Peng Jing1, Zeng Fang, He Yu-heng, Tang Yong, Yin Hai-yan, Yu Shu-guang

1 Department of Nursing, Leshan Vocational and Technical College, Leshan 614000, China

2 College of Acumox and Tuina, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China

BASIC STUDY

Influence of Electroacupuncture on COX Activity of Hippocampal Mitochondria in Senescenceaccelerated Mouse Prone 8 Mice

Peng Jing1, Zeng Fang2, He Yu-heng2, Tang Yong2, Yin Hai-yan2, Yu Shu-guang2

1 Department of Nursing, Leshan Vocational and Technical College, Leshan 614000, China

2 College of Acumox and Tuina, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, China

Author: Peng Jing, master degree candidate, lecturer

Objective: To observe the effect of electroacupuncture (EA) on cytochrome c oxidase (COX)activity of hippocampal mitochondria in senescence-accelerated mouse prone 8 (SAMP8) mice, and to explore the EA mechanism on Alzheimer disease (AD) in improving energy metabolic disorder.

Methods: Twelve SAMP8 mice were randomly divided into a model group and an EA group, with six in each group. Six senescence-accelerated mouse resistance 1 (SAMR1) mice were prepared as blank group. Mice in the EA group received EA on Baihui (GV 20) and Yongquan (KI 1), once a day for 7 d as a course, altogether 3 courses with one day interval between two courses. Mice in the model group and the blank group were manipulated and fixed as those in the EA group. After interventions, Morris water maze was employed to test spatial learning and memory ability to evaluate EA effect; spectrophotometry was used to detect the activity of hippocampal mitochondria COX.

Results: Compared with the blank group, mean escape latencies of the EA group and model group were prolonged significantly in Morris water maze tests (P＜0.01), the residue duration in the former platform quadrant significantly decreased (P＜0.01). Compared with the model group, mean escape latencies on 1 d, 2 d and 3 d of the EA group were significantly reduced (P＜0.05), and those on 4 d and 5 d continued the decreasing tendency (P＜0.01), the residue duration on the former platform quadrant was significantly prolonged (P＜0.05). The COX activity tests showed that, compared with the blank group, COX activities of the model group and the EA group were significantly decreased (P＜0.01); compared with the model group, COX activity of the EA group was significantly elevated (P＜0.01).

Conclusion: It’s plausible that EA improves AD learning and memory ability by increasing mitochondria COX activity, protecting the structure and function, and improving energy metabolism.

Electroacupuncture; Acupuncture Therapy; Alzheimer Disease; Electron Transport Complex IV; Mice

Alzheimer disease (AD) is a progressive degenerative neurological disease with an insidious onset. The clinical manifestations are comprehensive performance of dementia, such as memory impairment, aphasia, apraxia, agnosia, visuospatial skills damage, executive dysfunction, and changes in personality and behavior. The etiology and pathogenesis of AD are complex. Studies have shown that energy metabolism disorder is closely related toAD as the common pathway leading to neuronal degeneration, and links to three major pathological features of AD: the loss of brain neurons, senile plaque formation[1], neurofibrillary tangles[2]. Mitochondrion plays an important role in energy metabolism. Studies have shown that changes in mitochondrial morphology and function are key factors in energy metabolism disorders of AD. Therefore, protecting the mitochondria structure and improving the function would help improving energy metabolism and AD symptoms. Numerous clinical and animal experiments show that acupuncture treatment of AD has a curative effect[3-6]. But there are few reports on whether acupuncture produces the curative effect through improving energy metabolism. Based on our previous research[7], we have observed its effect on cytochrome c oxidase (COX) activity of hippocampus mitochondria in eightmonth senescence-accelerated mouse prone 8 (SAMP8),from the aspect of protecting mitochondrion and improving energy metabolism.

1 Materials

1.1 Animal and grouping

Twelve SAMP8 mice and six eight-month senescence-accelerated mouse resistance 1 (SAMR1) mice, male, weight of (20±2) g, provided by Senile Encephalopathy Laboratory Animal Center of First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (Qualified Number: W-J Tianjing Experimental Animals Quality Approved No. 006). Animals were adapted for one week before the experiment, with temperature maintained at 22-26 ℃ and humidity at about 55%, normal light. Water and feed were sterilized with high temperature steam. Using computer random grouping, SAMP8 mice were randomly divided into a model group or an electroacupuncture (EA) group, six in each; six SAMR1 mice in a blank group.

1.2 Main instruments and drugs

Morris water maze (Chengdu Tai Meng Science and Technology Co., Ltd.), G6805-1 type EA device (Qingdao Xinsheng Industrial Co., Ltd.), Huatuo Brand sterile acupuncture needles (0.30 mm in diameter, 13 mm in length, Suzhou Medical Supplies Factory Co., Ltd.), CR22G refrigerated centrifuge (Hitachi Koki Shares Commune), 721 spectrophotometer (Shanghai 3rd Spectral Instrument Factory), Mitochondria isolated (active) kit (Shanghai Genmed Gene Pharmaceutical Technology Co., Ltd.), COX activity assay kit (Shanghai Genmed Gene Pharmaceutical Technology Co., Ltd.).

1.3 Statistical methods

The SPSS 12.0 version statistical software was used for data analysis. Measurement data were expressed as mean ± standard deviationRepeated measurement data analysis of variance was used for place navigation test data, One-way ANOVA for space exploration and COX activity data.

2 Treatment Methods

2.1 EA group

Points: Baihui (GV 20) and Yongquan (KI 1).

Operation: Mice were fixed on a homemade device. Baihui (GV 20) was obliquely punctured to 3-5 mm, and Yongquan (KI 1) was perpendicularly punctured to 2-3 mm (one side at one time and alternatively).

Needles were connected with G6805-1 EA device, sparse-dense wave with frequency at 2-100 Hz, intensity of 2-4 V, making the lower limbs slightly shake, for 20 min, once a day, seven days as a course, altogether three courses (21 d) with one day interval between two courses.

2.2 Model group and blank group

Mice of these two groups were not treated with EA, but were fixed and manipulated in the same way at the same time.

3 Observations on Results

3.1 Detection index

3.1.1 Spatial learning and memory ability

Place navigation test: After 21 d of EA treatment, mice were tested by Morris water maze twice a day (once in the morning and once in the afternoon) for 5 d. Escape latencies were recorded, and the arithmetic mean of the two tests each day were then statistically analyzed[8].

Space exploration test: After place navigation test, the platform was removed and mice were placed at the midpoint of the opposite quadrant to the original platform, swimming freely for 120 s. The maze system automatically recorded the mouse residue duration in the original platform quadrant.

3.1.2 COX activity detection

At the end of the experiment, all mice were sacrificed; the hippocampus was rapidly removed according to the stereotaxic atlas of mice, washed twice with clean fluid, and put into the liquid nitrogen tanks. All the operations were conducted at 0-4 ℃conditions. The next day, the hippocampus tissue was grinded. Ice-cold lysis working solution, coldenhanced liquid and precooled solution were in turn added and mixed. The mixture was centrifuged at 4 ℃ and 1 500 r/min, the supernatant was removed and the precipitate was centrifuged again at

10 000 r/min. The precipitate particles were the mitochondria. In an ice slot, buffer, diluent, mitochondria and work fluid were added in a cuvette, mixed and put into the spectrophotometer (wavelength 550 nm) for readings. COX activity was calculated according to the formula.

3.2 Results

3.2.1 Spatial learning and memory ability

Results of place navigation test: In the 5-day test, compared with the blank group, escape latencies of the model group and the EA group were significantly longer (bothP＜0.01); compared with the model group, average escape latencies on 1 d, 2 d and 3 d of the EA group significantly decreased (bothP＜0.05), and those on 4 d and 5 d continued the decreasing tendency (P＜0.01), (Table 1).

Table1. Comparison of daily average escape latency of mice in each group in place navigation tests

Table1. Comparison of daily average escape latency of mice in each group in place navigation tests

Note: Compared with the blank group, 1) P＜0.01; compared with the model group, 2) P＜0.05)，3) P＜0.01

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Results of space exploration: Compared with the blank group, the residue durations of the model group and the EA group in the former platform quadrant were significantly shortened (bothP＜0.01); compared with the model group, the residue duration of the EA group in the original platform quadrant was significantly prolonged (P＜0.05), (Table 2).

Table 2. Comparison of the residue durations in the former platform quadrant of mice in each group

Table 2. Comparison of the residue durations in the former platform quadrant of mice in each group

Note: Compared with the blank group, 1) P＜0.01; compared with the model group, 2) P＜0.05

3.2.2 Effects of EA on hippocampal mitochondrial COX activity

Compared with the blank group, COX activities of the model group and the EA group were significantly decreased (P＜0.01); compared with the model group, that of EA group was increased (P＜0.01), (Table 3).

Table 3. Comparison of COX activities in hippocampus of mice

Table 3. Comparison of COX activities in hippocampus of mice

Note: Compared with the blank group, 1) P＜0.01; compared with the model group, 2) P＜0.05

4 Discussion

Mitochondria are cellular energy plants, providing energy for cellular activities through the process of oxidative phosphorylations. More and more evidences have shown that mitochondrial defects are closely associated with neurodegenerative diseases. In the neuronal mitochondria of AD, there are not only morphological changes such as swelling, degeneration[9], DNA mutations[10], as well as functional abnormalities like decreased mitochondrial membrane potential, increased permeability[11], decreased enzymatic activity[12-13], decreased ATP synthesis[14], abnormal excessive free radical generation[15], but also the consequent emergence or worsening of cell ‘calcium overload’[16], apoptosis[17]and the excessive release of excitatory amino acids[18].

COX is a key enzyme in the respiratory chain, and its activity can affect the function of the whole respiratory chain. Meanwhile, COX is the most unstable and vulnerable enzyme[19]. On one hand, its decreased activity could influence the mitochondrial respiratory function, thereby affecting the energy metabolism, and causing deficiency in ATP generation and acetylcholine, which will affect the function of cholinergic neurons, leading to impaired learning and memory; on the other hand, impairment of COX will cause electron leakage and more oxygen free radicals, which will worsen neuronal oxidative damage of the brain. In this experiment, after EA treatment, the COX activities of hippocampus mitochondrial in SAMP8 mice were significantly enhanced, and the learning and memory ability were improved.

In the previous study we have found that EA can improve succinic acid dehydrogenase (SDH) and ATPenzyme activity of the hippocampus mitochondrial respiratory chain[7]. With the results of this experiment, we speculate that the EA effect in improving learning and memory ability of AD may be associated with reducing mitochondria structural damage, enhancing mitochondrial function, and thus promoting energy metabolism to protect the brain function.

Conflict of Interest

The authors declared that there was no conflict of interest.

Acknowledgments

This work was supported by National Natural Science Foundation of China (No.30472235); Scientific Science Foundation for the Youth of the Education Department of Sichuan Province (No. 2006B023).

Statement of Human and Animal Rights

In this experiment, the treatment of animals conformed to the ethical criteria.

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Translator: Feng Xiao-ming

Yu Shu-guang, researcher, doctoral supervisor.

E-mail: pengjing1983@sina.com

R2-03

: A

Date: February 25, 2014