| BackgroundAlzheimer’s disease(AD),also known as senile dementia,is an irreversible neurodegenerative disease characterized by cognitive dysfunction,memory impairment,and abnormal behavioral changes.Alzheimer’s disease accounts for 60%-80%of the total number of dementia.According to relevant epidemiological studies,the number of people with dementia worldwide is about 47 million.It is expected that the incidence of dementia will triple by 2050,and the investment in global dementia medical services is increasing.Therefore,the study of Alzheimer’s disease has urgent practical significance.The main pathological features of AD are the senile plaque(SP)formed by abnormal aggregation of beta amyloid(Aβ),neurofibrillary tangles(NFT)formed by abnormal phosphorylation of tau protein,neuronal apoptosis,and synaptic dysfunction.It is believed that Aβ amyloid deposition is the most important pathogenesis of AD.Beta-amyloid cleaving enzyme(BACE1),a key protein involved in the production of Aβ peptide,the over expression of which may lead to excessive production of neurotoxicity.Previous studies have shown that BACE1 not only cleaves Amyloid Precursor Protein(APP),but also may interfere with the physiological functions of proteins other than APP,such as protein kinase A(PKA),which is considered closely related to long-term potentiation(LTP)memory,can effectively improve cognitive impairment.Therefore,revealing the potential mechanism of BACE1 in the pathogenesis of AD may have a good prospect for the treatment of dementia.ObjectiveIn this study,the vessel meridian electroacupuncture was performed to stimulate APP/PS1 mice,and the 7-month-old male mice were employed as dementia model.The spatial learning and memory ability was tested to detect the behavior change after electroacpuncture intervention.The proteins expression APP,BACE1 and p-PKA in hippocampus and cortex of APP/PS1 dementia mice were investigated by various detection methods.To explore the effect of electroacupuncture intervention on learning and memory ability of dementia mice and its underlying mechanism,and to further provide theoretical basis for the clinical practice of electroacupuncture treatment of AD.MethodsIn this study,total number of 18 of 7-month-old APP/PS1 double transgenic mice were randomly divided into model(M)group and electroacupuncture(EA)group,9 in each group.Nine male C57BL/6 male mice,with the same genetic background and age were employed as normal control(C)group.The mice were placed in the single-cage feeding system of the animal center of Beijing University of Chinese Medicine,under the room temperature 20-22℃,humidity 40%-60%,adaptive feeding for 1 week,and free access to water.The acupoints"Baihui","Yintang"and"Shuigou"(referring to Eleventh Five-Year"Acupuncture and Moxibustion",second edition),and binds with self-made mouse sleeves to routinely disinfect acupuncture points and needles.Using the sterile acupuncture needles(0.18*25mm)to insert into the skin."Baihui"and"Yintang"are flat-punched into the needle,and the two needles do not touch each other to prevent short circuit,then connected the needle handle with HANS-LH202 type electric apparatus,with sparse wave,and frequency at 2/100 Hz,voltage is 2V,input intensity is 1mA.While the electric stimulation is slightly vibrated by local muscles,and the mouse’s quiet tolerant is the appropriate stimulation.After intervention for 20min,the mice is quickly pricked to"Shuigou".The whole experiment lasted for one month,once every other day.The C and M groups did not receive any acupuncture intervention,but was bound once while the EA group receiving intervention,20 min/day,once every other day for 15 times.After 24 hours of the electroacupuncture treatment,Morris water maze test was performed to detect the behavioral changes after electroacupuncture intervention.The morphological changes of CA1 region of hippocampal cells in each group were observed by HE staining.The expressions of APP,BACE1,PKA in hippocampus and cortex of each group were observed by immunohistochemistry,immunofluorescence and western blot respectively.Results1 Morris Water Maze(MWM)test1.1 Navigation test resultsWith the training time prolonged,the escape latency and swimming distance of the fourth quadrant into the pool of each group showed a downward trend,but the mice in the M group did not show statistical significance.There is no significant difference in swimming speed between the mice in each group.Compared with the C group,on the 3rd,4th and 5th day of the experiment,the escape latency of the M group increased significantly,and the difference was statistically significant(P<0.05,P<0.01);compared with the M group,the escape latency of the EA group on day 4 and 5 was shortened,and the difference was statistically significant(P<0.05,P<0.01).Compared with the C group,on the 2nd,3rd,4th and 5th day of the experiment,the swimming distance of the M group increased significantly(P<0.01);compared with the M group On the 3rd,4th and 5th day of the experiment,the swimming distance of the EA group was significantly shortened(P<0.05,P<0.01).The swimming showed no statistical difference.1.2 Space exploration experiment resultsCompared with the C group,the time to first across the original platform in M group was increased,and the average distance from the original platform increased as well.The difference was statistically significant(P<0.01).Compared with the M group,the EA group shortened the time of the original platform for the first time,and the average distance from the original platform was decreased,which was statistically different(P<0.05,P<0.01).2 HE stainingIn the C group,the nerve cells in the CA1 region of hippocampus were stained clearly and evenly,the cells were arranged neatly,the number of neurons were large,and the nucleus were round and clear;the nerve cells in the M group were scattered and the nucleus were not clear;Compared with the M group,the cells in EA group were arranged regularly.3 ImmunohistochemistryThe results of immunohistochemical staining of BACE1 in hippocampus of CA1 region were more common in cytoplasmic expression.Compared with the C group,the neuronal staining of the model group was deep,and the positive expression of BACE1 was significantly increased.Compared with the M group,the nerve cells of the EA group were lightly stained and the expression of BACE1 positive cells was decreased.Compared with the C group,the mean optical density of the M group increased,the difference was statistically significant(P<0.01).Compared with the M group,the average optical density of the electroacupuncture group decreased significantly(P<0.01).Immunohistochemical staining of APP in hippocampus of CA1region was more common in cytoplasm and nucleus.Compared with the C group,the neuronal cells in the M group were stained deeply,and the positive expression of APP was significantly increased.Compared with the M group,the neurons in the EA group were lightly stained and the expression of APP was decreased.Compared with the Cl group,the mean optical density of the M group increased,the difference was statistically significant(P<0.01).Compared with the M group,the average optical density of the EA group decreased significantly(P<0.01).The results of Aβ immunohistochemical staining in hippocampus of CA1 region were more common in cytoplasmic expression.Compared with the C group,the neuronal cells in the M group were deeply stained,and the positive expression of Aβ was significantly increased.Compared with the M group,the nerve cells in the EA group were lightly stained and the expression of Aβ was decreased.Compared with the C group,the mean optical density of the M group increased,the difference was statistically significant(P<0.01).Compared with the M group,the average optical density of the EA group decreased significantly(P<0.01).The results of immunohistochemical staining of p-PKA in hippocampus of CA1 region were more common in cytoplasmic expression.Compared with the C group,the neuronal cells in the M group were lightly stained,and the positive expression of p-PKA was significantly reduced.Compared with the M group,the nerve cells in the EA group were stained deeper and the expression of p-PKA was increased.Compared with the C group,the mean optical density of the M group decreased,and the difference was statistically significant(P<0.01).Compared with the M group,the average optical density of the EA group increased significantly(P<0.41).5 Western blotCompared with the C group,the expression of BACE1 in the cortex of APP/PS1 mice was significantly increased(P<0.05).Compared with the M group,the expression of BACE1 in the EA group was decreased significantly(P<0.05).Compared with the C group,the BACE1 level in the hippocampus was significantly higher,and the difference was statistically significant(P<0.01).Compared with the M group,the BACE1 level in the EA group was decreased significantly(P<0.05).Compared with the C group,the expression of APP protein in the cortex of APP/PS1 mice was significantly increased(P<0.01).Compared with the M group,the expression level of APP protein in the EA group was decreased significantly(P<0.05).Compared with the C group,the expression of APP protein in the hippocampus was significantly increased(P<0.01).Compared with the M group,the expression level of APP protein in the EA group was decreased significantly(P<0.05).Compared with the C group,the expression of p-PKA in the cortex of the M group was significantly lower,and the difference was statistically significant(P<0.01).Compared with the M group,the expression of p-PKA in the EA group was increased significantly(P<0.05).Compared with the C group,the expression of p-PKA in the cortex of the M group was significantly lower,and the difference was statistically significant(P<0.05).Compared with the M group,the expression of p-PKA in the EA group was increased significantly(P<0.05).conclusions1 Morris water maze test shows that vessel meridian electroacupuncture can effectively improve the learning and memory impairment of AD.2 Vessel Meridian electroacupuncture can reduce the deposition of BACE1 in the cortex and hippocampus of APP/PS1 double transgenic mice,and reduce the abnormal cleavage of APP,thereby exert regulatory effects on PKA and its related substrates,such as LTP,to improve memory and learning.ability. |
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