg·kgí1)
SOD (U·mL1) 88.20 ± 12.11
199.9 ± 16.45
21.78 ± 5.074
50
105.0 ± 10.34*
220.4 ± 10.70*
14.65 ± 2.080*
No treatment control Low isoflavones dose
GSH-Px (U·L1)
MDA (ȝmol·L1)
Middle isoflavones dose
100
106.4 ± 5.021*
226.9 ± 14.89*
13.12 ± 2.089*
High isoflavones dose
200
114.6 ± 11.85*
245.5 ± 29.32*
13.00 ± 1.698*
115.0 ± 13.25*
235.5 ± 10.88*
13.63 ± 2.508*
Estradiol positive control
0.298
*P < 0.05 vs control group
Table 3
Comparison of the E-secretase activity (fluores-
cence units) in hippocampus of AD mice ( x ± s, n = 10) Group
Dose (mg·kgí1)
No treatment control Low isoflavones dose Middle isoflavones dose High isoflavones dose Positive control Substrate control Sample control
Fluorescence unit 22 729 ± 125.1
50 100 200 0.298
22 577 ± 50.62* 22 563 ± 26.79* 22 526 ± 44.08* 22 532 ± 119.8589* 758.7 ± 27.16 477.7 ± 22.23
*P < 0.05 vs control group
isoflavones treatment group was more effective than the estradiol positive control group. 4.4 Effect of isoflavones on the AD mouse hippocampal structure As shown in Fig.1, the no treatment control group possessed reduced hippocampal neuron numbers with many of the cells showing decreased volume and increased nuclear condensation. However hippocampal neurons from the isoflavones treated mice showed distinct cell membrane, highly-ordered arrangement, complete composition and a much higher cell number than the no treatment control group.
Low isoflavones dose (50 mg·kg1)
No treatment control
Meddle isoflavones dose (100mg·kg1)
High isoflavones dose (200 mg·kg1)
Estradiol positive control
Fig. 1. Histological section of hippocampus from AD mice treated with various amounts of isoflavones (HE, 40×)
YANG Hong, et al. /Chinese Journal of Natural Medicines 2010, 8(4): 280284
The treatment groups also showed similar structures and cell numbers to the estradiol positive control group.
5
Discussion
Soybean isoflavones have multiple phenolic hydroxyl groups, therefore its anti-oxidative activity is far superior than that of the glycosides. In this research, we significantly improved the percentage of aglycones in soybean isoflavones through optimized extraction process, making its biological activity more potent in vivo. Researches in recent years demonstrated that soybean isoflavones had good treatment outcomes for improving cognition and memory. These researches were mostly based on cholinergic neural damage models induced by hippocampal injection of D-galactose or Aȕ. However, these models could only reflect a single aspect of pathological symptoms and the corresponding detection methods were also limited, thereby unable to fully reveal the anti-AD mechanism of isoflavones. In this study, we selected a currently more-ideal model that can better reflect the pathological characteristics of AD patients, the SAM-P/8 senescence-accelerated mice model. These mice showed large amount of amyloidҏ (A) deposition in their brains [9], alteration of neural transmitters related to learning and memory (such as acetyl choline in cortex and hippocampus of the brain) [10-11], increased lipid peroxide levels in the brain compared to SAMR1 mice of the same age, disturbed anti-oxidation system, detectable oxidative stress damage [12-13] , and aging-related learning-memory function degeneration. Through systematical studies, this model can provide direct back-up evidence for the anti-AD mechanism of isoflavones. It has been reported that a large number of cholinergic neurons in AD patients’ basal forebrains were damaged or dead, causing significantly decreased ability to acquire choline. Interestingly, the extent of these effects was positively correlated with damage of patients’ cognitive functions [14-15]. Acetylcholinesterase (AchE) is an Ach specific hydrolase. Increased AChE activity may lead to expedited hydrolysis of the hippocampal cholinergic neural transmitter, Ach. When the Ach content was reduced, the neural transmitter’s function also dramatically decreased, therefore causing decreased learning and memory performance. Results from this study showed that isoflavones-treated AD mice could significantly reduce the cortex AchE activity. The results implicated that isoflavones could regulate AchE activity in the brain and indirectly increase the content of cholinergic neural transmitter Ach, which in turn improved the central cholinergic function and increased mouse learning and memory ability. Researches in recent years discovered that oxidative damage played an important role in neuron degeneration and cell apoptosis. The free radical theory of AD pointed out that imbalance of oxidation-reduction in the body could increase the production of lipid peroxide, cause alteration of numerous cellular enzyme activities, and finally speed up the neural
degeneration process [16-17]. In this study, we examined serum levels of three biochemical anti-oxidation indicators, MDA, SOD and GSH-Px. Results demonstrated that isoflavones could increase the activities of SOD and GSH-Px, while decreasing the level of MDA. In other words, isoflavones strengthened the anti-oxidation ability of the body, reduced neural damages from free radicals, and effectively postponed the degeneration and apoptosis of neurons. Among the many causation factors of AD, the most commonly recognized initiator is deposition of AESenile plaques found in the brain of AD patients are considered as a consistent characteristic of Alzheimer’s disease, and likely the causation of AD [18-19]. Senile plaques are mainly made up of AE. Results in this study indicated that the isoflavones could directly inhibit E-secretase activity, effectively decrease the formation and deposition of AE, reduce the AE mediated neural toxicity and apoptosis, improve the neuronal cell number and morphology, thereby protecting the important tissue structure of hippocampus. Among the many proposed etiologic mechanisms of AD, the most convincing hypothesis is the amyloid waterfall theory. According to this theory, genetic or environmental factors cause abnormal accumulation of Aȕ ҏin cells, forming amyloid plaques; meanwhile, these factors activate neuroglia cells and produce many inflammatory mediators; Through oxidative reaction, apoptosis and other pathways, some mediators directly damage neurons and synapses, while others further promote the synthesis of Aȕ, leading to neuron degeneration and chronic pathogenesis of AD. Combined data in this study suggested that isoflavones could target several factors of AD, such as the cholinergic neural function, anti-oxidation ability and Aȕ formation. Therefore, isoflavones has neural protective functions from multiple etiological angles of AD, improving the chronic degeneration of the nervous system. This study provides a thorough theoretical basis for the anti-AD effect of isoflavones, and suggests a bright future for isoflavones to replace estrogen in AD treatment.
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䅍㵋䢶䊋ⰵ㌍㯺㎰⿐㾂㭔㪒㈎⡄⿅⭥〛䐧䁱㈠ ཷ
܃1, 2, ࠡ ֹک2, ఉՋՋ 2, ࡇ 2, ᄻඟ ۿ1*
1
᰽Ӗ༰ಓࢳ༰ޏ೬༰ၝ, ڜᄼ 510632
2
ڜՊྐ༰ၝ҄ݮ༰ၝ, ڜᄼ 510006
ᨬ 㽕Ⳃⱘ˖൰ඉ࿓ܻර፣၍ճࣙമच໌ܤ೪ಊ࠼ͬܙԅᆴဈݯᄥdᮍ⊩˖ྻϢලޔ२࿓ܻර፣၍ڙวᄭ९ SAM-P/8 ࣙമच໌ܤ೪; Љ໌೪ଣЊྐྵᱟө߁ᱡਝ(AchE)ݣ; Љ໌೪༳ூбཾܤ୦ܤਝ(SOD)cځᣧإᣔܤཾڶਝ(GSH-Px)ݣ ॏۤτ֝ (MDA) ૉէ; ֥ڛЉՇں৴ᆦᄎ ȕ-דਿਝݣॏ; ມฑࡄڔГں৴ᆦᄎಊ࠼၍ͦԅέܤd㒧ᵰ˖ᄭ९ᆦ໌೪ଣЊ AchE ݣc༳ூ SOD ۤ GSH-Px ԅݣॏੜມغဟ੦ᆦ(P < 0.05); MDA ഃ଼cں৴ᆦᄎ ȕ-דਿਝݣॏ࢈Ԏဟ੦ᆦ(P < 0.05), ں৴ಊ࠼၍२ۤͦൟੜມ؟d㒧䆎˖࿓ܻර፣၍ඹڶԸࠋ AchE ݣ, ࠄඔغө߁ટಊ࠼Ԟᄩ ACh ۃ२, ؟ᄯ ө߁ટٟટ, ႙ஜ༰ޚ࿋ટॏ; யပஜࢦཾݣܤ, ωඹߦڶԎں৴ ȕ-דਿਝݣ, ߈Ϣగ Aȕ ാଫԅСಓۤцݲ, ߈ ாୣӽᄡԅಊ࠼՝ۤಊ࠼၍ͦԅ, ပ໒݈֟ಊ࠼ͬܙᆴဈd ݇䬂䆡 ࿓ܻර፣၍; ࣙമच໌ܤ೪; ྐྵᱟө߁ᱡਝ; ȕ-דਿਝ
䞥乍Ⳃ
ڜՊಘࢳޏද(ંົܣޙNos. 2004B30101001, 2009B030801343)ĢڜՊಘᄯྤྐڕसࡥࢳཙົં(No. 103079)