Exploring The Mechanism Of Electroacupuncture In Improving Learning And Memory Ability In AD Model Rats Based On TLR4/MyD88/NF-κB Signaling Pathway

【Objective】The TLR4/NF-κB signaling pathway plays an important role in the immune and inflammatory mechanisms of AD.Learning and memory is an important index to evaluate the overall development of the nervous system.In this study,the acupuncture points of shenshu and Fenglong were selected according to the treatment of tonifying kidney and eliminating phlegm,and the effects of electroacupuncture at Shenshu and Fenglong points on the learning and memory ability of AD rats were observed,and the mechanism of the effects of electroacupuncture at Shenshu and Fenglong points on the learning and memory ability of AD rats was explored based on TLR4/My D88/NF-κB signaling pathway.【Method】Sixty 2-month-old SPF male SD rats（body weight 250±20g）were randomly divided into sham operation group（A）,model group（B）,kidney-tonifying group（C）,phlegm-reducing group（D）and kidney-tonifying and phlegm-reducing group（E）,with 12 rats in each group.Rats in group B,C,D and E were injected with Aβ_25-355μL in each ventricle for modeling,and group A was given the same dose of normal saline.Group A and B were fed normally without intervention.Groups C,D and E were given 20min/time electroacupuncture intervention,once per day,and 6d was a course of 2courses,with an interval of 1d between them.After treatment,Morris Water Maze test（MWM）,open field test and new object recognition test（NOR）were used to detect behavioral changes of rats in each group.The pathological changes of hippocampal area were observed by HE staining and Nessie staining.TUNEL staining was used to detect cell apoptosis in rat brain.The concentrations of TNF-α,IL-1β,IL-4,IL-6 and IL-10 in serum of rats were determined by ELISA.The positive expressions of TNF-α,IL-1β,IL-4,IL-10,TLR4,My D88 and NF-κB were detected by immunofluorescence.The m RNA expressions of TLR4,My D88,NF-κB,TNF-α,IL-1βand IL-6 in hippocampus of rats were detected by real-time quantitative PCR.The expression of TLR4,My D88 and NF-κB protein in hippocampus of rats were detected by WB.【Results】1.Behavioral changes of rats in each group:（1）MWM test showed that in the water maze test,the average escape latency of rats in groups A,C,D and E was lower than that in group B,and the average times of crossing the platform was higher than that in group B;the average escape latency of rats in group E was lower than that in group C and D,and the average times of crossing the platform was higher than that in group C and D.（2）The open field experiment showed that the activity time of rats in group A,C,D and E was longer than that in group B,and the activity time of rats in group E was longer than that in group C and D.（3）NOR test showed that the new object recognition coefficient of rats in groups A,C,D and E was higher than that in group B,and the new object recognition coefficient of rats in group E was higher than that in groups C and D.2.Pathological changes of rats in each group:（1）HE staining results showed that neurons in the hippocampal area of group A were orderly arranged with complete structure,and the nuclei were round with clear staining and normal shape;The neurons in the hippocampus of group B were not arranged in order,with incomplete structure,compact arrangement,nuclear shrinkage,obvious marginalization and lobulation,and the number of apoptotic neurons increased.The cell morphology of group C,D and E tended to be normal,and only a few nuclear vacuoles were lost.（2）Compared with group A,the cells in group B were characterized by unstable structure,fuzzy hierarchy and loose arrangement of neurons,reduction or disappearance of some intracellular Nisi bodies,and decreased relative expression of neurons.Compared with group B,the cell structure of rats in groups C,D and E was more stable,the neuron layers were clearer and more closely arranged,the number of Nisseni corpuscle was not decreased significantly,and the relative expression of neuron number was increased.Compared with groups C and D,the relative expression of neuron number in group E was increased.3.Changes of apoptosis in the brain of rats in each group:TUNEL staining results showed that compared with group A,the relative expression of apoptotic cells in group B was increased;Compared with group B,the relative expression of apoptotic cells in groups C,D and E decreased;Compared with groups C and D,the relative expression of apoptotic cells in group E was decreased.4.Expressions of TNF-α,IL-1β,IL-4,IL-6 and IL-10 in each group:（1）ELISA results showed that compared with group A,serum concentrations of TNF-α,IL-1βand IL-6 in group B were increased,while concentrations of IL-4 and IL-10 were decreased;Compared with group B,the serum concentrations of TNF-α,IL-1βand IL-6 in groups C,D and E were decreased,while the concentrations of IL-4 and IL-10 were increased.Compared with groups C and D,the concentrations of TNF-α,IL-1βand IL-6 in serum of group E were decreased,while the concentrations of IL-4 and IL-10 were increased.（2）Compared with group A,the relative expression levels of TNF-αand IL-1βin hippocampus of rats in group B were increased,while the relative expression levels of IL-4 and IL-10 were decreased.Compared with group B,the relative expressions of TNF-αand IL-1βin the hippocampus of rats in groups C,D and E were decreased,while the relative expressions of IL-4 and IL-10 were increased.Compared with groups C and D,the relative expressions of TNF-αand IL-1βin the hippocampus of group E were decreased,while the relative expressions of IL-4 and IL-10 were increased.（3）Real-time quantitative PCR showed that the m RNA expressions of TNF-α,IL-1βand IL-6 in hippocampus of rats in group B were increased compared with that in group A;Compared with group B,the m RNA expression of TNF-α,IL-1βand IL-6 in hippocampus of rats in groups C,D and E decreased.Compared with groups C and D,the m RNA expressions of TNF-α,IL-1βand IL-6 in hippocampus of group E were decreased.5.Expression of TLR4,My D88 and NF-κB in each group:（1）immunofluorescence results showed that compared with group A,the relative expression of TLR4,My D88 and NF-κB in hippocampus of group B was increased;Compared with group B,the relative expression levels of TLR4,My D88 and NF-κB in hippocampus of rats in groups C,D and E were decreased.Compared with groups C and D,the relative expression levels of TLR4,My D88 and NF-κB in hippocampus of group E were decreased.（2）Real-time quantitative PCR showed that compared with group A,m RNA expression of TLR4,My D88 and NF-κB in hippocampus of group B was increased;Compared with group B,m RNA expression of TLR4,My D88 and NF-κB in hippocampus of rats in groups C,D and E decreased.Compared with groups C and D,m RNA expression of TLR4,My D88 and NF-κB in hippocampus of group E decreased.（3）WB results showed that compared with group A,the relative expression levels of TLR4,My D88 and NF-κB in hippocampus of group B were increased.Compared with group B,the relative expression levels of TLR4,My D88 and NF-κB in hippocampus of rats in groups C,D and E were decreased.Compared with groups C and D,the relative expression levels of TLR4,My D88 and NF-κB in hippocampus of group E were decreased.【Conclusion】In behavioral aspect,the learning and memory ability of group B was decreased,and negative emotions such as anxiety and depression appeared.In terms of pathology,the neurons in the hippocampus of rats in group B were not arranged in order,with incomplete structure and compact arrangement,and the number of neurons was decreased,some intracellular Nite bodies were reduced or disappeared,and the expression of apoptotic cells was increased.The expression of TNF-α,IL-1β,IL-6 increased,the expression of IL-4,IL-10decreased,the expression of TLR4,My D88,NF-κB increased,suggesting that learning and memory ability,AD,inflammatory factors are closely related to each other.Electroacupuncture can effectively prevent and treat AD by down-regulating the expression of TLR4/My D88/NF-κB signaling pathway protein,affecting the expression of TNF-α,IL-1β,IL-4,IL-6,IL-10 and other inflammatory factors,reducing the expression of apoptotic cells,increasing the number of neurons and improving the learning and memory ability of AD rats.And according to the statistical results,it is found that the combination of points selected by the idea of"tonifying kidney and eliminating phlegm"has better efficacy than single point.1.Electroacupuncture at Shenshu and Fenglong points can improve the learning and memory ability of AD model animals established by Aβ_25-35.2.The combined acupoints selected by the treatment idea of"tonifying kidney and eliminating phlegm"have better efficacy than single acupoints;3.Electroacupuncture at Shenshu and Fenglong points can improve the pathological damage of hippocampal neurons,the loss of Nisseni bodies and apoptosis of AD model animals.4.Electroacupuncture at Shenshu and Fenglong points may improve the animal behavior and pathological damage of AD model by inhibiting TLR4/My D88/NF-κB signaling pathway and expression of downstream cytokines.