| ObjectivesBased on the high correlation between epigenetic modification disorder and the pathogenesis of Alzheimer's disease(AD),and the decrease of cyclin-dependent kinase 5(CDK5)DNA methylation level,which leads to overactivation of tau protein and hyperphosphorylation of tau protein,the purpose of this study was to explore the effects of electroacupuncture on learning and memory ability,neuronal and synaptic ultrastructure of SAMP8 mice,and also to elucidate the effect of electroacupuncture on the prevention and treatment of learning and memory impairment in SAMP8 mice and its epigenetic mechanism,in order to provide scientific experimental basis for electroacupuncture intervention in AD.MethodA total of 72 SPF grade 5-month-old male SAMR1 and SAMP8 mice with a weight of(13±3)g were selected.After a week of adaptive feeding,the Morris water maze test was conducted to select qualified mice and randomly divided into 12 SAMR1 normal control groups.60 SAMP8 mice were randomly divided into model group,sham electroacupuncture group,2 Hz electroacupuncture group,inhibitor group and electroacupuncture + inhibitor group,12 mice in each group.The control group and the model group did't do any treatment.While the treatment group was treated,the same specifications of the fixed table were used for grabbing and bundling;the electroacupuncture group chooses "Baihui" and "Shenshu" to intervene.The front flat puncture was 3 ~ 5 mm,and the "Shenshu" punctures 5 mm obliquely at a 45° angle to the skin at the midline.Connected to HANS-200 A electro-acupuncture treatment instrument,a pair of wires are connected to Baihui and Shenshu points,and the left and right Shenshu points were used alternately.Continuous wave,frequency 2Hz,current 1m A,local micro-tremor at the acupuncture point is preferred,needle retention for 20 min,once a day,7 days as a course of treatment,a day of rest between treatment sessions,4 courses of treatment for a total of 31 days;sham electroacupuncture group received electroacupuncture but no electricity;inhibitor group and electroacupuncture + inhibitor group from the fourth at the beginning of the course of treatment,DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine treatment was given for 7 consecutive days(intraperitoneal injection of 0.2 mg/kg /day).After the intervention of mice in each group,Morris water maze experiment was conducted to evaluate the spatial learning and memory ability of mice in each group.After the end of the water maze experiment,fresh hippocampal tissues were taken and perfused and the indicators were detected.The transmission electron microscope was used to observe the changes of neuron morphology,number of synapses,and ultrastructure of synapses in the hippocampus CAI area;tau-5,tau-p S262,tau-p S404 PHF-1,CDK5,p25,and DNMT1 protein expression levels in the hippocampus were observed by Western-blot;the number of CDK5 positive neurons were observed by immunohistochemistry;fluorescent quantitative PCR was used to detect the expression level of CDK5 m RNA in the hippocampus of each group of mice;methylation-specific PCR The methylation level of the CDK5 DNA promoter region in the hippocampus of mice in each group was detected.Results1.The effect of electroacupuncture on the learning and memory ability of SAMP8 mice:(1)Observation of the signs of SAMP8 mice in each group: the normal group has bright hair,good mental state,normal response to eating,drinking and external stimuli;Compared with the normal group,the model group had slightly worse hair color,slight hair loss and reduced diet,and were slightly slower to respond to external stimuli such as pain and sound;Compared with the model group,the inhibitor group had yellow hair,hair loss,reduced diet,slow action and slow response to external stimuli,while the electroacupuncture group,sham electroacupuncture group,electroacupuncture + inhibitor group had better coat color,mental state and response to external stimuli than the model group,and the electroacupuncture + inhibitor group was better than the inhibitor group.The acupuncture group was better than the sham electroacupuncture group.(2)Results of Morris water maze navigation test of SAMP8 mice in each group: Compared with the normal group,the average escape latency of the model group mice was significantly prolonged(P<0.01);Compared with the model group,the average escape latency of mice in the electroacupuncture group,the sham electroacupuncture group and the electroacupuncture + inhibitor group was significantly shortened(P<0.05),and the average escape latency of mice in the inhibitor group was significantly prolonged(P<0.05);Compared with the sham electroacupuncture group,the average escape latency of mice in the electroacupuncture group was significantly shortened(P <0.05).(3)Results of Morris water maze space exploration experiment of SAMP8 mice in each group: Compared with the normal group,the time of the model group mice crossing the platform for the first time was significantly prolonged and the number of crossing platform was significantly reduced and the duration of swimming in the original platform quadrant was shorter(P<0.01);Compared with the model group,the time of crossing the platform for the first time in the inhibitor group was significantly longer and the number of times crossing the platform was significantly decreased and the duration of swimming in the original platform quadrant was shorter(P<0.05),However,the time of crossing the platform for the first time in electroacupuncture group and sham electroacupuncture group was significantly shortened and the number of times was significantly increased and the duration of swimming in the original platform quadrant was extended(P<0.01);Compared with the inhibitor group,the time of crossing the platform for the first time in the electroacupuncture + inhibitor group was significantly shorter and the number of times crossing the platform was significantly increased and the duration of swimming in the original platform quadrant was extended(P<0.05).(4)Results of spatial discrimination of SAMP8 mice in Morris water maze in each group: Compared with the normal group,the number of correct responses in the model group decreased(P<0.01);Compared with the model group,the number of correct responses in the electroacupuncture group and the sham electroacupuncture group increased(P<0.01);Compared with the inhibitor group,the number of correct reactions in the electroacupuncture + inhibitor group increased(P<0.05),but lower than that in the electroacupuncture group(P<0.05).2.The effect of electroacupuncture on neurons and synapses in the hippocampal CA? area of SAMP8 mice:(1)The ultrastructure of neurons in hippocampal CAI region of each group showed that the morphology of neurons in normal group and electroacupuncture group was complete,the structure of nucleus and membrane was clear,a large number of organelles could be seen,and the morphology of endoplasmic reticulum,mitochondria,lysosome and Golgi apparatus were normal.In the model group,the shape of neurons was irregular,the structure of nucleus and membrane was blurred,the volume of neurons decreased,the number of mitochondria decreased and swelled,accompanied by mitochondrial crest-like changes,while in the inhibitor group,the morphology of neurons was irregular,the structures of nucleus and membrane were blurred,the volume of neurons was significantly reduced,the number of mitochondria decreased,swelling and accompanied with mitochondrial crest-like changes,and the morphology of endoplasmic reticulum,lysosome and Golgi apparatus changed.The morphology of neurons in the sham electroacupuncture group was more regular,the number of organelles was less than that in the electroacupuncture group,cytoplasmic edema was serious,nuclear pyknosis,endoplasmic reticulum,ribosome and mitochondria were not significantly abnormal.In the electroacupuncture + inhibitor group,the morphology was regular,slightly blurred,the volume of neurons decreased,and the number of mitochondria decreased compared with normal.(2)Synaptic number changes in the hippocampal CAI area of SAMP8 mice in each group: Compared with the normal group,the number of synapses per unit area of the model group decreased(P<0.01);Compared with the model group,the number of synapses per unit area decreased in the inhibitor group(P<0.05),while the number of synapses per unit area in the electroacupuncture group,the sham electroacupuncture group and the electroacupuncture + inhibitor group increased(P<0.01 or P<0.05),and the number of synapses in the sham electroacupuncture group decreased(P<0.05).(3)Quantitative analysis of the thickness of the hippocampal CAI area and the width of the synaptic gap in the SAMP8 mice of each group: Compared with the normal group,the thickness of the hippocampal CAI area in the model group decreased and the width of the synaptic gap was slightly widened(P<0.01;P<0.05);Compared with the model group,the thickness of the hippocampal CAI area in the inhibitor group was reduced,and with no significant difference in the width of the synaptic gap(P>0.05),However,the thickness of postsynaptic dense zone in hippocampal CAI region increased and the width of synaptic space narrowed in electroacupuncture group and sham electroacupuncture group(P<0.01;P<0.05);Compared with the inhibitor group,the thickness of the postsynaptic dense zone in the hippocampal CAI region was thickened(P<0.01)and the width of synaptic space was slightly narrowed(P<0.05)in the electroacupuncture + inhibitor group.3.Effect of electro-acupuncture on the expression level of Tau-5 and phosphorylation sites ser262 and ser404 and PHF-1 in the hippocampus of SAMP8 mice:(1)Effect of electro-acupuncture on the expression level of Tau-5 protein in the hippocampus of SAMP8 mice: Compared with the normal group,the level of Tau-5 protein in the model group's hippocampus increased(P<0.05);Compared with the model group,the level of Tau-5 in the inhibitor group's mouse increased(P<0.05),while the electroacupuncture group and the sham electroacupuncture group decreased(P<0.05),and the sham electroacupuncture group Tau-5 protein level decreased less than the electroacupuncture group(P<0.05).(2)The effect of electroacupuncture on the expression levels of ser262 and ser404 in the phosphorylation sites of hippocampus in SAMP8 mice: Compared with the normal group,the expression levels of ser262 and ser404 in the phosphorylation sites of hippocampus in model groups increased(P<0.01);Compared with the model group,the expression level of ser404 increased in the inhibitor group(P<0.05),and there was no statistically significant difference in the expression level of ser262(P>0.05).The expression levels of ser262 and ser404 in the electroacupuncture group and the sham electroacupuncture group decreased(P<0.05).The expression level of ser262 and ser404 in the sham electroacupuncture group was smaller than that in the electroacupuncture group(P<0.05).Compared with the inhibitor group,the expression level of ser262 and ser404 in the electroacupuncture + inhibitor group decreased(P<0.01).(3)Effect of electroacupuncture on the expression level of PHF-1 in the hippocampus of SAMP8 mice: Compared with the normal group,the expression level of PHF-1 in the hippocampus of the model group was increased(P<0.01);Compared with the model group,there was no significant difference in the relative expresstion level of PHF-1 in the inhibitor group(P>0.05),the relative expression level of PHF-1 in the electroacupuncture group and the sham electroacupuncture group decreased(P<0.05),and the decrease in the sham electroacupuncture group was lower than that in the electroacupuncture group(P<0.05);Compared with the inhibitor group,the relative expression level of PHF-1 in the electroacupuncture + inhibitor group decreased(P<0.01).4.The effect of electroacupuncture on the expression level of P25 and CDK5 protein in hippocampus of SAMP8 mice:(1)The effect of electroacupuncture on P25 of hippocampus in SAMP8 mice: Compared with the normal group,the expression of P25 in the hippocampus of the model group increased(P<0.01);Compared with the model group,there was no significant difference in the level of P25 in the hippocampus of mice in the inhibitor group(P>0.05),while the expression of P25 in the hippocampus of the electroacupuncture group,sham electroacupuncture group,electroacupuncture + inhibitor group decreased(P<0.01).The average descending water in the sham electroacupuncture group and the electroacupuncture + inhibitor group was lower than that in the electroacupuncture group(P<0.05).(2)The effect of electroacupuncture on the expression level of CDK5 protein in the hippocampus of SAMP8 mice:The results of immunohistochemistry found that compared with the normal group,the model group had more CDK5 positive neurons in the hippocampus(P<0.05);Compared with the model group,the CDK5 positive neurons in the hippocampus increased in the inhibitor group(P<0.05),while in the electroacupuncture group and the sham electroacupuncture group,the CDK5 positive neurons decreased(P<0.01;P<0.05);Compared with the electroacupuncture group,the number of CDK5 positive neurons in the hippocampus increased(P<0.01)in the sham electroacupuncture group.WB results found that compared with the normal group,the CDK5 level in the model group's hippocampus increased(P <0.01);Compared with the model group,the level of CDK5 increased in the hippocampus of mice in the inhibitor group(P>0.05),while the electroacupuncture group and the sham electroacupuncture group decreased(P<0.01;P<0.05);Compared with the electroacupuncture group,the CDK5 level increased in the hippocampus of mice in the sham electroacupuncture group and electroacupuncture + inhibitor group(P<0.05).Compared with the inhibitor group,the level of CDK5 in the hippocampus of mice in the electroacupuncture + inhibitor group decreased(P<0.05).5.The effect of electroacupuncture on the level of DNA methyltransferase DNMT1,the methylation level of CDK5 DNA promoter region and the m RNA level of CDK5 in the hippocampus of SAMP8 mice:(1)The effect of electroacupuncture on the level of DNMT1 in the hippocampus of SAMP8 mice: Compared with the normal group,the relative expression level of DNMT1 in the hippocampus of the model group decreased(P<0.01);Compared with the model group,the relative expression level of DNMT1 in the inhibitor group decreased slightly(P<0.05).Howevever,the relative expression level of DNMT1 in the hippocampus of electroacupuncture group,the sham electroacupuncture group and the acupuncture + inhibitor group increased significantly(P<0.01 or P<0.05);Compared with the electroacupuncture group,the relative increase of DNMT1 in the sham electroacupuncture group was slightly lower(P<0.05).(2)The effect of electroacupuncture on the methylation level of the CDK5 DNA promoter region in the hippocampus of SAMP8 mice: Compared with the normal group,the methylation level of the CDK5 DNA promoter region in the model group decreased(P<0.01);Compared with the model group,the methylation level of the CDK5 DNA promoter region decreased in the inhibitor group(P<0.05),while the methylation level of the CDK5 DNA promoter region in the electroacupuncture group,sham electroacupuncture group,electroacupuncture + inhibitor group mice significantly increased(P<0.01 or P<0.05),and the methylation level of CDK5 DNA promoter region in mice in sham electroacupuncture group,electroacupuncture + inhibitor group was lower than that in electroacupuncture group(P<0.05).(3)The effect of electroacupuncture on the CDK5 m RNA level in the hippocampus of SAMP8 mice:It was found by fluorescent quantitative PCR that compared with the normal group,the CDK5 m RNA level in the model group mice hippocampus increased(P<0.01);Compared with the model group the CDK5 m RNA level in the hippocampus tissue of the inhibitor group increased(P<0.05),while the CDK5 m RNA level in the hippocampus tissue of the electroacupuncture group and the sham electroacupuncture group decreased(P<0.01;P<0.05),and the level of CDK5 m RNA in the hippocampus of mice in the sham electroacupuncture group was higher than that in the electroacupuncture group(P<0.05).Conclusion1.Electroacupuncture can improve the spatial learning and memory impairment of SAMP8 mice.It may be one of the important mechanisms of electroacupuncture to improve the damage of learning and memory of SAMP8 mice by improving the damage of neurons and synaptic ultrastructure in hippocampus.2.Electroacupuncture can inhibit the excessive phosphorylation of tau protein and reduce the formation of neurofibril tangles by inhibiting the excessive activation of CDK5,thus reducing the damage of neurons and synaptic ultrastructure.3.Electroacupuncture can upregulate the expression of DNMT1 in the hippocampus,increase the methylation level of the CDK5 gene promoter region,thereby inhibiting the transcription and expression of the CDK5 gene,and then inhibit the excessive phosphorylation of tau protein,it may be one of the important mechanisms for electroacupuncture to reduce the ultrastructural damage of neurons and synapses in SAMP8 mice and to improve the impairment of learning and memory abilities. |
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