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THE ANATOMICAL RECORD 294:1003–1014 (2011)

Protective Effect of Extract of Astragalus on Learning and Memory Impairments and Neurons Apoptosis Induced by Glucocorticoids in 12-Month Male Mice WEI-ZU LI, WEI-PING LI,* WEN ZHANG, YAN-YAN YIN, XIANG-XIANG SUN, SU-SU ZHOU, XIAO-QIONG XU, AND CHUN-RONG TAO Department of Pharmacology, Basic Medical College, Anhui Medical University, Hefei 230032, People’s Republic of China

ABSTRACT Alzheimer’s disease (AD) is a chronic neurodegenerative disorder marked by a progressive loss of memory and cognitive function. Stresslevel glucocorticoids are correlated with dementia progression in patients with AD. In this study, 12-month male mice were chronically treated with stress-level dexamethasone (DEX, 5 mg/kg) and extract of Astragalus (EA, 10, 20, and 40 mg/kg) or Ginsenoside Rg1 (Rg1, 6.5 mg/kg) for 21 days. We investigated the protective effect of EA against DEX injury in mice and its action mechanism. Our results indicate that DEX can induce learning and memory impairments and neuronal cell apoptosis. The mRNA levels of caspase-3 are selectively increased after DEX administration. The results of immunohistochemistry demonstrate that caspase-3 and cytochrome c in hippocampus (CA1, CA3) and neocortex are significantly increased. Furthermore, DEX treatment increased the activity of caspase-9 and caspase-3. Treatment groups with EA (20 and 40 mg/kg) or Rg1 (6.5 mg/kg) significantly improve learning and memory, downregulate the mRNA level of caspase-3, decrease expression of caspase-3 and cytochrome c in hippocampus (CA1, CA3) and neocortex, and inhibit activity of caspase-9 and caspase-3. The present findings highlight a possible mechanism by which stress level of DEX accelerates learning and memory impairments and increases neuronal apoptosis and the potential neuronal protection of EA. C 2011 Wiley-Liss, Inc. Anat Rec, 294:1003–1014, 2011. V

Key words: Alzheimer’s disease; memory and learning impairment; dexamethasone; apoptosis; extract of Astragalus

The glucocorticoids (GCs) response to stressful stimuli is regulated by the hypothalamic–pituitary–adrenal (HPA) axis, which triggers the adrenal cortex to release glucocorticoids (cortisol in primates and corticosterone in mice and rats). Rodent and primates studies suggest that chronic exposure to elevated glucocorticoids has neurotoxic effects and lowers the threshold for hippocampal neuronal degeneration and loss (Uno et al., 1989; Sapolsky et al., 1990). Glucocorticoids-related neuronal injury has been proposed as a mechanism in neurodegenerative disorder such as Alzheimer’s disease (AD). There is ample evidence implicating HPA axis dysfunction in AD, reflected by markedly elevated basal level of circulating cortisol (Swanwick et al., 1998) and a C 2011 WILEY-LISS, INC. V

Grant sponsor: Nature Science Foundation of Anhui Province; Grant number: 00144414; Grant sponsor: Nature Science Foundations of Anhui Province Education; Grant numbers: 2005hbz18, KJ2009A81. *Correspondence to: Wei-Ping Li, Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education; Key Laboratory of Chinese Medicine Research and Development, State Administration of Traditional Chinese Medicine, Anhui Medical University, Hefei 230032, People’s Republic of China. Fax: þ86-5515161133. E-mail: [email protected] Received 10 May 2009; Accepted 17 February 2011 DOI 10.1002/ar.21386 Published online 28 April 2011 in Wiley Online Library (wileyonlinelibrary.com).

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failure of cortisol suppression after a dexamethasone (DEX) challenge (Molchan et al., 1990; Nasman et al., 1995). Also, plasma levels of the stress hormone, cortisol, are correlated with the rate of dementia progression in patients with AD (Csernansky et al., 2006). Genetic studies indicate a link between glucocorticoids function and the risk for AD, because a rare haplotype in the 50 regulatory region of the gene encoding 11-hydroxysteroid dehydrogenase type 1 was associated with a sixfold increased risk for sporadic AD (Dominique et al., 2004). Epidemiological evidence further supports a role for stress as a risk factor for AD because elderly individuals prone to psychological distress are more likely to develop the disorder than age-matched, nonstressed individuals (Wilson et al., 2005). Nerve cell loss is extensive in brains of AD patients, and little is known about its cause, time course, and mechanisms. Recently, programed cell death or apoptosis has been implicated as a mode of cell death in AD (Stadelmann et al., 1999). Apoptosis is characterized by plasma membrane blebbing, nuclear fragmentation, and cell shrinkage and is initiated by the proteolytic enzymes, such as caspases (Nicholson, 1999). Numerous studies have documented the activation of caspases in the AD brain as well as the cleavage of critical cellular proteins (Rohn et al., 2001; Su et al., 2002). These studies suggest that it is the caspase-mediated cleavage of important cellular proteins, per se, and not the full execution of apoptosis that may be important for driving the pathology associated with AD. The extract of Astragalus (EA) was extracted from the root of a kind of traditional Chinese herb Astragalus membranaceus (Fisch) Bge. One of its active parts of EA is astragalosides (AST). Previous studies from our laboratory showed that AST possessed an antiaging and immunomodulatory effect, probably being related to its antioxidative properties (Lei et al., 2000, 2003). Our previous study also demonstrated that AST had protective effect on cerebral ischemia injury (Yin et al., 2005). However, the protective effect and mechanism of EA on learning and memory impairments and neurons apoptosis induced by glucocorticiods is unknown. This study was, therefore, designed to explore the potential mechanism of glucocorticoids on learning and memory impairments and neuronal cell apoptosis and the protective effect and mechanism of EA. We investigated stress-level DEX administration on the pathological consequences, learning and memory impairments, and neuronal cell apoptosis in 12-month male mice. Therefore, in this study, we used a DEX-induced neuron injury model in the mouse to further explore the neuroprotective effect of EA and its mechanism. Our results indicated that EA has protective effects on DEX-induced learning and memory impairments and neuronal cell apoptosis.

MATERIALS AND METHODS Animals All rodent experiments were performed in accordance with animal protocols approved by Anhui Medical University Animal Care Committee, which follows the protocol outlined in The Guide for the Care and Use of Laboratory Animals published by the US National Institute of Heath (NIH publication number 85-23; revised 1996). Experimental protocols described in this study

were approved by the Ethics Review Committee for Animal Experimentation of Anhui Medical University. Healthy 12-month male mice (50  5 g) were housed in a room at a constant temperature of 22 C  3 C with 40% humidity under a 12-hr light–dark cycle. They had free access to tap water and food. They were allowed 1 week to acclimatize before experimentation.

Drugs and Reagents The extract of Astragalus (EA, brown powder; content of astragalosides >63%), extracted from the root of Astragalus membranaceus (Fisch) Bge, was obtained from the Institute of Medicine of Hengxing, Hefei. Ginsenoside Rg1 (Rg1, white powder; content of Rg1 >98%) was provided by the Institute of Pharmaceutical Research of Xiehe Medical University of China. Dexamethasone (DEX) and Hoechst 33258 were provided by sigma company. Primer DNA was synthesized by the Shanghai Bioengineering Technical Service Limited Company. Trizol reagent was purchased from Invitrogen Company. Hexamer primers and PrimescriptTM RTase were provided by TakaRa Biotechnology Limited Company. Reverse transcription-polymerase chain reaction (RT-PCR) test kits were obtained from Promega Company. The antibody of caspase-3 and cytochrome c (rabbit anti-mouse) was purchased from ABZOOM Company. Streptavidin– biotin complex kit and diaminobenzidine (DAB) staining kit were purchased from Boster Biological Engineering Company. Caspase-3 and caspase-9 activity assay kit was purchased from Beyotime Institute of Biotechnology. Drugs were dissolved in distilled water, and all other chemicals were of the highest analytical grade available.

Treatment of Animals The animals were randomly divided into six groups: control group, DEX (5 mg/kg)-treated group, EA (10, 20, and 40 mg/kg) groups, and Rg1 (6.5 mg/kg) group. We performed all experiments between 09:00 and 12:00 hr. DEX group received dexamethasone (intragastric administration (i.g.), 5 mg/kg, 21 days), EA groups were treated with EA and dexamethasone (EA, i.g., 10, 20, and 40 mg/kg, DEX, 5 mg/kg, 21 days), the Rg1 group was treated with Rg1 and dexamethasone (Rg1, i.g., 6.5 mg/kg, DEX, 5 mg/kg, 21 days), and control group received equivalent volumes of vehicle. The body weight of all mice was weighed every 2 days.

Spontaneous Motor Activity Test Spontaneous motor activity (SMA) was measured (at 10 and 20 days after DEX treatment) in a computer-controlled SMA apparatus (Institute of Pharmaceutical Research of Chinese Academy of Medical Sciences). The apparatus consisted of four sensory boxes that registered any activity of the animals through an infrared system, an interface, and a computer that allowed the acquisition and storage of data from the sensory boxes. The SMA was measured for 2 min after mice were placed in the box 1 min to adapt to the environment.

Morris Water Maze Test The water maze apparatus (Institute of Pharmaceutical Research of Chinese Academy of Medical Sciences),

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TABLE 1. Effect of EA on body weight induced by DEX in 12-month male mice

Group Control DEX Rg1 EA

Dose (mg/kg)

Number of samples

– 5.0 6.5 10.0 20.0 40.0

12 11 7 10 8 10

Before DEX treatment 47.75 48.18 49.29 47.80 49.25 48.00

     

3.89 3.82 4.50 4.21 3.81 4.40

DEX treatment 10 days 47.58 41.82 43.29 43.20 42.75 42.80

     

3.85 4.83##,* 3.50# 3.01# 5.06# 3.49#

20 days 45.75 37.73 40.86 39.80 40.38 41.10

     

4.14 4.52##,** 4.02## 4.24##,* 4.57## 3.98##

Twelve-month male mice were treated with 5 mg/kg DEX daily for 21 days. Compared with before DEX treatment, the mice of DEX, Rg1, and EA (10, 20, and 40 mg/kg) had significant weight loss (Student’s t-test, #P < 0.05, ##P < 0.01). Compared with control group, DEX-treated group (10 and 20 days) and EA (10 mg/kg, 20 days) had significant weight loss (one-way ANOVA, Day 10, F5,52 ¼ 1.896; Day 20, F5,52 ¼ 4.494. Student’s t-test, *P < 0.05, **P < 0.01). Compared with DEX group, EA and Rg1 had no significant effect on the body weight induced by DEX. Data are mean  SD.

TABLE 2. Effect of EA on SMA induced by DEX in 12-month male mice

Group Control DEX Rg1 EA

DEX treatment

Dose Number of (mg/kg) samples – 5.0 6.5 10.0 20.0 40.0

12 11 7 10 8 10

were stained with HE (Hematoxylin and Eosin) and examined under a light microscope.

10 days 79.33 50.64 59.29 58.80 65.63 75.00

     

28.38 21.32* 33.32 32.32 27.82 26.16~

20 days 59.83 39.91 49.00 40.80 52.00 54.30

     

22.62 21.19* 30.05 24.26 25.32 23.07

Spontaneous motor activity (SMA) was detected at 10 and 20 days after DEX treatment. Compared with control group, SMA of DEX-treated group was significantly decreased. (One-way ANOVA, 10 days, F5,52 ¼ 1.608; 20 days, F5,52 ¼ 1.151. Student’s t-test, 10 days, t21 ¼ 2.721, *P < 0.05; 20 days, t21 ¼ 2.175, *P < 0.05). EA (40 mg/kg) could significantly increase the SMA at 10 days (t19 ¼ 2.349, ~P < 0.05). Data are mean  SD.

mice handling, and general testing procedure were described elsewhere (Janus et al., 2000). All mice underwent a reference memory training with a hidden platform placed in the center of one quadrant of the pool (southeast) for 5 days (from 17 days till 21 days after DEX treatment), with four trials (90 sec per trial) per day. After the last trial of 5 days, the platform was removed from the pool, and each mouse received one 60sec swim ‘‘probe trial.’’ Escape latency (seconds) was recorded using an online image video tracking system, which expresses the learning and memory results. For the probe trials, the number of crossing the platform site (NCP) and swimming time in the quadrant of platform (STP) were recorded, which express the spatial place preference (Gass et al., 1998). Behavioral data were analyzed using analysis of variance.

Histological Examination At 24 hr after the final DEX administration, the animals were killed and the brains were removed. The brains were immediately dissected in half along the coronal line; half were frozen (80 C) for biochemical analysis, and the other half were fixed in 4% paraformaldehyde and embedded in paraffin. The brains were sliced into 5-lm sections using a microtome. The sections

Hoechst 33258 Staining For nuclear staining, paraffin sections were deparaffinized with xylene two times for 15 min each and then rinsed with PBS. The sections were incubated with 25 mM Hoechst 33258 for 15 min at 37 C, washed with PBS, mounted onto slides using antifade mounting medium, and then examined by fluorescence microscopy (Olympus Opticals, Tokyo, Japan) (Ex/Em: 352 nm/461 nm). Morphologically, cells undergoing apoptosis appear smaller than normal and in which the chromatin appears condensed, deeply stained, and cellular fragmentation into apoptotic bodies (Qiu et al., 2000). Four sections per group and three high-power fields (400) of CA1, CA3 and neocortex per section were utilized for quantitative analysis. The total numbers of neuron cells and nuclear condensed cells in three high-power fields of CA1, CA3 of hippocampus and neocortex in each section were counted. The percentage of nuclear condensed cells was counted in each field.

RT-PCR The brain total RNA was extracted using TRIzol according to the instructions of the manufacturer and resuspended in 20 mL of DEPC-treated water (Liu et al., 2009). RNA concentration was determined using a biophotometer (Shanghai Scientific). Four micrograms of RNA was reverse transcribed to generate cDNA using random hexamer primers and PrimescriptTM RTase. PCR was conducted using a RT-PCR kit. Primer sequences and annealing temperatures were designed as follows: caspase-3, sense primer 50 ACTGGAATGTCATCTCGCTCTG30 , antisense primer 50 CCACGACCCG TCCTTTGA30 , anneal temperature 56 C, PCR target 468 bp, cycles 38. b-Actin, sense primer 50 AGC ATTTGC GGTGCA CG ATGGAGGG30 , antisense primer 50 ATGCC ATCCTGCGTC TGGA CCT GGC30 , anneal temperature 52 C, PCR target 606 bp, cycles 38. PCR products and a 1-kb DNA molecular weight marker (Promega, USA) were then electrophoresed on a 1% agarose gel, and the gel was visualized and photographed by the gel imaging system (Biosens SC810X, Shanghai Bio-Tech). The intensity of the PCR products generated by the b-actin and target sequences (caspase-3)

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TABLE 3. Effect of EA on mean escape latencies induced by DEX in 12-month male mice

Group

Dose (mg/kg)

Number of samples

– 5.0 6.5 10.0 20.0 40.0

12 11 7 10 8 10

Control DEX Rg1 EA

Mean escape latencies (sec) Day 1 71.82 81.38 75.49 76.07 75.01 75.53

     

14.41 7.82 7.92 10.52 12.73 12.14

Day 2 65.64 78.22 68.03 66.89 66.84 64.82

     

15.43 12.17* 15.41 14.54 13.18 19.73

Day 3 54.18 72.09 64.58 66.48 66.32 59.76

     

20.77 11.47* 15.36 16.02 19.09 14.13~

Day 4 52.25 69.91 60.10 63.83 62.68 56.51

     

18.27 13.53* 14.25 15.42 17.09 15.02~

Dexamethasone treatment resulted in learning and memory impairment in 12-month male mice. The mean escape latency shown in the results is the mean latency of four trials per day in learning and memory training experiment. Compared with DEX-treated group, EA (40 mg/kg) could significantly shorten the mean escape latency. Data are mean  SD, Student’s t-test, Day 2, t21 ¼ 2.157; Day 3, t21 ¼ 2.526; Day 4, t21 ¼ 2.614, *P < 0.05, DEX group versus control group. Day 3, t19 ¼ 2.206; Day 4, t19 ¼ 2.150, ~P < 0.05, EA 40 mg/kg versus DEX group.

TABLE 4. Effect of EA on STP and NCP in DEX-induced 12-month male mice

Dose Number of Group (mg/kg) samples Control DEX Rg1 EA

– 5.0 6.5 10.0 20.0 40.0

12 11 7 10 8 10

Swimming time in the quadrant of platform (STP, sec) 22.99 13.52 19.35 16.23 18.62 20.88

     

7.59 6.23** 3.93~ 6.11 3.13~ 5.67~~

Number of crossing the platform site (NCP) 2.92 1.55 2.14 1.80 2.38 2.60

     

1.73 1.13* 1.57 1.14 1.19 0.97~

Dexamethasone treatment resulted in impairment of memory and learning in 12-month male mice. In the probe trial in Morris water maze test, swimming time in the quadrant of platform (STP, one-way ANOVA, F5,52 ¼ 3.604) and the average number of crossing the platform site (NCP, F5,52 ¼ 1.639) were significantly different for DEX group and control group. Compared with DEX group, EA and Rg1 could significantly increase the STP and NCP. Data are mean  SD. Student’s t-test was used within group comparison. STP, t21 ¼ 3.252, **P < 0.01 DEX versus control; t16 ¼ 2.201, ~P < 0.05 Rg1 versus DEX; t17 ¼ 2.118, ~ P < 0.05 EA (20 mg/kg) versus DEX; t17 ¼ 2.820, ~~P < 0.01 EA (40 mg/kg) versus DEX. NCP, t21 ¼ 2.228, *P < 0.05 DEX versus control; t19 ¼ 2.289, ~ P < 0.05 EA (40 mg/kg) versus DEX.

was quantified by a computer-assisted, linear scanning densitometric analysis of the photograph in reflectance mode. A ratio of target to b-actin PCR product units was used to calculate the relative intensity of caspase-3 mRNA expression.

Immunohistochemistry Paraffin sections from mice brains were used for immunohistochemical analysis. Paraffin sections were cut at 5 lm and affixed to slides to ensure adhesion. For immunohistochemistry, all sections were blocked in dilute (3%) hydrogen peroxide to inactivate endogenous peroxidase and nonimmune goat serum and then were processed for immunohistochemistry. In all cases, the primary antibody of caspase-3 (1:100) and cytochrome c (1:100) was left to react overnight at 4 C. Immunostaining was visualized by the peroxidase method with a bio-

tinylated anti-rabbit secondary antibody and DAB oxidation. Sections were resin mounted and observed under a bright field microscope. The positive cells were stained brown. Analyses were carried out with an observer blinded to the experimental protocol. Four sections per group and the three high-power fields of CA1, CA3 and neocortex per section of the same magnification (400) were utilized for quantitative analysis. The areas of caspase-3 and cytochrome c-positive neurons in the CA1, CA3 of hippocampus and neocortex were observed under the microscope and measured using Image-Pro Plus 6.0 analysis system. The area percentage of caspase-3 and cytochrome c-positive neurons of three fields in the CA1, CA3 and neocortex was counted in each section to indicate the expression of caspase-3 and cytochrome c.

Caspase-3 and Caspase-9 Activity Assay The activity of caspase-3 and caspase-9 was measured by cleaving acetyl-Asp-Glu-Val-Asp p-nitroanilide (AcDEVD-pNA) and acetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHD-pNA), respectively, a selective substrate for caspase-3 and caspase-9. Tissues were homogenized in a homogenizing buffer containing 15 mM Tris-HCl, pH 7.4, 320 mM sucrose, 1 mM EGTA, 2 mM EDTA, 50 mM NaF, 1 mM MgCl2, 1 mM Na3VO4, and 30 mM sodium pyrophosphate plus protease inhibitors and centrifuged at 14,000g for 60 min at 4 C. Protein concentration of supernatants was measured by Bradford’s method, and equal amounts of proteins (10 lL) were incubated in a total volume of 100 lL comprised of 80 lL detection buffer. The reaction was started by addition of caspase-3 and caspase-9 substrate Ac-DEVD-pNA (10 lL) and AcLEHD-pNA (10 lL). After incubation for 60 min at 37 C, cleavage of the substrate was detected using a Microplate Reader (SPECTRAMAX 190, USA) with 405-nm wavelengths. Activities of caspase-3 and caspase-9 were expressed as changes in DEVDase and LEHDase activity.

Statistical Analysis Statistical differences between the groups were analyzed using either Student’s t-test or one-way ANOVA. Results are expressed as the means  SD, and the level of significance was P < 0.05.

PROTECTIVE EFFECT OF EA AGAINST DEX INJURY IN MICE

Fig. 1. Histopathological observation of the hippocampus and neocortex (HE stain). No remarkable neuronal abnormalities in the hippocampus (CA1, CA3) and neocortex of the control group were observed, whereas DEX-treated group mice showed degeneration of neurons in the hippo-

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campus and neocortex and disorder of the array of neurons. EA and Rg1 clearly improved pathomorphological change of hippocampal (CA1, CA3) and neocortex neurons. (A) Control group; (B) DEX group; (C) Rg1 group; (D) EA 10 mg/kg; (E) EA 20 mg/kg; and (F) EA 40 mg/kg. 400.

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Fig. 2. Photomicrographs of hippocampal neurons and cortical cells staining with Hoechst 33258 after treatment with DEX (400, N ¼ 4). Nuclear condensation and fragmentation were prominent 21 days after treatment with 5.0 mg/kg DEX (B) when compared with control group (A). The percentage of nuclear condensed cells in CA1, CA3 and neocortex was counted. Compared with control group, DEX

treatment significantly increased the percentage of nuclear condensed cells. EA and Rg1 could decrease the percentage (G). A, control; B, DEX; C, Rg1; D, EA 10 mg/kg; E, EA 20 mg/kg; and F, EA 40 mg/kg. Data are mean  SD. Neocortex, F5,18 ¼ 3.373; CA1, F5,18 ¼ 9.239; CA3, F5,18 ¼ 2.888. Student’s t-test, **P < 0.01 DEX versus control group. #P < 0.05 Rg1, EA versus DEX group.

PROTECTIVE EFFECT OF EA AGAINST DEX INJURY IN MICE

RESULTS Effect of EA on Body Weight and SMA Induced by DEX in 12-Month Male Mice We measured the body weight and SMA at 10 and 20 days after DEX treatment. Our results indicated that 21-day DEX exposure induced weight loss in mice (Table 1). Compared with before DEX treatment, the mice of DEX, Rg1, and EA (10, 20, and 40 mg/kg) had significant weight loss. Compared with control group, DEX-treated group (10 and 20 days) and EA (10 mg/kg, 20 days) had significant weight loss. Compared with DEX group, EA and Rg1 had no significant effect on the body weight induced by DEX. In SMA experiment, compared with control group, SMA of DEX-treated group was significantly decreased. EA (40 mg/kg) could significantly increase the SMA at 10 days (Table 2).

Effect of EA on Learning and Memory Impairments Induced by DEX in 12-Month Male Mice To examine the relationship between stress-level DEX exposure and cognitive deficits and the protective effects of EA in mice, we assessed learning and memory ability by using Morris water maze test as described above. Twenty-one-day DEX exposure induced impairment of learning and memory in mice. In learning and memory training experiment, the mean escape latencies of DEXtreated group (Days 2, 3, and 4) were significantly

Fig. 2. Continued.

Fig. 3. Mice were treated with DEX (5 mg/kg) for 21 days followed by RNA extraction and RT-PCR. PCR products were then run on an agarose gel to determine the expression level of caspase-3 (A). The ratios of caspase-3 gene expression relative to b-actin are presented

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lengthened versus control group mice. Compared with DEX group, EA (40 mg/kg) significantly shortened escape latencies (Table 3). In probe trial, the average number of crossing the platform site (NCP) and swimming time in the quadrant of platform (STP) were also significantly different (1.55  1.13 vs. 2.92  1.73 for NCP and 13.52  6.23 vs. 22.99  7.59 for STP for the DEX-treated mice and control group mice, respectively). EA (20 and 40 mg/kg) and Rg1 (6.5 mg/kg) significantly increased the swimming time in the quadrant of platform, and EA (40 mg/kg) increased the number of crossing the platform site (Table 4).

Effect of EA on Neuronal Degenerative Changes and Apoptosis in the Hippocampus and Neocortex Induced by DEX in 12-Month Male Mice To investigate the possibility of links between memory impairment severity and neuronal degeneration and apoptosis, we examined the neuropathology and apoptosis. No remarkable neuronal abnormalities in the hippocampus (CA1, CA3) and neocortex from mice of the normal control group were observed, whereas brains of DEX model group mice showed degeneration of neurons in the hippocampus (CA1, CA3) and neocortex, disorder of the array of neurons. The neuronal cell body became small and deeply stained with dye. Treatment with EA at different dose or Rg1 clearly improved pathomorphological change of hippocampal and neocortex neurons, such as reduction of condensed chromatin of neurons (Fig. 1). We further studied the nuclear morphology of hippocampal neurons and cortical cells by staining with Hoechst 33258. Hoechst 33258 binds to chromatin, allowing fluorescent visualization of normal and condensed chromatin (Qiu et al., 2000). Morphologically, cells undergoing apoptosis show chromatin condensation, become small, deeply stained, and cellular fragmentation into apoptotic bodies (Qiu et al., 2000). Quantitation of nuclear condensed cells detected with Hoechst showed that DEX model mice have a significant increase in the percentage of condensed cells in hippocampus (CA1, CA3) and neocortex. EA or Rg1 significantly decreased the percentage of condensed cells in hippocampus (CA1, CA3) and neocortex (Fig. 2).

in the bar graph (B). Data are mean  SD of caspase-3 mRNA to bactin. One-way ANOVA, F5,24 ¼ 4.617; Student’s t-test, **P < 0.01 versus the control; #P < 0.05 versus the DEX group. M, marker.

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Fig. 4. Effect of EA on percentage of cytochrome c immunoreactive cells in area in the hippocampal (CA1, CA3) and neocortex of DEX-exposure mice (400, N ¼ 4). The area of cytochrome c-positive cells was analyzed in three fields of the same magnification with Image-Pro Plus 6.0 analysis system software (G). Data are mean  SD. One-way

ANOVA, neocortex, F5,18 ¼ 5.968; CA1, F5,18 ¼ 4.16; CA3, F5,18 ¼ 2.361; Student’s t-test, **P < 0.01 versus the control; #P < 0.05 versus the DEX group. A, control; B, DEX; C, Rg1; D, EA 10 mg/kg; E, EA 20 mg/kg; and F, EA 40 mg/kg.

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parison with DEX group, the activity of caspase-9 and caspase-3 was significantly decreased by EA (40 mg/kg) or Rg1 (Table 5).

DISCUSSION

Fig. 4. Continued

Effect of EA on the mRNA Levels of Caspase-3 in the Brain of DEX-Exposure Mice We monitored caspase-3 mRNA levels after DEX treatment. We found that 21-day DEX treatment significantly increased caspase-3 mRNA levels (0.774  0.088 vs. 0.547  0.071 for DEX and control group). Compared with DEX-treated group, EA (20 and 40 mg/kg) and Rg1 (6.5 mg/kg) could significantly decrease the mRNA level of caspase-3 (0.598  0.071, 0.587  0.075 for EA 20 and 40 mg/kg and 0.608  0.067 for Rg1 6.5 mg/kg, respectively) (Fig. 3A,B).

Effect of EA on Cytochrome c and Caspase-3 Immunoreactivity in the Hippocampus and Neocortex Cells in the Brain of DEX-Exposure Mice As we have shown, DEX treatment increases mRNA levels of caspase-3 in 12-month male mice. We next investigate the influence of EA on cytochrome c and caspase-3 immunoreactivity in the hippocampus (CA1, CA3) and neocortex cells in the brain of mice. There were few cytochrome c and caspase-3 immunoreactive neuronal cells in the hippocampus (CA1, CA3) and neocortex of control group mice. The staining was light, and the immunoreactive cells were fewer. In the hippocampal CA1, CA3 region and neocortex of DEX-treated mice, the areas of cytochrome c and caspase-3 immunoreactive cells increased significantly in comparison with control group mice. Compared with DEX group, EA at different dose or Rg1 group significantly decreased the areas of cytochrome c and caspase-3 immunoreactive neuronal cells (Figs. 4, 5).

Effect of EA on the Activity of Caspase-9 and Caspase-3 in the Brain of DEX-Exposure Mice Immunohistochemistry analysis of caspase-3 revealed that 5 mg/kg DEX treatment enhanced caspase-3 immunoreactivity within cell bodies of the hippocampus as well as in neurons of the neocortex. We next investigated whether DEX administration increases the activity of caspase-9 and caspase-3 and the effects of EA. Our results revealed a marked increase in activity of caspase-9 and caspase-3 in the DEX-treated group. In com-

Stress is an unavoidable condition of the human experience and includes both major life events and the problems of daily life (McEwen, 2002). Chronic psychosocial stressors trigger increase in the levels of glucocorticoids stress hormones that, in turn, have deleterious effects on the structure and function of CNS structures, especially the hippocampus (Hibberd et al., 2000; Csernansky et al., 2006). The hippocampus is a primary target for neuronal degeneration in the brains of patients with AD. Because hippocampal cells express glucocorticoid receptors, they are the principal target sites for glucocorticoids, the adrenocortical hormones secreted during stress. It has been reported that an altered response of the HPA system occurs in patients with AD and that these alterations may increase glucocorticoids levels (Hatzinger et al., 1995). At present, the mechanism by which increased HPA axis activity could accelerate the AD process is unknown (Jeong et al., 2006). In our previous study, DEX could strongly increase the vulnerability of the hippocampal neuron to amyloid b-protein in vitro (Yao et al., 2007). This study was further to research the effect of stress-level glucocorticoids on learning and memory impairments and neurons apoptosis in 12-month nontransgenic mice, which may promote the development of AD. We simultaneously studied the neuroprotective effect of EA. This study demonstrates that 21-day DEX exposure induced weight loss, SMA decrease, and impairment of memory and learning, accompanied by severe histological damage in the CA1, CA3 region of the hippocampus and neocortex neurons in 12-month male mice. EA could significantly increase the SMA, shorten mean escape latencies, and increase the swimming time in the quadrant of platform and the number of crossing the platform site. These findings suggest that long-term glucocorticoids elevations may mediate the exacerbation of hippocampus and neocortex damage, which may be an important role in AD genesis. EA could effectively improve learning and memory behavior and showed a potential protective effect against injury by DEX insult. Neuronal death is an important characteristic of AD. Therefore, we further investigated the effects of stresslevel glucocorticoids and EA on neuronal apoptosis in hippocampus and neocortex. Neuronal cell death is the final pathological consequence of many CNS diseases, including AD. Apoptosis is a subtype of cell death that is involved in diverse physiological and pathological processes, including AD (Yang et al., 2008). In this study, histological examination showed that DEX treatment induced degeneration of neurons in the hippocampus (CA1, CA3) and neocortex. The neuronal cell body became short and deeply stained with dye. Nuclear staining with Hoechst 33258 showed nuclear condensation and fragmentation in dead cells. EA treatment inhibited the apoptosis in the hippocampus (CA1, CA3) and neocortex neurons in 12-month male mice. This may be an important mechanism by which EA exerts a protective effect on DEX insult.

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Fig. 5. Effect of EA on percentage of caspase-3 immunoreactive cells in area in the hippocampal (CA1, CA3) and neocortex of DEXexposure mice (400, N ¼ 4). The area of caspase-3-positive cells was analyzed in three fields of the same magnification with Image-Pro Plus 6.0 analysis system software (G). Data are mean  SD. One-way

ANOVA, neocortex, F5,18 ¼ 6.782; CA1, F5,18 ¼ 3.963; CA3, F5,18 ¼ 2.916; Student’s t-test, **P < 0.01 versus the control; #P < 0.05 versus the DEX group. A, control; B, DEX; C, Rg1; D, EA 10 mg/kg; E, EA 20 mg/kg; and F, EA 40 mg/kg.

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TABLE 5. Effect of EA on the activity of caspase-9 and caspase-3 in the brain of DEX-exposure mice (x 6 s, N 5 6) Group Control DEX Rg1 EA

Fig. 5. Continued

Mitochondria have been found to be essential in controlling at least certain apoptosis pathways (Green and Reed, 1998). The mechanisms by which they exert this function include release of caspases activators as cytochrome c and apoptosis-inducing factor (Liu et al., 1996), and disruption of electron transport and oxidative phosphorylation (Adachi et al., 1997; Garcia-Ruiz et al., 1997). Cytochrome c has been reported to be released from mitochondria into the cytosol of many cell types undergoing apoptosis (Kluck et al., 1997). Once in the cytosol, cytochrome c presumably binds to Apaf-1 and procaspase-9 and forms a functional apoptosome. Various stimuli that induce apoptosis lead to the release of cytochrome c from mitochondria, which plays a key role in a common pathway of activation of caspases (Mancini et al., 1998; Mulugeta et al., 2007). It has been demonstrated that cytosolic cytochrome c can bind Apaf-1 and subsequently trigger the sequential activation of caspase-9 and caspase-3 (Mancini et al., 1998). Activation of caspase-3 has been shown to be a key step in the execution process of apoptosis, and its inhibition can block apoptotic cell death. Activated caspases cleave a variety of target proteins, thereby disabling important cellular processes and breaking down structural components of the cell, such as lamin, and eventually causing cell death (Thornberry et al., 1997). We investigate the influence of DEX and EA on the mRNA level of caspase-3, immunoreactivities of cytochrome c and caspase-3. In our study, the immunoreactivities of cytochrome c and caspase-3 in the hippocampus and neocortex were found to be extensively elevated in the DEX-treated group, and this group was also found to have a higher caspase-3 mRNA level versus the control group. We further investigated the activity of caspase-3 and caspase-9 in the brain homogenate. The results showed that the activity of caspase-3 and caspase-9 was significantly increased in DEX-treated group. In comparison with DEX treatment group, EA significantly decreased the mRNA level of caspase-3, the expression of cytochrome c and caspase-3 in CA1, CA3 region of hippocampus and neocortex, and the activity of caspase-3 and caspase-9. These observations suggest that EA can decrease the expression and activity of caspase-3 and caspase-9 and may have an important role in protecting hippocampus and neocortex neurons against DEX-induced injury. In summary, our study suggests that stress-level glucocorticoids could accelerate learning and memory

Dose (mg/kg) – 5.0 6.5 10.0 20.0 40.0

Caspase-9 (U/g prot) 12.12 18.83 13.56 14.69 13.22 12.67

     

3.44 4.99* 3.87 3.17 4.22 3.61~

Caspase-3 (U/g prot) 8.96 13.85 9.69 11.04 9.73 9.58

     

3.44 3.09* 2.69~ 3.18 2.58~ 2.6~

The activity of caspase-9 and caspase-3 was measured by cleaving acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVDpNA) and acetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHDpNA), respectively. Compared with control group, DEX exposure could significantly increase the activity of caspase-9 and caspase-3 in the brain of mice. In comparison with DEX group, the activity of caspase-9 and caspase-3 is significantly decreased by EA (40 mg/kg) or Rg1. Data are mean  SD. Student’s t-test was used within group comparison. Caspase-9, t10 ¼ 2.476, *P < 0.05 DEX versus control; t10 ¼ 2.237, ~P < 0.05 EA (40 mg/kg) versus DEX. Caspase-3, t10 ¼ 2.363, *P < 0.05 DEX versus control; t10 ¼ 2.271, ~P < 0.05 Rg1 versus DEX; t10 ¼ 2.290, ~P < 0.05 EA (20 mg/kg) versus DEX; t10 ¼ 2.364, ~P < 0.05 EA (40 mg/kg) versus DEX.

impairments and increase neurons injury. EA treatment, via improvement of learning and memory, and downregulation of cytochrome c and caspase-3 levels, produces a protective effect against injury in response to DEX exposure.

ACKNOWLEDGMENTS The authors thank Zhensheng Wu (Department of Pathology, Basic Medical College, Anhui Medical University) and Dake Huang and Li Gui (Department of Morphology, Basic Medical College, Anhui Medical University) for their excellent technical assistance.

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