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EXPERIMENTAL WORK AND RESEARCH Effect of Tiaoxin Recipe on Spatial Memory and Energy Metabolism of Oxidation Injured Alzhelmer's Disease Rats* QIU Hong ( ~ ~)1, JIN Guo-qin (:~1~i~) 1, ZHAO Wei-kang ( ~ j ~ ) z , and ZHANG Xue-li (~@~L) 1 Objective: To observe the effect oI Tiaoxin Recipe (TXR) on the spatial memory, brain mitochondrial energy metabolism oI oxidation injured Alzheimer's disease (AD) rats, and to explore the mechanism of TXR in treating AD. Methods: Eighty-eight SD rats were randomly divided into five groups (normal group, operative group, "AD" model group, TXR group and Aricept group). An oxygen free radical generation system (dihydroxy fumaric acid-trichloroierric-adenosine diphosphate, DHF-FeCls-ADP) was used to create oxidation injured rat models mimic to AD; spatial learning and memory impairment (Morris water maze method), the activity of Succinate-oxidase, NADH-oxidase, CytC-oxidase (Clark oxygen electrode method) and the expression of cytochrome oxidase (CO)1T mRNA (in situ hybridization method) were observed. ReHlt~: Compared with the normal group, the spatial memory, activity of CytCoxidase and CO ITmRNA expression of oxidation injured "AD" rats were obviously decreased; TXR, however, could improve these functions in "AD" rat models obviously. Coneluion: The mechanism of the action oI TXR in treating AD was partly related to its effect on anti-oxidation which could improve brain mitochondrial energy metabolism. KEY WORDS Tiaoxin Recipe, oxidation injury, spatial memory, energy metabolism

ABSTRAOT

Alzheimer's disease (AD) is an age related disorder characterized by progressive degeneration of learning and memory function. Oxidation stress and mitochondrial dysfunction are implicated in the neurodegenerative process in AD. According to the theory of traditional Chinese medicine ( T C M ) , one of the main causes of AD is "Xinqi (heart-qi) deficiency", which will lead to impairment of brain function (1>. Tiaoxin Recipe (N,t~,~,TXR) has been clinically used via oral administration for the treatment of AD with good curative effect (z>. In this experiment, an oxygen free radical generation system (dihydroxy fumaric acid, DHF, plus trichloroferric-adenosine diphosphate, FeCla-ADP) to create oxidation injured rat models mimic to AD was used, and compared with Aricept to detect the effect of TXR in modulating the spatial memory and brain mitochondrial metabolism of the rat models, which could help us to interpret the mechanism of the action of TXR in treating AD.

METHODS

Animals Experiments were carried out on altogether eighty-eight male SD rats (from the Animal Center of Shanghai University of TCM, weighing 200-I-10 g). Drugs Tiaoxin Recipe. Composed of Tiaoxin (Xin-regulating) herbs, such as Radix Codonopsis pilosula, Ramulus Cinnamomi, Poria, Rhizoma Acori, Radices Polygalae, roast Radix Glycyrrhizae, etc. TXR extract with 7. 45 g crude drug per milliliter was provided by the Research Institute of Gerontology of Shanghai TCM University9 Aricept. Also named Donepezil. Confirmed by FDA in treating AD clinically, it is a new 9This program was supported by National Natural Science Foundation of China (No. 39830450) 1. Institute of Basic Medicine, Shanghai University of TCM, Shanghai (200032)~ 2. The Research Institute of Gerontologyof Shanghai TCM~ Correspondenceto. QIU Hong, Tel: 021--54231881, Email:qiu-hong(~eitiz,net

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kind of acetylcholinesterase (ACHE) inhibitors, manufactured by BCM Limited, UK, repacked by Pharmtech ( H K ) Co., Limited, with registration No. HK-42885.

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Isolation of Cerebral Mltochondrla Slightly modified Clark method was used (4) .

Determination of Enzyme AoUvlty Reagents end Equipment DHF, Succinate, cytochrome C and nicotinamide adenine dinucleotide ( N A D H ) were purchased from Sigma Company. Morris water maze was manufactured by the Research Institute Gerontology of Shanghai University of TCM. Oxygen electrode (SP-2 type), manufactured by Shanghai Research Institute of Plant Physiology of CAS. Freezing sledge microtome, made in Germany

Experimental Design The rats were randomly divided into 5 groups, normal group (16 rats), operative control group (12 rats), "AD" model group (20 rats), TXR group (20 rats) and Aricept group (20 rats). Rats in the TXR group were orally given TXR extract (3.3 ml/kg, 10 times the dose given to a human adult), while the rats in the Aricept group were orally given Aricept solution (0. 083 mg/kg, the same amount as the dose to a human adult). Administration began one week before operation. All drugs were given orally once a day for consecutively 3 weeks.

Establishment of "AD" Models Rats were anaesthetized with pentobarbital sodium (40 mg/kg) through intraperitoneal injection. DHF (2.4 mmol/L)-FeC13 (43nmol/L)-ADP ( 1 . 5 6 / ~ m o l / L ) , total 5/~1 at a time was infused into cerebral ventricle (P 0. 9mm, L 1. 4mm, H 3. 9ram) with mini-osmotic pump. Rats in " A D " model group, TXR group and Aricept group had DHF-FeC13-ADP infused into them every other day, altogether 3 times. Rats in operative control group were given normal saline instead.

Determination of Spatial Leamlng and Memory Function Morris water-maze method was used (3~.

Slightly modified Clark oxygen electrode method was used (s) . It was necessary to destroy mitochondrial membrane before detection. Incubation media were as follows. Detection media I is 10 mmol/L K2HPO4-KH2PO4, pH 7. 4. Detection media 11 is 40 mmol/L KzHPO4-KHzP04, pH 7.4. Enzyme activity was calculated by way of the oxygen consumption rate at the beginning of the reaction. Succinate (Suc)oxidase was determined in 2.5 ml of detection media I at 37"C with I mg protein content of mitochondria. After 1 min pre-incubation, the reaction was started by the addition of 20 ill of succinate ( 0 . 5 mol/L) and 20 t~l of cytochrome C (1. 75 m m o l / L ) . NADH-oxidase was determined in 2. 5 ml of detection media 11 at 37*Cwith 1 mg protein content of mitochondria. After 1 min pre-incubation, the reaction was started by the addition of 20 t~l of NADH (50 mmol/L) and 20 t~l of cytochrome C (1.75 mmol/L). Cytochrome C (CytC) oxidase was also determined in 2. 5 ml of detection media I at 37"C with 1 mg in protein content of mitochondria. After 1 min of preincubation, 10 t~l of tetramethyl-p-phenyldiamine TMPD (175 m m o l / L ) , 10 ~1 of VitC (1.75 mol/L) and 20/~1 of cytochrome C (1. 75 mmol/L) were added.

Frozen 811de Preparation Brain tissues were fixed at paraform fixative solution [-4% paraform, 0.1 mol/L phosphate buffered saline ( P B S ) , pH7.2-] for 3 - 6 hours after the whole body was perfused with normal saline arid paraform fixative solution, and then immerged in 30 % sucrose in 0.1 mol/L PBS ( p H 7 . 2 ) at 4"C. Thirty #m sections were cut on a freezing sledge microtome and stored in ISH protection media (300 g sucrose, 300 ml ethylene glycol, 500 ml 0. 1 mol/L PBS, 0. 1% diethyl pyrocarbonate to 1000 ml) at --20"C

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Changes of Enzyme Activities In Cortex

until they were used.

Oytochrom Oxlclase11 mRNA Expression Referring to literature (s) , in situ hybridization was used. Oligonucleotide probe of cytochrome oxidase ]1 (CO lI ) is 5 " GCTCT T C T A T AATAG GGGAT GTGGC G T C T T - - 3 " , which is labeled with digoxin. The sequences were synthesized by Boster Company of Wuhan. The kit was purchased from Boster Company. The procedure of experiment followed the instructions of the kit. CO II mRNA expression in cortex and hippocampus was detected.

Compared with the normal group, the activity of CytC-oxidase in cortex of oxidation injured rats was significantly decreased. The same tendency appeared in those of Succinate-oxidase and NADH-oxidase. TXR and Aricept, however, could increase the activity of CytC-oxidase obviously (all P 0 . 0 5 ) . The results were shown in Fig. 2.

Statistical Analysis All data were analyzed by ANOVA and followed by SNK test to compare their differences. Values less than 0.05 were considered as significant difference. RESULTS

Changes of Spatial Leaming and Memory Function Compared with the normal group and operative control group, the latency of "AD" model rats was obviously prolonged, ( P ~ 0 . 0 5 ) suggesting that TXR and Aricept could improve this function (all P ~ 0 . 05). See Fig. 1. 9

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Normal g r o u p

~0perative control group A "AD~group

40 N~.__

k

30

X

--"N

TXR g r o u p Arlcept

group

20 10 0

[

I

1

I

2

I

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Changes of OO II mRNA Expression In Cortex and Hlppocempue Compared with the normal group, the number of CO ]] positive neurons of "AD" rats was obviously reduced, showing that CO [[ mRNA expression in cortex and hippocampus of " A D " model rats was lowered. TXR treated rats showed high CO ]] mRNA expression, suggesting that TXR could obviously improve the above-mentioned parameters ( P ~ 0 . 0 5 ) while Aricept had no effect on them, P ~ 0 . 0 5 . (Fig 3,4). DISCUSSION

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Fig. 2 Effect of TXR on Mitochondrial Respiratory Enzyme Activity of Oxidation Injured "AD" Rats (nmoi/min 9 mg 9 Pro)

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Training Day Fig. 1. Effect of TXR on Latency of Oxidation Injured "AD" Rats

There are rich mitochondria in brain tissues, which is the important site for cell respiratory and energy metabolism. At the same time, mitochondria is the main place

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Fig. 3. Effect of TXR on Hippocampus CO IJ mRNA Expression of Oxidation Injured "AD" Rats N o t e s : A : Normal g r o u p ; B: Operative control group; C: "AD" g r o u p ; D: T X R g r o u p ; E : A r i c e p t group; the same as in Fig 4

Fig. 4. Effect of TXR on Cortex CO II mRNA Expression of Oxidation Injured "AD" Rats

for generating reactive oxygen species (ROS). If mitochondrial respiratory function or the activity of respiratory complex was destroyed, energy metabolism of the brain would be reduced. It is easy for brain tissues to suffer from oxidation injury of ROS, for the accumulation of unsaturated fatty acid with large oxygen consumption and its anti-oxidation activity are comparatively low. Large number of ROS could destroy the structure and function of mitochondria, which could affect the activity of pyruvate dehydrogenase and the synthesis of

acetylcholine(7~. So the function of the cholinergic neurons is destroyed gradually. At the same time, large amount of ROS also could damage mitochondrial DNA (mtDN A ) , causing a reduction in mRNA expression for the mtDNA-encoded CO lI , and the decreasing activities of enzyme which could also harm the energy metabolism ~s~. Morris water maze is the commonly used equipment to detect spatial learning and memory function of rats. From our experiment, it is inferred that spatial learning and

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memory function in oxidation injured "AD" model rats was impaired, and TXR, showing no difference from Aricept, could improve this function. Suc-oxidase, NADH-oxidase and CytCoxidase are the main composition of respiratory complex. Moreover, CytC-oxidase is the key enzyme whose activity could influence the function of the whole respiratory chain directly. At the same time, it is easy for CytC-oxidase to be hurt by ROS, resulting in electron loss during the electron transport. Our results showed that compared with normal group, the activity of CytC-oxidase in cortex of " A D " rats was significantly decreased, and the same tendency appeared in the activity of Suc-oxidase and NADH-oxidase, indicating that oxidation injury could impair the electron transport in inner mitochondrial membrane, and reduce the ability of mitochondria in utilizing oxygen. However, TXR and Aricept could increase the activity of CytC-oxidase obviously. CO lI is a cytochrome oxidase subunit encoded by mtDNA (6). CO [[ mRNA expression in cortex and hippocampus was observed with in situ hybridization method. The results indicated that CO 1] mRNA expression in cortex and hippocampus in "AD" model rats was lowered, which further confirmed that oxidation injury could impair mtDNA and its encoded subunit, and then lead to damage of energy metabolism mechanism. TXR-treated rats showed high CO lI mRNA expression. But Aricept had no effect on CO [[ mRNA.

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In these experiments, the results showed that the spatial memory, the activity of CytC-oxidase and the expression of CO 1] mRNA in the oxidation injured "AD" rats were distinctly decreased. Both TXR and Aricept could improve the spatial memory function and the activity of CytC-oxidase in "AD" model rats, indicating that T X R ' s effect in treating AD might be related to its effect on improving cholinergic system. However, TXR could also increase the CO [[ mRNA expression, showing that TXR's effect might be partly due to its anti-oxidation directly or indirectly. REFERENCES 1. LI YM, ZHANG CY, LIN SM. Theoretical exploration on Xin-benefiting and Shen-tonifying method in treating AD. J Gansu College TCM 2000;17(2) : 1--3. 2. LIN SM. Clinical research on the effect of TXR on Alzheimer's disease. J Shanghai Univ TCM 1997 ; 11(1) : 44. 3. Morris RJ. Developments of a water-maze procedure for studying spatial learning in the rats. Neurosci Methods 1984~11 " 47--60. 4. Zini R, Morin C, Bertelli A, et al. Effect of resveratrol on the rat brain respiratory chain. Drugs Exptl Clin Res 1999~XXV(2/3) : 87--97. 5. Heinz S, John JL, Matthias H, et al. Liposome-mitochondrial inner membrane fusion. J Bioehem 1980~ 255(8) : 3748--3756. 6. Nancy A, Simonian NA, Hyman BT, et al. Functional alterations in Alzheimer's disease: selective loss of mitochondrial-encoded eytoehrome oxidase mRNA in the hippocampal formation. J Neuropathol Exp Neurol 1994~ 53 (5) : 508--512. 7. Meier Ruge W, Iwangoff P, Bertoni Frecldari C, et al. What is primary and what is secondary for amyloid deposition in Alzheimer's disease. Ann NY Acad Sei 1994~ 719 : 23--27. 8. Mecocei P, MaeGarvey U, Kaufman AE, et al. Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain. Ann Neurol 1993~34 : 609 --616. (Received November 25, 2002)