| 1 BackgroundAlzheimer's disease is a degenerative disease of the central nervous system,characterized by disorders of learning function and the decline in memory.It is common and frequently occurring among the elderly,with a relatively high incidence of disease and a large association between the incidence and age,the incidence of the disease increases with age.According to the report issued by the international Alzheimer's disease association,the number of people suffering from AD in the world is about 47 million,and the number of AD patients worldwide is expected to reach 113 million 500 thousand by the year 2050s.Such a large group of patients will bring heavy economic burdens and social burdens to all countries of the world.Therefore,AD is highly valued by the world medical scientific research community and has become a difficult problem to be tackled in the modern medical field.However,the pathogenesis of AD is very complex.Each pathogenesis is not single.Various mechanisms are interwoven and interrelated,which lead to the occurrence of AD.And,with the gradual deepening of the research on AD in recent years,the single mechanism can not explain the occurrence of AD.More and more scholars believe that inflammatory reaction is the main mechanism of AD,which plays an important role in the development and development of AD.In the brain of AD patients,the inflammatory reaction exists in various stages of the disease.The previous study of our group found that the 'Tongdu Qishen'acupuncture method has the effect of treating AD,and its possible mechanism is to inhibit the activation of microglia and reduce the damage to the brain by the inflammatory mediators.The activation of microglia and the release of related inflammatory mediators are closely related to the pathogenesis of AD.This study is used as a breakthrough point in the treatment of model mice by pulsed electroacupuncture and music electroacupuncture.Morris water maze,immunohistochemical and Western blot techniques were used to observe the changes of inflammatory factors and behavioural changes in the hippocampus microglia activation from the different levels of living body to pathological tissue to explore the possible mechanism of different electroacupuncture treatment of AD under the guidance of 'Tongdu Qishen' theory,and compare the difference of curative effect between music electroacupuncture and pulse electroacupuncture in the treatment of AD.2 ObjectiveIn this study,7 month old APP/PS1 double transgenic AD mice were treated by using the 'Tongdu Qishen' method with different electroacupuncture methods.The spatial learning ability and spatial memory ability of the model mice were observed by Morris water maze,and the expression level of COX-2 and TGF-beta 1 in the hippocampus was detected by molecular biology methods to explore the possible mechanism of different electroacupuncture treatment of AD under the guidance of'Tongdu Qishen' theory,and compare the difference of curative effect between music electroacupuncture and pulse electroacupuncture in the treatment of AD.3 MethodGroup:40 male APPswe/PS1dE9 double transgenic mice of SPF grade were randomly divided into model group,drug group,pulse electroacupuncture group and music electroacupuncture group.10 C57BL/6 male mice of the same age and same genetic background were used as normal control group,with a weight of 24.6±4.3g.Intervention:normal control group:without any treatment under the same feeding condition,the control group was caught in the homemade rat when the other groups were treated.After 20min,the rats were dismantled and put into the cage to ensure that the intervention conditions were in accordance with the other groups and were captured once a day for 15 days.Model group:the treatment method is the same as the normal control group.Drug group:the dosage of donepezil was administered to the stomach.In order to maintain the accuracy of the dosage of the drug,the dosage of each mouse was determined according to the weight of the stomach,the dose was 0.92 mg/kg.Before the drug was used,the drug was crushed into powder,then dissolved in distilled water and gavage was treated once a day for 15 days.When other groups were treated,they were caught in the homemade rat.After 20min,the rats were disassembled and put back into the squirrel cage to ensure that the intervention conditions were consistent with the other groups.Pulse electroacupuncture group:in the treatment of electroacupuncture group,it is taken into the homemade rat set and fixed,and then treated.The selected acupoint is the prescription of 'Tongdu Qishen' method,that is,Baihui,Yintang and Renzhong points.The selected needle is a disposable aseptic acupuncture needle of Zhongyan Taihe brand.First we chose Renzhong ueing pricking needing,and then chose Baihui and Yingtang hoeizontal needing with the direction downward and upword respectively.The puncture depth is 0.5cm.After the acupuncture,in order to prevent the mice from struggling to lead to the needle body out of the acupoint,the needle was fixed with tape,then the two needle handles were connected with the two poles of the HANS-LH202 electroacupuncture instrument.The density wave was selected,the frequency was 2-50Hz,each time 20min,once a day for 15 days.Music electroacupuncture group:the method of fixation,acupoint selection and acupuncture were the same as that of the pulse electroacupuncture group.Two needle handles were connected with the two poles of the ZJ-12H Musical electroacupuncture instrument.Rhythm clear,moderate speed and intensity of the music prescription was selected,each time 20min,once a day for 15 days.Detection methods and indicators:after 15 days of treatment,the Morris water maze hidden platform experiment was used to test the spatial learning ability of mice.Morris water maze space exploration experiment was used to test the spatial memory ability of mice.The expression of COX-2 and TGF-beta 1 in hippocampus of mice was measured by immunohistochemical method,and Western blot prescription was used.Methods the expression level of COX-2 and TGF-beta 1 in hippocampus of mice was detected.4 Results4.1 MWM Result4.1.1 Results of MWM hidden platform experimentAfter a period of training,the mice in each group can shorten the escape latency in varying degrees.Therefore,the Morris water maze hidden platform experiment is suitable for this study.The decrease trend of the normal control group is the most obvious,indicating that the learning and memory ability of the model group is better.In contrast,the decline trend of the escape latency is relatively slow and the learning and memory ability is poor.The decrease trend of the escape latency in the drug group,the pulse Electroacupuncture group and the music electroacupuncture group is between the normal group and the drug group.The three different treatment methods can improve the spatial learning ability of the model mice to a certain extent.The decrease tendency of the escape latency of the pulse electroacupuncture group is more obvious than that of the drug group,and the decrease tendency of the evasion latency in the music electroacupuncture group is more obvious than that of the pulse electric acupuncture group.On the first day of training,the escape latency of the model group had been statistically different from that of the normal group(P<0.05).The difference in escape latency between the two groups was further increased in the second to 5 days of the training(P<0.01),indicating that the 7 month old APP/PS1 double transgenic mice had a significant obstacle to spatial learning ability,and the animal model was successful.In the third to 5 days of training,the escape latency of the drug group,the pulse electroacupuncture group and the music electroacupuncture group were shorter than the model group(P<0.01),indicating that after 15 days of treatment,drugs,pulse electroacupuncture and music electroacupuncture could improve the learning ability of the mice.In the fourth to 5 days of training,the escape latency of the pulse electroacupuncture group and the music electroacupuncture group was significantly shorter than that of the drug group(P<0.05),but there was no statistical difference between the pulse electroacupuncture group and the music electroacupuncture group(P>0.05),The escape latency of music electroacupuncture group was shorter than that of pulse electroacupuncture group,but there was no significant difference between them(P>0.05).4.1.2 Results of MWM space exploration experimentThe proportion of the target swimming time in the model group was significantly lower than that of the normal control group(P<0.01),indicating that the APP/PS1 double transgenic mice model was successful,and there was a significant obstacle to spatial memory ability.The ratio of the target swimming time in the drug group,the pulse electroacupuncture group and the music electroacupuncture group were significantly higher than the model group(P<0.05,P<0.01,P<0.01).It showed that after 15 days of treatment,drugs,pulse electroacupuncture and music electroacupuncture could improve the memory ability of the AD model mice,and the music electroacupuncture group was superior to the drug group(P<0.01),there was no statistical difference between the drug group and the pulse electroacupuncture group,the pulse electroacupuncture group and the music electroacupuncture group(P>0.05),The proportion of swimming time in the target quadrant of music electroacupuncture group was higher than that of pulse electroacupuncture group,but there was no significant difference between them(P>0.05).The number of cross platform in the model group was significantly less than that of the normal control group(P<0.01),indicating that the APP/PS1 double transgenic mice model was successful,and there was a significant disorder of spatial memory ability.The number of cross platform of drug group,pulse electroacupuncture group and music electroacupuncture group were more than model group(P<0.05,P<0.05,P<0.01).It showed that after 15 days of treatment,drugs,pulse electroacupuncture and music electroacupuncture could improve the spatial memory ability of AD model mice,including drug group and pulse electroacupuncture group,drug group and music electroacupuncture group,pulse group,pulse group,pulse group,pulse electroacupuncture group,pulse electroacupuncture group,pulse electroacupuncture group,pulse electroacupuncture group,pulse electroacupuncture group,pulse electroacupuncture group,pulse electroacupuncture group,pulse electroacupuncture group,pulse electroacupuncture group,pulse electroacupuncture group,and pulse group.There was no statistical difference between pulse electricacupuncture group and music electroacupuncture group(P>0.05).4.2 Results of Immunohistochemical DAB stainingCOX-2 is mainly expressed in the cell membrane and cytoplasm of microglia in the hippocampus of mice.The positive cells were brown,mainly stained on the cell membrane,and a slight amount of coloring in the cytoplasm.TGF-beta 1 was mainly expressed in the hippocampus of mice.The expression site was the cell membrane of microglia,cytoplasm and axon,and the positive cells were yellow granules.Compared with other groups,the number of positive cells in the normal control group was less than that of the normal control group,and the number of positive cells in the model group was significantly higher than that in the normal control group,and the color was deeper.Compared with the model group,the number of the drug group and the pulse electroacupuncture group is relatively small,and the color is also lighter.The statistical analysis of positive cells of COX-2 and TGF-beta 1 in the hippocampus of each group:the number of positive cells in the model group was significantly more than that of the normal control group(P<0.01),indicating that there was an obvious inflammatory response in the brain of 7 month old APP/PS1 double transgenic mice.The expression of inflammatory factors COX-2,TGF-beta 1 increased,and the APP/PS1 double transgenic mice were increased.The model is successful.The positive cells in the drug group,the pulse electroacupuncture group and the music electroacupuncture group were significantly less than the model group(P<0.01),indicating that after 15 days of treatment,drugs,pulse electroacupuncture and music electroacupuncture could reduce the expression of COX-2 and TGF-beta 1 in the brain of AD model mice.There was no statistical difference between the drug group,the pulse electroacupuncture group and the music electroacupuncture group(P>0.05),The number of crossing platform in music electroacupuncture group was more than that in pulse electroacupuncture group,but there was no significant difference between them(P>0.05).4.3 Results of Western blot detectionThe relative expression of COX-2 in the model group was more than that of the normal control group(P<0.01),indicating that 7 month old APP/PS1 double transgenic mice had obvious inflammatory response in the brain,and the relative expression of inflammatory factor COX-2 increased,and the APP/PS1 double transgenic mice model was successful.The relative expression of COX-2 in the drug group,the pulse electroacupuncture group and the music electroacupuncture group were significantly less than that of the model group(P<0.05,P<0.01,P<0.01),indicating that after 15 days of treatment,drugs,pulse electroacupuncture and music electroacupuncture could reduce the expression of COX-2 in the brain of AD model mice.There was no statistical difference between the drug group,the pulse electroacupuncture group and the music electroacupuncture group(P>0.05),The relative expression of COX-2 in music electroacupuncture group was less than that in pulse electroacupuncture group,but there was no significant difference between them(P>0.05).The relative expression of TGF-beta 1 in the model group was more than that of the normal control group(P<0.01),indicating that the 7 month old APP/PS1 double transgenic mice had obvious inflammatory response in the brain,and the relative expression of inflammatory factor COX-2 increased,and the APP/PS1 double transgenic mice model was successful.The relative expression of TGF-beta 1 in the drug group,the pulse electroacupuncture group and the music electroacupuncture group were significantly less than that of the model group(P<0.05,P<0.05,P<0.01),indicating that after 15 days of treatment,drugs,pulse electroacupuncture and music electroacupuncture could reduce the expression of TGF-beta 1 in the brain of AD model mice.There was no statistical difference between the drug group,the pulse electroacupuncture group and the music electroacupuncture group(P>0.05),The relative expression of TGF-beta 1 in music electroacupuncture group was less than that in pulse electroacupuncture group,but there was no significant difference between them(P>0.05).5 Conclusions(1)The APPswe/PS1dE9 double transgenic AD model mice selected in this experiment have been learning and memory dysfunction,the activation of microglia and the release of inflammatory factors at the time of 7 month old,which is an ideal animal model for the study ofAD.(2)Under the guidance of the 'Tongdu Qishen' theory,pulse electroacupuncture and music electroacupuncture can improve the spatial learning ability and spatial memory ability of the model mice,but there is no statistical difference between them.Only from the numerical point of view,the music electroacupuncture group is superior to the pulse electroacupuncture group.(3)The expression of inflammatory cytokines COX-2 and TGF-beta 1 in the hippocampus of AD model mice can be reduced by 'Tongdu Qishen' pulse electroacupuncture and music electroacupuncture,but there is no statistical difference between them. |
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