Network pharmacological analysis on the active ingredients of Yigan Powder in treating Alzheimer' s disease with depressive disorder

Chi Zhao ,Rong-Juan Guo ,Fei-Fei Ren ,Yao Yu ,Jun-Nan Li ,Yang Li

1.Beijing University of Chinese Medicine,Beijing 100029,China

2.DongFang Hospital,Beijing University of Chinese Medicine,Beijing 100078,China

3.Beijing Hospital of Traditional Chinese Medicine,Beijing 100010,China

Keywords:Yigan Powder Alzheimer’ s disease Depressive disorder Network pharmacology

ABSTRACT Objective:To study the potential therapeutic effect of Yigan Powder on Alzheimer’s disease(AD) comorbid Depressive Disorder with Network Pharmacology. Methods:The active ingredients of Yigan Powder were screened from Traditional Chinese Medicine Systems Pharmacology database and analysis platform (TCMSP) by ADME parameters,targets are predicted by Swiss Target Prediction database,disease targets are downloaded from GeneCards,OMIM and PharGKB database.Using R to run GO terms enrichment and KEGG pathway enrichment analysis.Protein interaction data is downloaded from the String database.Cytoscape software is used to build network.AutoDock Vina and PyMOL software are used for molecular docking. Results:There are 125 active ingredients in Yigan Powder with 953 predicted targets,85 of predicted targets are related to Alzheimer's Disease comorbid Depressive Disorder,shows highly enriched signaling pathways and biological processes.PPI network shows APP,MAPK1 and STAT3 may be important potential treatment of Alzheimer's Disease comorbid Depressive Disorder.The results of AutoDock Vina docking showed that the active ingredients of Yigan Powder had good binding activity with important receptors.Conclusion:Yigan Powder shows effect on neurotransmitter metabolism,synaptic transmission,neuroinflammation and other aspects in the treatment of Alzheimer's Disease comorbid Depressive Disorder.

1.Introduction

Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by progressive cognitive impairment,which is a major cause of disability among people over 65 years old[1-2].Mental disorders may occur during the process of AD,depression disorder is one of common psychiatric disorders.Epidemiological studies showed that nearly 40% patients with dementia had depressive disorder during dementia progression[3],while persistent depression among patients ranged from 30% to 85%,whereas conventional antidepressants had poor effects on depression therapy for patients with Alzheimer's disease[4].

Yigan powder was first included in “Baoyingcuoyao” by Xue Kai,a famous doctor in Ming Dynasty,including Chaihu,Gouteng,Danggui,Chuanxiong,Gancao,Fuling and Baizhu.Yigan Powder was frequently used in treating behavioral and psychological symptoms of dementia (BPSD) in Japanese Kampo Medicine[5].Chinese medicine doctors also used them for treatment of neurological disorders and gastrointestinal disorders[6-7].

Experimental studies indicated that Yigan powder could inhibit amyloid β-protein (Aβ) formation and glutamate-mediated excitatory toxicity,promoted 5-HT and dopamine levels in prefrontal cortex of aged rats to reverse cognition impairment,showing antidepression,anti-anxiety and neuroprotection efficacy.

Hopkins[10] firstly proposed the concept of network pharmacology based on network biology and systematic pharmacology.It’s known that Chinese medicine compounds can reveal complex mechanism and regulation network through the interaction between components,targets and diseases.This study used network pharmacology methodology to explore targets and signaling pathways of Yigan powder on AD comorbid depressive disorder and elucidate potential therapeutic mechanism[11].

2.Materials and methods

2.1 Chemical compounds collection

The ingredients of Yigan powder were collected and screened via TCMSP database (http://lsp.nwsuaf.edu.cn/index.php,Version 2.3).The screening standards were oral-bioavailability (OB) ≥ 30%,Blood-brain-barrier (BBB) ≥ 0.3,drug-like (DL) ≥ 0.18.

2.2 Target prediction

The predicted targets of Yigan Powder was retrieved via PubChem database (https://pubchem.ncbi.nlm.nih.gov/),downloaded the structure of compound protein and predicted reverse target via SwissTargetPrediction server (http://www.swisstargetprediction.ch/).Targets protein correction were searched via UniProt database (http:/www.uniprot.org/ Update in 2014-04-10).

2.3 Target genes for disease

target prediction were obtained by retrieving Genecards database(https://www.genecards.org/ Version 4.14) OMIM database (https://omim.org/ Update in 2020-05-08) and PharmGkb database (https://www.pharmgkb.org/).

2.4 Network conduction and analysis

Network was conducted and analyzed using Cytoscape-v3.7.2,constructing drug-active component-predicted target network and active component-target-disease network.Cytoscape analysis function was used to analyze nodes and edges of networks.

2.5 Targets Enrichment analysis

Go terms enrichment analysis and KEGG pathway analysis were performed using clusterProfiler package[12],Biological Process and KEGG Pathway were analyzed by the package,setting P-value ≤0.01.Then resultswere visualized by enrichplot package.

2.6 PPI analysis

Protein-protein interaction (PPI) network helps to explore important efficacy targets of Yigan powder.Searching and predicting proteinprotein interactions via STRING database (https://string-db.org/)[13].Download PPI information in STRING database then using Cytoscape software to draw PPI network diagram and analysis.MCODE was used to analyze PPI network and conduct Cluster analysis.

2.7 Molecular docking

AutoDock Vina is mainly used for studying structural biology and protein function,which can predict binding activity between important active components and protein receptors.Structures of target protein were searched and downloaded from PDB(http://www.rcsb.org/),using AutoDock Vina to run molecular docking,using PyMOL to visualize.

3.Results

3.1 Active ingredients screening

Active ingredients of Yigan powder were screened from TCMSP.1097 active compounds were collected and were screened by OB,DL and BBB parameters,other compounds were supplemented by related literature,such as kaempferol quercetin[14],ferulic acid[15],ligustilide[16] and Atractylodes macrocephala[17],which were proved having anti-depression or anti-dementia pharmacological effects.125 bioactive compounds were obtained eventually (Table 1).

Table 1Information of active ingredients of Yigan Powder

3.2 Target prediction

Predicted targets were obtained based on reverse target prediction results of SwissTargetPrediction platform..It was screened out by Probability >0 and UniprotPKB retrieval function was used to correct and transform into human targets.953 target genes were obtained eventually.

3.3 Disease target collection

Disease targets were collected via GeneCards,OMIM and PharmGKB.1016 AD-related genes and 1248 depression-related genes were found.Using R to draw Venn diagram (Fig.1).166 genes were closely related to AD and 179 were closely related

to depression,85 genes involved in two diseases simultaneously which were likely to be important targets in treating AD comorbid depressive disorder.

Figure 1 Venn Diagram of targets of Yigan Powder-Alzheimer’s Disease-Depressive Disorder

3.4 Network conduction and analysis

3.4.1 Active ingredients-predicted targets network

The information of 125 active ingredients and 953 predicted targets was imported into Cytoscape software,conducting active ingredients-predicted targets network.This network is constructed by 1081 nodes and 8591 edges,every node was associated with 15.895 nodes in average.There are 45 active ingredients with more than 100 predicted targets among 125 active ingredients,which may be the potential therapeutic ingredients of Yigan powder (Fig.2).

Figure 2 Network of herbs-active ingredients-targets of Yigan Powder

3.4.2 Active ingredients-targets-disease network

125 active compounds and potential targets were used to conduct active ingredients-targets-disease network (Fig.3).Degree is defined as number of edges connected with nodes,higher degree of active ingredients indicated more targets were correlated with each other,as it can be recognized by color.Targets with higher degree were close to red,otherwise blue.The degree of 8 active ingredients were more than 40 in this network,including 4 flavonoids and 2 alkaloids,mostly from Gancao and Gouteng,may be potential therapeutic ingredients of Yigan powder in treating AD comorbid depreesive disorder (Table 2).

Figure 3 Network of active ingredients-targets-disease of Yigan powder

Table 2Information of main active ingredients and nodes in Yigan Powder targeted network

Among 85 overlap genes,60 target genes were associated at least with 5 active ingredients.The higher degree meant the higher relevance of AD comorbid depressive disorder (Table 3).

Table3Information of main target protein and nodes in Yigan Powder targeted network

3.5 Enrichment analysis

GO terms and KEGG pathway enrichment analysis results showed that biological processes mainly associated with cognition,learning,memory,neurotransmitter metabolism regulation,synaptic transmission,MAPK activity,glial regeneration and differentiation,reactive oxygen species biosynthesis metabolism,calcium homeostasis etc (Fig.4).

KEGG pathway enrichment analysis involves results showed that pathways mainly associated with AD signaling pathway,neuronal activity ligand receptor action,dopaminergic neuron synapse,synaptic signal pathway,Rap 1 signaling pathway,cancer associated protein polysaccharide,multidrug resistance and AGE-RAGE signaling pathway in diabetic complications (Fig.5).

Among them,AD related pathways include oxidative phosphorylation,autophagy,apoptosis,insulin signaling pathway,calcium signaling pathway,etc.APP and PSEN,tau phosphorylation and NFTs (NFTs) and regulation of N-methyl-aspartic acid receptor(NMDAR) are the essential parts of AD pathway.Pathological processes were closely associated with neuronal apoptosis,autophagy,mitochondrial dysfunction,axonal transport dysfunction,synaptic plasticity impairment and neurodegenerative disorders (Fig 6).

Figure 4 GO Biological Process Enrichment Analysis

Figure 5 KEGG Pathway Enrichment Analysis

Figure 6 Potential targets for the regulation of Alzheimer's disease pathways by Yigan Powder

3.6 PPI network and Cluster module analysis

3.6.1 PPI network

The node information of combined score ≥ 0.9 in network data is imported into cytoscape to conduct PPI network.The area and color of nodes are correlated with degree,the color and thickness of edge are correlated with Combined score.191 interactions exist among 67 proteins (Fig.7).APP (degree=20),MAPK1 (degree=19),STAT3(degree=17) are the central parts of PPI networks,showing close correlation with potential efficacy mechanisms of Yigan powder.

3.6.2 Cluster module

MCODE plug-ins of Cytoscape software was utilized to analyze Cluster modules for PPI networks,with 5 Cluster modules eventually(Fig.7).Further understand the biological functions involved in 5 Cluster modules,Cluster 1 is highly correlated with synaptic signal transduction processes,calcium signaling pathways and PI3k/Akt signaling pathways.Cluster 2 is associated with glutamate receptor activity and Cyclic adenosine monophosaphate (cAMP) signaling pathway.Cluster 3 related to biosynthesis of TNF signaling pathway,Toll-like receptor (TLR) signaling pathway,nuclear transcription factor kappa B (NF-κB) signaling pathway and interleukin-6 (IL-6),IL-17.Cluster 4 is associated with neurotransmitter metabolism such as DA and 5-HT.Cluster 5 is associated with steroid metabolism.

Figure 7 PPI network and Cluster analysis

3.7 Molecular docking

Wallichilide and 7-Acetoxy-2-methylisoflavone were used as ligands.Amyloid Precursor Protein (APP),Mitogen-Activated Protein Kinase 1 (MAPK1),Signal Transducer and Activator of Transcription 3 (STAT 3),Acetylcholinesterase (AchE),and Serotonin Transporter (SERT) were used as receptors.Donepezil and Citalopram were used as control drug.Docking results were visualized by PyMol software (Fig8).

Figure 8 Results of AutoDock Vina molecular docking

There were multiple docking conformations between 2 active ingredients and 5 target proteins respectively.The results showed that the lowest binding energies were selected as final docking results (Table 4).Lower binding energy represented better binding ability between ligands and receptors.Wallichilide and 7-Acetoxy-2-methylisoflavone had good binding capacity to 5 target proteins but not as good as control drugs.

Table 4Results of ligand-receptor protein molecular docking

4.Discussion

Alzheimer disease is a complex neurodegenerative disease.Both depressive disorder and Alzheimer disease are associated with multiple hypothesis but with common pathophysiology features such as decreasing levels of neurotransmitter,decreasing synaptic number,decreasing BDNF level,mitochondrial dysfunction and neuroinflammation[18].There is a traditional concept “same treatment for different diseases” as a very important treating view in traditional Chinese medicine.Herb compounds can regulate complex disease networks as multiple ingredients-multiple targets-multiple pathways,Clinical trails results indicated that Yigan powder showed effect on improving cognition,reducing behavioral and psychological symptoms[19].Yigan powder combined regular treatment could reduce dosage of antipsychotics[20].

Based on database and network analysis,this study found that the intervention of Yigansan in AD comorbid depressive disorder is related to 125 main active ingredients.The target prediction and network analysis results showed that APP,MAPK,ESR,MAO,GSK3B and other targets played a key role in the pharmacological function.APP is cleaved by proteases in the human body to produce Aβ,which eventually causes synaptic dysfunction,neuroinflammation and cell death,which are the core pathological processes of AD.MAPK is a widely expressed serine/threonine protein kinase,mainly composed of extracellular regulated protein kinases (ERK),p38 and c-Jun N-terminal kinase (c-Jun N-terminal kinase,JNK).The composition,in which p38 plays an important role,participates in pro-inflammatory signaling transduction and can regulate the synthesis of cytokines,causing different degrees of neuroinflammation,and involves tau-protein hyperphosphorylation and apoptosis[21].Estrogen has a protective effect on memory cholinergic neurons,can inhibit the production and accumulation of Aβ by activating ESR,and improve synaptic transmission by regulating biological processes such as cytoskeleton and protein transport[22],affecting memory,Learning and other behavioral processes.At the same time,estrogen response can increase the sensitivity of DA nerves and increase the content of serum 5-HT,which plays a role in the process of affective disorders and cognitive disorders.MAO is the main enzyme involved in the metabolism of monoamines in the body,and it is involved in the decomposition process of various neurotransmitters such as 5-HT and DA.SRC is the main tyrosine kinase of NMDAR tyrosine phosphorylation.The glutamatergic neurotransmission dysfunction and changes in neurosynaptic plasticity caused by the phosphorylation of NMDARs are related to the pathophysiological process of AD and depression[23] .GSK3B participates in the phosphorylation of tauprotein and the formation of neurofibrillary tangles,and can also increase the synthesis of Aβ in the brain and induce neuronal apoptosis,which is closely related to the pathogenesis of AD[24].At the same time,Yigan powder can also act on AchE alone to treat AD.By inhibiting the activity of AchE,it can enhance the level and transmission efficiency of ACh between cholinergic neurons and achieve the purpose of improving the clinical symptoms of AD patients.It can be seen that Yigan powder can act on the central nervous system through a variety of targets and signaling pathways,exerting anti-dementia,anti-depression,anti-oxidation,antiinflammatory and neuroprotective effects.

The results of GO enrichment analysis showed that the biological process mainly associated with neurotransmitter metabolism,synaptic transmission,MAPK cascade activation,and the regulation of calcium ion homeostasis.The results of KEGG pathway enrichment analysis show that the pathways mainly associated with oxidative phosphorylation,cell apoptosis,calcium ion signaling pathway,etc.Abnormal neurotransmitter metabolism is a common pathophysiological manifestation of AD and depression.The active ingredients of Yigan powder can regulate AchE and SERT,and improve the metabolism of neurotransmitters such as 5-HT and ACh in the brain.Under the action of extracellular stimuli such as stress,MAPK participates in processing and regulating various cell activities.Some scholars have found that in the early stage of AD,the activation of p38 MAPK exists in the central nervous system[25],which is one of the important mechanisms of AD,and ERK can bind to a variety of neurotransmitter receptors to induce depressive symptoms[26].At the same time,inhibiting the activity of p38 MAPK can block the NF-κB signaling pathway,reduce the release of IL-1β,IL-18 and other kinds of cytokine,thereby reducing neuroinflammation.Due to reactions such as metabolism,oxidation,and stress,the imbalance of calcium ion homeostasis in damaged neuronal cells can induce pathological processes such as Aβ aggregation,tau-protein phosphorylation,and mitochondrial dysfunction,which ultimately affect learning and memory[27].Disorders of calcium homeostasis can also cause excessive activation of NMDAR,trigger glutamate toxicity,and can lead to neuronal cell apoptosis.Yigan powder can regulate the calcium ion signal pathway through a variety of ways,inhibit tauprotein hyperphosphorylation,improve mitochondrial function,and inhibit neuronal cell apoptosis[28].The PPI network can observe the interaction and regulation relationship between proteins.PPI network results of Yigan powder in the treatment of AD comorbid depressive disorder show that APP,MAPK1,STAT3,SRC,etc.have more protein interaction relationships,which may be the efficacy targets of Yigan powder.The key utility target is mainly associated with the regulation of neurotransmitters,neuroinflammation and apoptosis.

In summary,this study used network pharmacology methods to analyze the traditional Chinese medicine and disease targets,and explored the complex relationship network and potential mechanisms of Yigan powder in the treatment of AD comorbid depressive disorder.The results show that the target,biological process and related pathways of Yigan powder not only aim at the core target of AD,reduce the deposition of Aβ,but also reduce neuroinflammation,improve neuroplasticity,and regulate neurotransmitter metabolism.It can improve mood disorders in the process of AD on the basis of first-line drugs.Combined with regular treatment can show better curative effects,and can provide new ideas for the treatment of AD comorbid depressive disorder,but the results still need to be verified by further studies.The network pharmacology method is based on the information of the existing compounds,and cannot fully clarify the chemical reactions between the compounds and the changes in the human body metabolic process.It is also necessary to further study the material basis and mechanism of action of Yigan powder.

Author’s contribution

Zhao Chi,first author,is the plan designer and executor of this research.He has completed data collection,data analysis,writing of the first draft of the paper and review of the paper;Guo Rongjuan,corresponding author,guides research design and data analysis,and review the article criticaly;Ren Feifei,third author,participated in research design and result analysis,and reviewed the content of the article;Yu Yao,fourth author,participated in data collection,and provided certain technical support in statistical analysis;Li Junnan,fifth author,participated in the literature review and guided the revision of the paper;Li Yang,sixth author,participated in the literature review and reviewed the content of the article.