Based On The Microglia Polarization And The Stability Of Dendritic Spine To Explore The Mechanism Of Modified Shu Yu Pills Improving Learning And Memory Function In APP/PS1 Mice

ObjectiveThe theoretical part of this article summarizes the cognition of dementia by ancient and modern Chinese medicine doctors,and explores the theoretical feasibility of treating Alzheimer’s disease（AD）with spleen and kidney supplementation,Phlegm and blood stasis co-treatment;the experimental part is based on AD model mice observation of behavioral and hippocampal pathological morphology,to explore the therapeutic effect of Modified Shu Yu Pills on AD model mice.The mechanism of Modified Shu Yu Pills improving the cognitive function of AD mice is based on the Nlrp3/ASC/Caspase-1pathway of microglia phenotype transformation and the BDNF/Rac1/Cofilin pathway of dendritic spine stabilization.Methods1.Theoretical part:By consulting ancient medical books and searching modern databases,I combed the understanding of the pathogenesis of dementia by ancient and modern physicians,and summarized the comprehensiveness of the treatment of AD from spleen deficiency and kidney deficiency,and phlegm and blood stasis.Combining our team’s preliminary research foundation and modern pharmacological results,provide theoretical support for the treatment of AD with Modified Shu Yu Pills.2.Experimental Part:APP/PS1 mice were randomly divided into 3 groups,namely the model group,the Modified Shu Yu Pills group,and the Donepezil group,each with 10 mice,and another 10 mice of the same month and same background wild-type C57BL/6J was used as a normal group.The Modified Shu Yu Pills group was given 14 g/kg and the Donepezil group was given 1mg/kg by gavage,once a day.Daily gavage volume was 0.2ml/10g.We gavaged the mice for 35 days.The normal group and the model group were given the same volume of saline.After the gavage,the Morris water maze was used to observe the learning and memory ability of the mice in each group;the open field experiment was used to observe the anxiety and depression of the mice.After behavioral examination,fixed brain tissues were taken,and Nissl staining method was used to observe the pathological changes of hippocampal neurons in each mouse;immunohistochemical method was used to observe the hippocampal Aβdeposition,SYN,and PSD-95 protein expression in each group of mice;Golgi staining was used to observe changes of dendritic spines of mice in each group;immunofluorescence method was used to detect the co-expression of Iba1 and i NOS and the co-expression of Iba1 and CD36.After the behavioral test,fresh brain tissue was taken by decapitation method.The protein expression of Nlrp3,ASC,Caspase-1,IL-1β,BDNF,Trk B,rac1,p-LIMK1,LIMK,p-Cofilin,and Cofilin were detected by Western blot;qPCR was used to detect the relative expression of Nlrp3,Pro IL-1β,TNFα,IL-18,IL-4,IL-10,BDNF,LIMK1 mRNA.Results1.Morris water maze results:compared with the normal group,the escape latency of mice in the model group was significantly prolonged（P<0.01）;compared with the model group,the escape latency of the Modified Shu Yu Pills group was significantly shorter（P<0.01）;compared with the donepezil group,the difference in the escape latency of Modified Shu Yu Pills group was not statistically significant.Compared with the normal group,the results of the fixed flight experiment showed that the model group had significantly reduced,the number of platform crossings and the percentage of stay in the target quadrant was significantly reduced（P<0.05）.Compared with the model group,the number of times of crossing the platform and the percentage of stay in the target quadrant increased significantly after treatment in Modified Shu Yu Pills group（P<0.01）.There was no significant difference between Donepezil group and Modified Shu Yu Pills group.2.Open field test results:Compared with the normal group,the total movement distance,the central area movement distance percentage and the central area stay time percentage of the mice in the model group were significantly reduced（P<0.01）;compared with the model group,Modified Shu Yu Pills group can significantly increase the total movement distance of mice,the central area of the movement distance percentage and the percentage of stay time in the central area（P<0.01）;compared with the model group,The donepezil group was able to increase the total movement distance,the central area movement distance percentage,and the central area residence time percentage of APP/PS1 mice（P<0.01）.Compared with the donepezil group,there was no significant difference in Modified Shu Yu Pills group（P<0.01）.3.Nissl staining results:the normal group of mice has abundant Nissl bodies,complete cell membranes and obvious staining.Compared with the normal group,the number of hippocampal neurons in the model group was significantly reduced,the cell membrane was ruptured,the staining was lighter,and the number of Nissl bodies was reduced.Compared with the model group,the number of neurons of Modified Shu Yu Pills group increased,the Nissl bodies increased,and the staining became darker;Compared with the model group,the number of Nissl bodies and neurons in the hippocampus of the donepezil group increased.4.Immunohistochemistry results:For Aβ_1-42 immunohistochemistry,compared with the normal group,the deposition of Aβ_1-42 in the model group was increased（P<0.01）.Compared with the model group,Aβ_1-42 was reduced in the Modified Shu Yu Pills（P<0.05）.Compared with donepezil group,the deposition of Aβ_1-42 of Modified Shu Yu Pills decreased（P<0.05）.For SYN、PSD-95 immunohistochemistry,compared with the normal group,the protein expression of SYN and PSD-95 in the model group was significantly reduced（P<0.01）;compared with the model group,the Modified Shu Yu Pill’s expression of histones was significantly increased（P<0.01）.Compared with the donepezil group,the protein expression levels of SYN and PSD-95 in the Modified Shu Yu Pills group were increased（P<0.01）.5.Golgi staining results:Compared with the normal group,the dendritic spine of the model group was significantly reduced（P<0.01）;compared with the model group,the dendritic spine density of Modified Shu Yu Pills group increased（P<0.05）;compared with the donepezil group,the dendritic spines in the group of Modified Shu Yu Pills were increased（P<0.05）.6.Immunofluorescence results:Compared with the normal group,the expression of microglia markers Iba1 and i NOS in the model group increased significantly,while the co-expression of Iba1 and CD36 decreased.Compared with the model group,the Hippocampus Iba1 and i NOS positive cells were significantly reduced in Modified Shu Yu Pills group,while the co-expression of Iba1 and CD36 increased.Compared with the donepezil group,the number of Iba1 and i NOS co-expression cells in the Modified Shu Yu Pills group was significantly reduced,while the co-expression of Iba1 and CD36 increased.7.WB results:Compared with the normal group,the expression of Nlrp3,ASC,Caspase-1,IL-1βprotein in the model group showed increased,and the protein expression of BDNF,Arg-1 Trk B,Rac1,p-LIMK1/LIMK1,p-Cofilin/Cofilin decrease（P<0.01）.Compared with the model group,the expressions of Nlrp3,ASC,Caspase-1,IL-1βprotein in the group of Modified Shu Yu Pills and the expression of p-Cofilin/Cofilin was significantly increased（P<0.05）.Compared with donepezil,the Nlrp3,ASC,Caspase-1,IL-1βin the group of Modified Shu Yu pills significantly decreased,and the Protein expression of BDNF,Arg-1,BDNF,Trk B,Rac1,p-LIMK/LIMK,p-Cofilin/Cofilin increase（P<0.05）.8.qPCR results:Compared with the normal group,the expression of Nlrp3,IL-1β,TNFα,IL-18 mRNA in the model group was significantly increased,and the expression of IL-4,IL-10,BDNF,LIMK,and mRNA was significantly decreased in the model group.（P<0.05）.Compared with the model group,the expressions of Nlrp3,IL-1β,TNFα,and IL-18 mRNA in the groups of the Modified Shu Yu Pills were significantly reduced,and the expressions of IL-4,IL-10,BDNF,and LIMK mRNA were significantly reduced.（P<0.05）.Compared with donepezil group,the relative expressions of Nlrp3,IL-1β,TNFα,IL-18 mRNA in the Modified Shu Yu Pills group decreased,and the expressions of IL-4,IL-10,BDNF,and LIMK mRNA increased significantly.Conclusion1.Deficiency of the spleen and kidney,and mutual obstruction of phlegm retention are the basic pathogenesis of AD.Spleen and kidney supplementation,and co-treatment of Phlegm and blood stasis are effective treatments for AD.2.Modified Shu Yu Pills can improve cognitive function and relieve depression in APP/PS1 mice.3.Modified Shu Yu Pills can inhibit the Nlrp3/ASC/Caspase-1 pathway and promote the conversion of microglia to an alternatively activated phenotype,so as to increase neurotrophic and repair functions4.Modified Shu Yu Pills can promote the stability of dendritic spines and improve cognitive impairment through the BDNF/Rac1/LIMK/Cofilin pathway.