To Explore The Active Components,key Targets And Molecular Mechanisms Of Huangjing And Danggui Drug Pair In The Treatment Of Alzheimer’s Disease Based On Pharmacology And Experimental

Objective:The combination method of network pharmacology and animal experiments was used to investigate the effective components,key targets and molecular mechanism of Huangjing and Danggui drug pair(HDP)in the treatment of Alzheimer’s disease(AD),so as to provide experimental basis and reference for the clinical application of Huangjing pill in the prevention and treatment of AD.Methods:This study was divided into two parts,including experiment 1,network pharmacological prediction and molecular docking analysis;Experiment 2.Animal experiment verification.Experiment 1.Network pharmacological prediction and molecular docking analysis.1.Screening of active ingredients and targets of HDP: TCMSP and Pub Chem database were used to collect the information of HDP active ingredients and their targets,and Uniprot database was used to complete the verification of the target names of each active ingredient2.Screening of targets for the treatment of AD with HDP: Online databases such as human gene database,online human Mendelian genetics database,genetic pharmacology and pharmacogenomics database,drug bioinformatics and chemical informatics database,disease-related genes and mutation sites database were logged in to complete the collection of AD therapeutic targets.The targets of each database were merged and all duplicated data were eliminated.The intersection of the sorted AD targets and HDP active component targets could obtain the targets of HDP active component in the treatment of AD.3.PPI network construction:Firstly,Firstly,String11.0 database was used to obtain the protein interaction data of AD targets treated by HDP.The data results were then imported into Cytoscape3.8.0 software to visualize the interactions between the targets and analyze the key core targets.4.Gene ontology(GO)functional enrichment analysis and KEGG pathway enrichment analysis: Log in DAVID online database,input HDP treatment AD targets,and conduct GO functional enrichment and KEGG pathway enrichment analysis.5.Network diagram construction of drug components,target proteins and pathways: The information of active components of HDP,their therapeutic targets and pathways were input into Cytoscape3.8.0 software to construct the network diagram of "components,targets and pathways".The features of the network diagram were analyzed using the plug-in tool Analyze Network,and the main components,targets and the relationships among pathways of HDP in the treatment of AD were determined.6.Molecular docking analysis of the effective components and target protein of HDP: The files of drug molecules and core target proteins were downloaded from Pub Chem database and RCSB PDB database,respectively.The protein molecules and ligand molecules were converted to format and preprocessed before docking,then imported into Auto Dock4.2 software for molecular docking.Experiment 2.Animal experiment verification.1.Animals and Groups: Ninety SPF Kunming mice,males aged 4 weeks and weighing(22±2)g,were randomly divided into 6 groups with 15 mice per group,which were normal control group,AD model group,Donepezil hydrochloride(positive drug,0.65 mg/kg/d)group,HDP low dose(1.75 g/kg/d)group,HDP low dose(1.75 g/kg/d)group medium dose(2.5 g/kg/d)group,HDP high dose(7.5 g/kg/d)group.2.AD modeling and experimental drug intervention: The AD modeling process was divided into two parts.Except for the normal control group,mice in the other groups were first injected with 1% D-galactose solution(5 m L/kg)subcutaneously in the neck and back for 3 weeks,and then injected with scopolamine(2 mg/kg)peritoneally for 2 weeks,for a total of 5 weeks.One week after AD modeling,mice in the Donepezil hydrochloride group and in the low,medium and high dose groups of Huangjing and Danggui drug pair extract were intragastrically administrated with the corresponding dose of the test drug every day,and mice in the AD model group were intragastrically administrated with 0.5 m L sterile normal saline every day for 4 weeks.3.Detection of behavioral differences in learning and memory: In order to evaluate the improvement effect of HDP on the learning and memory ability of AD mice,Morris water maze test was used to detect the difference of escape latency within 5 days of each group.4.Detection of changes in cerebral cortex and hippocampus neurons: HE staining and Nissl staining were used to detect the number of neurons in cerebral cortex and hippocampus of mice in each group.5.Change detection of PI3K/AKT signaling pathway in hippocampus:Quantitative Real-time PCR(RT-q PCR)was used to detect the differences of PI3 K and AKT m RNA expression in the hippocampus of mice in each group.Western blot(WB)was used to detect the changes of PI3 K and AKT expression in the hippocampus of mice in each group.6.Detection of hippocampal nerve apoptosis: The expression difference of Bax,Bcl-2 and Caspase-9 positive neurons in the hippocampus of mice in each group was detected by immunohistochemical method.7.Differential detection of hippocampal neural stem cells(NSCs)proliferation:Immunofluorescence was used to detect the changes of Br DU-positive cells in the DG region of the hippocampus of mice in each group to detect the differences in NSCs proliferation.Results:Experiment 1.Results of network pharmacological prediction and molecular docking analysis1.Screening results of the effective components and targets of HDP : After screening,18 active ingredients with corresponding targets were obtained,with a total of 176 corresponding targets.There are 150 potential targets of this drug pair for the treatment of AD.2.Screening of targets for the treatment of AD by HDP: There are 150 potential targets of this drug pair for the treatment of AD.3.PPI network construction: The PPI network interaction map showed that 147 proteins had interaction,the number of edges was 1682,the average node degree was22.9,the average local clustering coefficient was 0.584,the expected number of edges was 597,and the PPI concentration p value was <1.0×10-16.Topological analysis showed that the top five core targets of HDP were AKT1,JUN,TNF,TP53 and INS.4.GO functional enrichment analysis: A total of 154 biological processes,77 cellular components and 132 molecular functions of GO were obtained.Among them,the biological processes mainly involve the positive regulation of drug response,RNA polymerase II promoter,signal transduction,and negative regulation of cell apoptosis.Cell components mainly involved plasma membrane,cell solute,cytoplasm,nucleus,etc.The molecular functions involved protein binding,enzyme binding,protein kinase binding,protein homodimer activity and so on.5.KEGG pathway enrichment analysis: A total of 161 signaling pathways were obtained for the treatment of AD by this drug pair,among which PI3K/AKT signaling pathway was the most enriched,and apoptosis-related targets were enriched including Bax,Bcl-2 and Caspase-9.6.Construction of network diagram of drug components,target proteins and pathway: Based on the construction and analysis of the network diagram of drug-target-pathway,it was concluded that the main components of HDP for the prevention and treatment of AD were stigmasterol,apigenin,isoliquiritigenin,baicalin and β-sitosterol.7.Molecular docking experiment: The active ingredients of HDP can be docked with all core targets,and the overall binding energy of AKT1 docking with all drug molecules in the core targets is the lowest;The binding energy of β-sitosterol to each core target was the lowest among the main active components.The docking results indicated that β-sitosterol was the most effective component of HDP in the treatment of AD.PI3K/AKT signaling pathway is the most effective signaling pathway for HDP treatment of AD.Experiment 2.Verification results of animal experiments.1.Behavioral difference detection such as learning and memory: Compared with the normal control group,the escape latency of mice in AD model group was significantly increased,suggesting that the mice showed obvious impairment in learning and memory ability(P<0.01).Compared with AD model group,the escape latency of mice in each drug group was significantly shortened,indicating that their learning and memory ability was significantly improved(P<0.05,P<0.01),and HDP showed a good dose-dependent relationship in the dose range of 1.75-7.5 g/kg/d.The above results showed that HDP effectively shortened the escape latency of AD mice,suggesting that HDP can improve the learning and memory disorders of AD mice,and can effectively treat AD.2.Detection of changes in cerebral cortex and hippocampus neurons: Compared with the normal control group,the number of neurons in S1 Tr region of cerebral cortex and CA1 and CA3 region of hippocampus in AD model group decreased,and the structure distribution was disordered.The cytoplasmic nuclei of neurons were unevenly colored,the cellular bodies were swollen and vacuole-like,the intracellular structures were disordered and loose,and the nuclei were shrunk(P<0.01).Compared with AD model group,the number of S1 Tr region in cerebral cortex and CA1 and CA3 region in hippocampus of mice in all drug groups increased,and the structure was arranged more neatly.The staining of neuronal cytoplasm and nucleus was deepened,and the phenomenon of cytoplasm loose and nucleolus degeneration and contraction were significantly reduced(P<0.05,P<0.01).In addition,the pharmacodynamic effect of HDP was enhanced with the increase of dosage in the range of 1.75-7.5 g/kg/d,indicating that the neuroprotective effect of the drug pair was better.3.PI3K/AKT signaling pathway detection of hippocampal nerve: Compared with normal control group,PI3 K and AKT m RNA and protein expressions in hippocampal of mice in AD model group were significantly decreased(P<0.01).Compared with AD model group,PI3 K and AKT m RNA and protein expressions in hippocampal of mice in all drug groups were significantly increased(P<0.05,P<0.01).In addition,the pharmacodynamic effect of HDP was enhanced with the increase of dose in the range of 1.75-7.5 g/kg/d,indicating that this drug can activate PI3K/AKT signaling pathway in AD animals.4.Hippocampal nerve apoptosis detection: Compared with the normal control group,the positive expression of Bcl-2 in the hippocampal CA1 and CA3 regions of the AD model group was significantly decreased(P<0.01),and the positive expression of Bax and Caspase-9 was significantly increased(P<0.01,P<0.01).Compared with the AD model group,the positive expressions of Bcl-2 in the hippocampal CA1 and CA3 regions of the mice in each drug group were significantly increased(P<0.05,P<0.01),and the positive expressions of Bax and Caspase-9 were significantly decreased(P<0.01,P<0.01).In addition,the effect of HDP was enhanced with the increase of dose when the dose was 1.75-7.5 g/kg/d,indicating that the drug pair could reduce the neuronal apoptosis in the brain of AD animals.5.Proliferation detection of hippocampal NSCs: Compared with normal control group,Brd U positive cells were significantly decreased in AD model group(P<0.01);Compared with AD model group,the number of Brd U positive cells in hippocampus of mice in all drug groups was significantly increased(P<0.05,P<0.01),and the efficacy of HDP was enhanced with the increase of dose in the range of1.75-7.5 g/kg/d,indicating that the drug can promote the proliferation of hippocampal NSCs in AD animals.Conclusions:1.HDP has a good therapeutic effect on AD.2.The main components of HDP in the treatment of AD are β-sitosterol,stigmasterol,apigenin,isoliquiritigenin,baicalin,etc.The main targets of regulation are AKT1,JUN,TNF,TP53,INS,etc.The regulated signaling pathways include PI3K/AKT,p53,TNF,Ca2+,inflammation and Wnt,etc.This drug has a multi-component,multi-target and multi-pathway mechanism for the prevention and treatment of AD.3.HDP can reduce neuronal apoptosis and promote the proliferation of hippocampal NSCs by activating PI3K/AKT signaling pathway and play a therapeutic role in AD.