| 1. BackgroundAlzheimer’s disease (AD), a kind of senile dementia, is a nervous system degenerative disease with progressive development that the aging level is exceeding over physiological aging level or premature aging which can lead to brain function disorder and acquired, consistent intelligence disturbance with characteristics of hidden onset and high disability rate. Memory and cognitive function disorder is often seen clinically with multiple functional changes including speech, comprehension, motion and emotion, and its onset is around 3 to 15 years, which is the main disease of senile dementia. AD has been researched for over a century since German Doctor Alois Alzheimer reported the first case in annual meeting of German Psychiatric Association in 1906. However, the etiology is unknown and the pathogencsis is unclear without effective treatment, and the progression of disease is suppressed and delayed by some cholinesterase. The global aging process is aggravated with the improvement of living conditions and medical and healthcare conditions. And the aging problem is especially highlighted in China due to historical and national conditions of China. The research data provided by American Scholar shows that AD morbidity in people of 65 years old to 74 years old is 1.6%, but that in people above 85 years old reaches to over 42%. It is seen that AD’s morbidity rises with rising of ages, which increases AD’s morbidity. It is estimated by WHO that patients with AD accounts for 0.441% of global population by 2015, but the rate wi 11 rise to 0.556% by 2030. AD has become the forth cause of death in elderly people after heart diseases, malignant tumor and apoplexy [1-3]. As a result, the research work of AD is extremely urgent, and it is one of medical workers’missions to serve the society.2. ObjectiveIn this research, SAMP8 mice were selected as research subjects interfered by pulse electric-acupuncture apparatus and music electric-acupuncture apparatus. "Tong du qi shen method" was put forward to treat AD based on supervisor professor Li zhigang’s long-term clinical experience and previous experimental work. This research explores influence of different electric-acupunctures on A β protein elimination pathway and compare curative effect difference of two kinds of electric acupuncture to provide experimental basis for clinical application of music electrie acupuneture in the treatment of AD, which is guided by "Tong du qi shen method" with A β protein in CA3 region of AD mice as point-cut through behavioristics and molecular biology.3. MethodsGroping:36 SAMP8 mice were randomly al located into 3 groups by random number table:Model Control Group (C), Pulse Electric-acupuncture Group (PE) and Music Electric-acupuncture Group (ME) with 12 mice in each.12 SAMR1 mice were divided into Blank Control Group. Interference:Mice in ME group were put in mice bags with their heads exposed. They were acupunctured at ’Baihui’ and ’yin tang’, then the needle handle was connected to the musi c electric-acupuncture apparatus with wires. Music therapy for cerebropathy treatment was used and current was adjusted till the needle handle was slightly vibrating and kept mice quiet without struggling. The treatment last 20 mins once a day. After being acupunctured, Renzhong was quick punctured. The treatment last 15 days. PE group was connected with pulse electric-acupuncture apparatus, and other interference was the same as that in ME group. B group and C group was grabbed and bounded with same methods. The treatment last 15days, once a day and 20min at a time. Testing:The behavioral ability was tested by Morris mater maze. Immunchistochemial imaging was used to test the expression of A β1-42, ECE1 and NEP in hippocampal CA3. Expression of hippocampal ECEI was tested by Western Blot and influence of different electric-acupunctures on A β elimination mechanism and the differences were compared.4. Results4.1 Testing result of Morris mater mazeThe result of place navigation test:1) The comparison among different groups in different time showed that the escape latencies of C group from day 1 to day five were significantly increased compared with than of B group (P<0.05). The escape latencies of PE group in 5 days were all shortened compared with that of C group, and the differences between groups was statistically significant from day 5 (P<0.05). The escape latencies of ME group in 5 days were all shortened, and the differences between groups was statistically significant from day 5 (P<0.01). The escape latency of ME group was shorter than that of PE group from day 2, but the difference of curative effect was not statistically significant (P>0.05).2) The comparison within different groups in different time showed that the escape latencies of B group and ME group were significantly decreased from 5th day’s training compared with that of day 1, and the difference was greatly significant (P<0.01). The escape latency of PE group was also significantly decreased from day 5, and the difference was statistically significant (P<0.05). The escape latency of M group had no significant difference during training period (P>0.05).3) The overall trend of escape latency among four groups was decreased. The trend of B group in the first three days was gentle, and it was gradually significant from day 4. The escape latencies of PE group and ME group showed downhill drop from day 3 without significant drop in day 4, and it showed downhill drop again in day 5. The down trend of C group was gentle with plateau in day 3, and the tread showed (descent phase-plateau-descent phase).The result of spatial probe test:The number of platform-site crossover of C group mice was significantly decreased compared with that of B group (P<0.01). Proportion of the target quadrant swimming distance in total distance was small and the difference was significant (P<0.01). The proportion of the time that spent on exploring the platform in target quadrant in total time was small, and the difference was statistically significant (P<0.05). The number of platform-site crossover of PE group mice was increased compared with that of C group, and the difference was statistically significant (P<0.05). The number of platform-site crossover of ME group mice was also significantly increased (P<0.01). Meanwhile, proportion of the target quadrant swimming distance in total distance of PE group and ME group was significantly increased after two kinds of interference (P<0.01). The proportion of the time that spent on exploring the platform in target quadrant in total time was increased, and the difference was statistically significant (P<0.05). The curative effect of ME group was better than that of PE group, but the difference was not statistically significant (P>0.05).4.2 Testing result of immunohistochmical imaging methodThe expression of Aβ 1-12 in CA3 region of SAMP8 mice was in cytomembrane and endochyrema and the positive cells were brown. The expression level of A β 1-42 in C group was increased significantly compared with that of B group (P<0.01). The expression level of A β 1-42 in PE group and ME group were significantly decreased compared with that of C group (P<0.05). The curative effect had no significant difference between that in ME group and PE group. The expression of ECE1 and NEP in CA3 region of SAMP8 mice were in cytomembrane and endochyrema, and the positive cells were brown. The expression level of ECE1 and NEP were both significantly decreased compared with that of B group (P<0.01). The expression level of ECE1 and NEP of PE group and ME group were both significantly increased compared with that of C group (P<0.01). The expression level of ME group was higher than that of PE group (P<0.05), but the difference of expression of NEP in two groups had no significant difference (P>0.05).4.3 Testing result of Western Blot methodThe expression level of ECE1 in CA3 region of SAMP8 mice in C group was significantly decreased compared with that of B group (P<0.01). The expression of PE group was increased after being interfered by pulse electric-acupuncture (P<0.05). The expression of ME group was significantly increased after being interfered by music electric-acupuncture (P<0.01). The expression of PE group and ME group had no significant difference (P>0.05).5, Conclusion(1) The research result shows that SAMP8 mice, the subjects of this research has obvious learning and memory disorder, and the expression of A β protein in hippocampal CA3 region which is a successful AD model.(2) The interference of pulse electric-acupuncture and music electric-acupuncture on AD model mice can improve their learning and memory ability, and the curative effect of music electric-acupuncture was slightly better than that of pulse electric-acupuncture.(3) The interference of pulse electric-acupuncture and music electric-acupuncture on AD model mice can decreased the expression of A β1-42 in hippocampal CA3 region of SAMP8 mice.(4) The interference of pulse electric-acupuncture and music electric-acupuncture on AD model mice can both increase the expression of two kinds of Aβ degrading enzyme ECE1 and NEP in hippocampal CA3 region of SAMP8 mice to promote Aβ degradation pathway, which can achieve goal of treating dementia. Music electric-acupuncture is better than pulse electric-acupuncture in the ability of improving the expression of ECE1, but they have no significant difference in increasing the expression of NEP.(5) This research proves that "Tong du qi shen method" has a positive significance in guiding treatment of AD.
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