Study On Material Basis And Mechanism Of Erjing Pills In Prevention And Treatment Of Alzheimer’s Disease

Objective:On the one hand,virtual screening of the material basis and mechanism of Erjing pills in preventing and treating AD was carried out by means of network pharmacology,which laid the foundation for later experiments.On the other hand,material basis,action mechanism and the effects of Erjing pills on learning and memory of AD rats induced by ovariectomy combined with D-galactose and Aβ_1-40injection and their substances were studied by means of behavioral,morphological,proteomics and cell experimental verification.Both of which confirm each other,together indicate the material basis and action mechanism of Erjing Pills in preventing and treating AD.Method:1 Study on Material Basis and Action Mechanism of Erjing Pill in Prevention and Treatment of AD Based on Network Pharmacology1.1 Acquisition and Screening of Erjing Pill’s CompoundsThe chemical constituents of Erjing pills were collected by TCMSP database,Taiwan TCM database,Chinese Academy of Sciences chemical database,related literature research and laboratory acquisition.In order to facilitate the later analysis,the compounds in Lycium barbarum L.and Polygonatum sibiricum were named uniformly,and the drug-like properties of the collected components were screened by ADMET Descriptors module in DS software.1.2 Target Prediction of Active Compounds in Erjing PillsAll compounds were converted into standard Canonical SMILES format by PubChem database.SMILES format file was imported into Swiss Target Prediction platform.Attributes were set as"homo sapiens".Potential targets of active compounds were predicted and known targets of unpredictable active compounds were supplemented according to literature reports.1.3 Collection of Targets for Erjing Pills in Prevention and Treatment of ADThe genes of AD were collected through OMIM,TTD and DiGSeE three disease target websites by the key word of"Alzheimer’s disease".The repetitive target genes were removed and compared with the target obtained in the previous step.Finally,the potential target of Erjing pills in preventing and treating AD was obtained.Using UniProtKB search function in UniProt database,the species are defined as"Homo sapiens",and the uniform target codes are"Q9Y5N1,Q9BXC0,Q96JD6"formats,which standardize the naming of target proteins.1.4 Enrichment Analysis of PathwayThe potential targets of Erjing Pills in preventing and treating AD were put into Metascape platform.After submitting,the input and analysis species were selected as"H.sapiens"and set P<0.01.GO annotation analysis and pathway analysis were carried out on the targets of Erjing Pills.The results were saved and sorted according to the number of targets involved in each item to screen the top biological processes and pathways.1.5 Building Component-Target Protein-Signaling Pathway NetworkComponent-target protein-signal pathway network was constructed by Cytoscape3.2.1 software,using selected compounds,targets of AD and signaling pathways of targets.The network analysis under the Tools panel was used to calculate the relevant network parameters.1.6 Molecular docking verificationUsing the FDA-certified drug’s name for clinical treatment of AD as the keyword,searching on GeneCards platform and ranking with"score"as the parameter,we obtained the most relevant targets and compared them with the potential targets of Erjing pills in preventing and treating AD.Using the results of the comparison with the active ingredients of Erjing pills and five positive drugs were made use of CDOCKEER of DISCOVERY STUDIO to module docking and screening and docking activity with candidate targets at least better than four positive control drugs.2 Pharmacodynamic Evaluation of Erjing Pills on Learning and Memory of AD Rats Induced by Ovariectomy Combined with D-galactose and Aβ_1-40 Injection2.1 Quality control of Erjing Pills:According to the requirements of Chinese Pharmacopoeia（2015 version）,determination of Glucose Content in Polygonatum sibiricum,determination of Glucose and Betaine in Lycium barbarum L.and glucose and betaine in Lycium barbarum L.was determined,the content of water in Polygonatum sibiricum and Lycium barbarum L.was determined by the second method of General Principle 0832.2.2 Drug preparation:Polygonatum sibiricum and Lycium barbarum L were taken1400 g each,soaked in tap water of 5 times volume for 30 minutes,boiled,decocted twice,decocted for 40 minutes for the first time,decocted for 50 minutes for the second time,filtered and merged for two times,concentrated in water bath at 80-100℃to obtain 2333 mL（containing 1.2 g/mL of crude drug）and stored in refrigerator at-20℃for reserve.2.3 Animal models,grouping and administration:model group,positive control group（Donepezil,1.0 mg/kg）,Erjing pills high dose group（9.0 g raw medicine/Kg）,Erjing pills medium dose group（4.5 g raw medicine/Kg）and Erjing pills low dose group（2.25 g raw medicine/kg）,11 in each group,12 sham operation groups.All rats except the sham-operated group were injected with D-galactose 100 mg/kg intraperitoneally one week after operation,once a day for 7 weeks.Four weeks after operation,Aβ_1-4010 ug/rat was injected locally into the brain.After 3 weeks of ovariectomy,rats in model group and sham operation group were given the same amount of saline,while those in other groups were given the corresponding drugs with a dose of 10 mL/kg once a day for 28 consecutive days.2.4 Test indicators:（1）Behavioral indicators:After 28 days of administration,the identification index was detected by new object recognition experiment,and the escape latency and positioning navigation ability were detected by Moriss water maze method.（2）Morphological indicators:HE staining was used to observe the morphology of neurons in hippocampal CA1 region,and Nissl staining was used to observe the distribution of Nissl substance in hippocampal CA1 region.（3）Pathological indicators:The distribution of Aβ_1-40-40 and p-tau⁴⁰⁴ proteins was detected by immunohistochemistry.（4）Estrogen level and receptor expression:ERβpositive cells in hippocampal CA1 region were detected by immunohistochemistry and serum estrogen level was detected by Elisa method.（5）The effect of Erjing Pills on proteomics of hippocampus of AD rats induced by ovariectomy combined with D-galactose and Aβ_1-40 injection:Extracting,reducing,alkylating,enzymatic hydrolysis and LC-MS detection of proteins in hippocampus tissue,GO analysis of differential proteins using PANTHER Classification System database,String database construction of protein interaction network map,KEGG database enrichment Signaling pathways.（6）DA level and expression of D1 receptor and TH:serum DA level was detected by Elisa method,and the expression of D1 receptor and tyrosine hydroxylase in CA1 region was detected by immunohistochemistry.3.Material Basic Research on Erjing Pills in Prevention and Treatment of ADMTT assay was used to detect the optimal concentration of PC12 cells induced by Aβ_25-35;MTT assay was used to detect the effect of Erjing pills monomer compounds on the survival rate of AD model induced by Aβ_25-35;Fluo-3 AM calcium ion detection kit was used to detect the effect of Erjing pill monomer compounds on Ca²⁺level of AD model of PC12 cells.Result:1 Study on Material Basis and Action Mechanism of Erjing Pills in Prevention and Treatment of AD Based on Network PharmacologyBy comparing the target databases of AD and Erjing Pills components,127selected targets were analyzed by GO annotation and KEGG pathway.It was found that synaptic signal transduction,transsynaptic signal transduction and anterograde transsynaptic signal transduction were the top ones in biological process,while dendrites,axons and postsynapses were the top ones in cell composition analysis,mainly concentrated in neurons and cell membranes.Molecular function analysis mainly includes phosphatase activity,protein kinase activity and reductase activity.Targets of Erjing pills for preventing and treating AD involve 145 pathways,including cancer signaling pathway,neuroactive ligand-receptor interaction,PI3K-Akt signaling pathway,5-HT synapse and calcium signaling pathway.The target of AD-positive drug was selected from the intersection of the target of AD-positive drug and the previous target,and the target of AD-positive drug was docked with 65 active ingredients in Erjing pills.It was found that 20 active ingredients such as polygonatine A and compound E in Erjing pills had better binding ability to disease-related proteins than at least four positive drugs.2 Pharmacodynamic Evaluation of Erjing Pills on Learning and Memory of AD Rats Induced by Ovariectomy Combined with D-galactose and Aβ_1-40 Injection2.1 Quality control of Erjing Pills:The average content of betaine in Lycium barbarum L.was 0.6924%.The content of glucose and water in Polygonatum and Lycium barbarum L.met the requirements of pharmacopoeia.2.2 Test indicators:（1）Behavioral indicators:In Morris water maze experiment,the escape latency of Erjing pills high and middle dose group was significantly shorter than that of model group from the 3rd day.In the space exploration experiment,the number of times of Erjing pills high dose group crossing the platform was also significantly increased compared with the model group.In the new object recognition experiment,compared with the model group,the high dose group and the positive drug group had higher new object recognition index.（2）Morphological indicators:HE staining showed that compared with sham-operated group,neurons in hippocampal CA1 area of model group were arranged loosely and disorderly,with deeper nucleus staining and fewer layers of neurons.Compared with model group,neurons in hippocampal CA1 area of high,middle,low dose group and positive control group were more complete,orderly,without damage and clear nucleolus.Nissl staining showed that compared with sham operation group,Nissl substance in hippocampal CA1 area of model group decreased with the loss of neurons;compared with model group,the number of Nissl substance in hippocampal CA1 area of high,middle,low dose group and positive control group was more,the shape was more complete and the arrangement was neat.（3）Pathological indicators:Compared with sham-operated rats,the number of Aβand p-tau⁴⁰⁴ positive cells in hippocampal CA1 area of model rats increased significantly,and the pericellular staining was deeper.Compared with model rats,the number of Aβand p-tau404 positive cells in hippocampal CA1 area of rats in positive drug group and high dose group decreased significantly,and the cell staining was lighter.（4）Estrogen level and receptor expression:Compared with sham-operated group,the level of serum estrogen and the number of ERβpositive cells in hippocampus were significantly reduced,and the cytoplasm and processes were lighter.Compared with model group,the number of ERβpositive cells in positive drug group,high-dose group,middle-dose group and low-dose group increased significantly,while the estrogen level in positive drug group and high-dose group increased significantly.（5）DA level and expression of D1 receptor and TH:Compared with sham-operated group,DA,D1 receptor and TH levels in model group were significantly lower;compared with model group,DA level and TH positive cells in positive drug group and high dose group were significantly higher,and the number of D1 positive cells in positive drug group,high dose group,medium dose group and low dose group was significantly increased.（6）The effect of Erjing Pills on proteomics of hippocampus of AD rats induced by ovariectomy combined with D-galactose and Aβ_1-40 injection:（1）The results of Protein Discovery and SIEVE software analysis showed that there were more than247 differentially expressed proteins in Erjing pills high and middle dose groups compared with model group,including microtubule-related proteins,heat shock proteins and brain protection-related proteins.AD-related proteins.（2）GO analysis and String analysis showed that there were 91 differential proteins with catalytic activity,56 binding functions and 12 transporter activities.Differential proteins were most distributed in cells,followed by organelles,macromolecule complexes and membranes.There are 109 differential proteins involved in cell processes,metabolic processes 95 kinds and stimulatory reactivity of 46 kinds.（3）KEGG database signal pathway enrichment results:The results showed that the above differential proteins were involved in 93 signal pathways,such as PI3K-Akt signaling pathway,Alzheimer’s disease.3 Material Basic Research on Erjing Pills in Prevention and Treatment of AD3.1 Effect of Erjing Pills Monomer Compounds on the Survival Rate of PC12 CellsThe optimal concentration of AD model Aβ_25-35 in PC12 cells was 60μmol/L,and the dose gradients of 4’,5-dihydroxyflavone,polygonatine A and compound E were 10μmol/L,20μmol/L and 40μmol/L.3.2 Effect of Erjing Pills Monomer Compounds on Survival Rate of PC12 Cell AD Model Induced by Aβ_25-35The effects of 4’,5-dihydroxyflavone,polygonatine A and compound E on the survival rate of AD model of PC12 cells induced by Aβ_25-35 were detected by MTT assay.Compared with the model group,the survival rate of PC12 cells in the dosage groups of polygonatine A 20 and 40μmol/L was significantly increased（P<0.05）,and the cell survival rate was proportional to the dosage of compound E 20μmol/L.The survival rate of PC12 cells in the dosage group of compound E 20μmol/L was also higher than that in the model group.The survival rate of 4’5-dihydroxyflavone group at 20 and 40μmol/L doses was significantly higher than that of model group.3.2 Effects of Erjing Pills Monomer Compounds on Ca²⁺in PC12 Cells Induced by Aβ25-35The levels of Ca²⁺in AD model of PC12 cells were significantly increased in the40μmol/L group,20μmol/L group and 20μmol/L group of 4’5-dihydroxyflavone.Conclusion:1 Erjing Pills can improve the learning and memory ability of AD rats.2 Erjing Pills may increase the expression of TH and DA by increasing estrogen level in blood and ERβcells in brain,resulting in the increase of D1 positive cells and the up-regulation of TH-DA-D1-Gq-PLC-IP3R-CaM-Camk2d protein in Dopaminergic synapse pathway,and the increase of intracellular Ca²⁺concentration,resulting in explosive calcium flow,thereby promoting intracerebral neurosynaptic transmission and decreasing the expression of Aβand p-tau⁴⁰⁴ in hippocampal CA1 region.Improve learning and memory ability,produce the pharmacological effect of prevention and treatment of AD.3 The active ingredients of Erjing Pills in preventing and treating AD is dibutyl phthalate,paeonol,tyrosine,apigenin,4’,5-dihydroxyflavone,polygonatine A and compound E.