Wei-Jie QIANGa, c, Ying CHENa, Mei-Feng XIAOb, Wei-Yan CAIa, Yi-Fei DAIa, Qing YANGa, Yu-Jie LIa, Xiao-Gang WENGa, Qi LIa, Ya-Jie WANGa, Xiao-Xin ZHUa*, Fu-Yuan HEb*
a. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700;
b. College of Pharmacy, Hunan University of Traditional Chinese Medicine, Hunan, Changsha, 410208;
c. Anhui University of Chinese Medicine, Anhui, Hefei, 230012.
Alzheimer’s disease (AD) and other chronic, progressive neurodegenerative diseases pose a serious threat to human health. Yuan Zhi powder is a traditional Chinese medicine recorded in the General Records of Sagely Benefaction that has demonstrated good therapeutic efficacy in treating learning and memory disorders. However, the exact molecular mechanisms underlying these effects remain unclear. This study constructed a database of chemicals in Yuan Zhi using existing databases and literature, and predicted potential targets of Yuan Zhi using reverse molecular docking and building an ingredients-targets network. Based on this, an AD-related target network map was created and analyzed to obtain “active ingredients–AD-related targets network” and screened for key targets. The rationality of the predicted targets was confirmed by comparison with targets reported in the literature. By analyzing the gene functions and biological processes associated with Yuan Zhi, we found that the therapeutic effects of Yuan Zhi powder on AD are mainly achieved through regulation of acetylcholine levels and stimulation of synaptic transmission.
- 2. Materials and methods
- 2.1. Construction of AD molecular target network
- 2.2. Construction of Yuan Zhi powder chemical library and creation of sdf-mol files
- 2.3. Reverse prediction of potential targets
- 2.4. Network construction and analysis
- 2.5. Screening, function analysis, and biological process analysis of key targets
- 2.6. Literature validation
- 3. Results and analysis
- 4. Discussion
- Funding Support
- Competing Interests
Aging of the population has increased the socioeconomic burden of neurodegenerative diseases and psychiatric disorders. According to a quantitative assessment of the World Health Organization, neuropsychiatric disorders account for 17.5% of the global burden of disease, exceeding the burden of cardiovascular diseases and cancer. In China, by 2020, neuropsychiatric disorders are expected to increase to one-quarter of the total burden of disease. It has been estimated that one-fifth of the elderly in China suffer from chronic mental disorders or neurodegenerative disease1. The future burden of brain diseases in China may reach RMB 1 trillion per year. For example, Alzheimer's disease (AD), a type of dementia, is one of the most common neurodegenerative diseases. The prevalence of dementia in China is 2–5%, which gradually increases with increasing age. Currently, the annual incidence of dementia is 1% overall and 10–20% in the elderly2. In the treatment of systemic diseases with complex pathogenesis, including neurodegenerative diseases and psychiatric disorders, multi-target drugs such as traditional Chinese medicines (TCMs), tend to have better therapeutic efficacy than single-target drugs. Although the mechanisms underlying these effects are unclear, multi-ingredient TCM formulas that focus on restoring and maintaining the balance of the body with multi-target and multi-pathology synergistic effects are naturally aligned with the systems theory for complex diseases used in modern treatments.
A large number of TCM formulas are available to prevent and treat encephalopathy. Liver and kidney deficiency, collateral obstruction due to blood stasis, and phlegm obstruction of the orifices are considered the main causes of delirium, dullness, forgetfulness, and depression, for which tonifying deficiency, dispersing stasis, and eliminating phlegm are the main treatments used in TCM. Yuan Zhi powder is a formula found in the General Records of Sagely Benefaction for tonifying deficiency, fortifying strength, and supplementing the will3. It is composed of Yuan Zhi (Radix polygalae), Huang Lian (Rhizoma coptidis), Bai Fu Ling (Sclerotium poriae cocos), Shi Chang Pu (Rhizoma acori tatarinowii), and Ren Shen (Radix ginseng). Its effects include: curing forgetfulness, tonifying the heart and qi, fortifying strength, and supplementing the till3. Modern studies have shown that Yuan Zhi powder and the various herbal medicines in this formula have significant contribute to the treatment of learning and memory impairments in AD patients and animal models4,5,6. However, the exact molecular mechanisms underlying its effects on dementia, forgetfulness, and other TCM encephalopathies are still unclear.
Based on high-throughput data analysis, computer-based virtual computing, and network database searching, in this study, we constructed molecular biology networks to systematically reveal the pharmacological effects and molecular mechanisms underlying the effects of Yuan Zhi powder7,8. Our aim was to uncover the molecular biological targets of Yuan Zhi powder in the treatment of AD and other TCM encephalopathies9, thereby providing a basis for further research and clinical applications of Yuan Zhi powder.
2. Materials and methods
2.1. Construction of AD molecular target network
The keyword, “Alzheimer's disease,” was entered into the drugbank database (http://www.drugbank.ca/) to identify the drugs used to treat AD and to confirm the targets of dementia treatment. Following the, the AD target-interaction target pairs were found using protein-protein interaction (PPI) databases such HAPPI, OPHID, IntAct, MINT, and DIP to generate an AD molecular biology network.
2.2. Construction of Yuan Zhi powder chemical library and creation of sdf-mol files
By searching through PubMed, TCMID10, TCM Database@Taiwan11, NCBI Pubchem, and the literature, we identified all of the active ingredients, including each Chinese herb, in Yuan Zhi powder, and standardized the active ingredients in the CAS format to construct the chemical library for Yuan Zhi powder. The chemical structure of all active ingredients was confirmed using the CAS database. Each structural diagram was drawn using Chembiodraw Ultra 12.0 and stored in tMDL Molfile (*.mol) format12. The generated MDL Molfile (*.mol) files were imported into OpenBabel and converted from the MDL Molfile format into the sdf-MDL MOL format.
2.3. Reverse prediction of potential targets
The sdf-mol files of Yuan Zhi powder active ingredients were uploaded to the PharmMapper Server, and “reverse pharmacophore matching” was performed to obtain virtual screening results13. Potential drug targets were obtained through a search using biologically active small molecules as probes, and the biological activity of the compounds was predicted. Because of the non-standardized naming of drug targets, the UniProtKB search function of the UniProt database was used to correct the names of all retrieved proteins to their official names by entering the protein names into the database and restricting the species to human. The above database search and conversion operations were performed to retrieve protein information related to the active ingredients.
2.4. Network construction and analysis
Network construction, analysis, and visualization were performed using Cytoscape v3.4.0 to construct the AD molecular target network and the ingredient-target network of Yuan Zhi powder. The nodes represent the chemical compounds and targets, and the edges represent the compound-target pairs and target-interaction target pairs. In the biosynthesis network, the Network Analyzer plug-in was selected for the topological analysis of the network to determine the main parameters of this network.
2.5. Screening, function analysis, and biological process analysis of key targets
Indicators of centrality are key indicators that measure the importance of a node in the network diagram; a larger value indicates that the node is more important in the overall network and has a greater impact on the overall network structure and function14. In this study, we adopted the degree centrality algorithm as the core. This was supplemented with closeness centrality and betweenness centrality algorithms to screen and comprehensively evaluate the key targets of AD treatment by Yuan Zhi powder. ClueGO was then used to perform the gene function analysis and biological process analysis for AD targets of Yuan Zhi powder action.
2.6. Literature validation
After reviewing the New Thoughts and New Targets in Pharmacology, a literature validation of active ingredients reported for Yuan Zhi (Radix Polygalae), Huang Lian (Rhizoma coptidis), Bai Fu Ling (Sclerotium poriae cocos), Shi Chang Pu (Rhizoma acori tatarinowii), and Ren Shen (Radix ginseng). The literature search was conducted using the China National Knowledge Instructure (CNKI) database, China Academic Journals database (Wanfang Data), and PubMed. The search terms included the following: the Chinese/English name of the Chinese medicine + name of the active ingredient + target/protein/receptor/expression/effects. Literature related to the efficacy, pharmacological effects, and targets of the five Chinese herbs were recorded. The time range of the search was between January 1, 1979, and December 30, 2016. Boolean operators “AND” and “OR” were used to customize the search: (SU = “name of Chinese medicine”) AND (FT = “target” OR FT = “protein” OR FT = “receptor”). The retrieved articles had to meet the following inclusion criteria: relevance to the topic of this study, namely, neurodegenerative diseases and psychiatric disorders; inclusion of target information for the five Chinese herbs, including name of the medicine, efficacy, ingredients, and targets in Chinese and English. Data that were not relevant to our research topic and that lacked the required TCM target information were excluded. Relevant Chinese and English articles that met the criteria were retrieved, and the results were stored in an xls file. The keyword information in the article citation was exported, and the TCM-related information was collated into information entries. Targets with effects were labelled with +1, targets without effects were labelled with −1, and targets with no recorded effect intensity were labelled with 0. Based on this, a database of the relationship between the drug ingredients and targets was established. Finally, the correlation coefficients between the active ingredients and targets were calculated to construct a network diagram.
3. Results and analysis
3.1. Chemical library of Yuan Zhi powder
From the databases and literature above, the authors collected the chemical compounds of the various Chinese herbs in Yuan Zhi powder, including 80 compounds in Yuan Zhi (Radix polygalae), 48 in Huang Lian (Rhizoma coptidis), 34 in Bai Fu Ling (Sclerotium poriae cocos), 105 in Shi Chang Pu (Rhizoma acori tatarinowii), and 190 in Ren Shen (Radix ginseng). Reverse molecular docking was performed to determine the drug effect targets. After mapping the AD molecular target network and eliminating ingredients that were not found to act on AD targets, we considered the following ingredients relevant: 10 compounds in Yuan Zhi (Radix polygalae), 27 in Huang Lian (Rhizoma coptidis), six in Bai Fu Ling (Sclerotium poriae cocos), 73 in Shi Chang Pu (Rhizoma acori tatarinowii), and 64 in Ren Shen (Radix ginseng). The specific names of the active ingredients are shown in Table 1.
|Yuan zhi(Radix polygalae)||Harmine, perlolyrine, onjixanthone I, norhyoscyamine, norharman, Harman, benzoic acid, trimethoxycoumarin, 1-oxyphenylacetyl, 1,6-dihydroxy-3,10-dimethoxyxanthone|
|Huang lian (Rhizoma coptidis)||Columbianadin, coptisine, quercetin, phellodendrine, p-coumaric acid, noroxyhydrastinine, methyl protocatechuate, magnoflorine, jatrorrhizine, isovanillin, hydroxytyrosol, groenlandicine, GENOP, FER, ethyl caffeate, epiberberine, rhein, isocorydine, coptisine, columbamine, lariciresinol, cinnamic acid, berlambine, berberrubine, berberine, 6-O-E-feruloylajugol, tetrahydroberberine|
|Bai fu ling (Sclerotium poriae cocos)||Palmitic acid, uridine, lauric acid, hederagenin, alpha-D-Methylglucoside, daucosterol|
|Shi chang pu (Rhizoma acori tatarinowii)||Panasinsene, cadinol, ZINC02040970, ZINC01849758, ZINC01609418, WLN: Q1R, veraguensin, vanillic acid, thymol, 4-allylanisole, 5-allylanisole, spathulenol, protocatechuic acid, melamine cyanurate, p-coumaric acid, patchoulene, palmitic acid, murolan-3,9(11)-diene-10-peroxy, methyl-eugenol, methyl-isoeugenol, marmesin, kaempferol, isohomoenol, hydroxymethylfurfural, guaiol, 2,4,5-trimethoxybenzaldehyde, 2,4,6-trimethoxybenzaldehyde, asarone, epishyobunone, emodin, elemicin, diethylbenzylamine, diethyl phthalate, camphene, cineole, calmodulin, calamendiol, calacorene, caffeic acid, cadinene, BZM, Bisasaricin, selinene, humulene, gurjunene, asarone, tripelennanmine hydrochloride, astragaloside, hydroxyphenylacetic acid, aristolone, apigenin, 9-aminoacridine, humulene, cubebene, asatone, isopentenyl kaempferol, 2”-O-methylisoliquiritigenin, 3”-O-methylisoliquiritigenin, 4”-O-methylisoliquiritigenin, trans-caryophllene, linalool, methylcinnamic acid, methyl acrylate, 1-Isopropyl-4-methylbicyclo[3.1.0]hex-3-yl acetate, (1R,3aS,4R,6aS)-1,4-bis(3,4-dimethoxyphenyl)-1,3,3a,4,6,6a-hexahydrofuro[4,3-c]furan, valencene, carveol, terpinen-4-ol, ()-ledene, caryophyllene oxide, alpha-longipinene, cedrane, allo-aromadendrene|
|Ren shen (Radix ginseng)||Elemene, vulgarin, trifolirhizin, galloylpaeoniflorin A, stigmasterol, pentadecanoic acid, panaxynol, palmitic acid, paeonol, N-salicylidenesalicylamine, nepetin, neohexene, neocnidilde, angeloylgomisin, methylselenocysteine, methyl palmitelaidate, methylhexadecanoic acid methyl ester, methyl linoleate, MAV, malvic acid, linoleic acid, isocitric acid, kaempferol 3-arabinoside. kaempferol, maackiain, hexanal, GUP, girinimbine, ginsenoyne E, ginsenoyne D, ginsenoyne B, ginsenoyne A, ginsenoside rh2, ginsenoside rh, selinene, protopine, frutinone A, cadinene, elemicin, dianthramine, dibutyl phthalate, dauricine cis-Widdrol alpha-epoxide, β-sitosterol, selinene, humulene, elemene, caryophyllene, bisabolene, arachidonic acid, aposcopolamine, alloaromadendrene, hexadecenoic acid, 5-[(3aS,6R,6aR)-2-keto-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-6-yl]valeric acid, 3-methylheptane, 3-ethyl-3-methylheptane, Α-bulnesene, anisic acid, farnesene, (Z)-2-methyl-5-[(1S,2R,4R)-2-methyl-3-methylene-2-norbornanyl]pent-2-en-1-ol, (1S,4E,8E,10R)-4,8,11,11-tetramethylbicyclo[8.1.0]undeca-4,8-diene, (1R,4S,4aR,8aR)-4-isopropyl-1,6-dimethyl-3,4,4a,7,8,8a-hexahydro-2H-naphthalen-1-ol, (1R,4E,7E,11R)-1,5,9,9-tetramethyl-12-oxabicyclo[9.1.0]dodeca-4,7-diene, elemol oxide.|
3.2. Analysis of topological parameters
The ingredient-AD target network diagram for Yuan Zhi powder was created using Cytoscape (Figure 1), and the topological parameters of this network were calculated (Table 2). Each active ingredient in Yuan Zhi powder had an average of 2.14 (383/179) AD targets, and each AD target was connected to 31.9 (383/12) ingredients, on average. This indicates that Yuan Zhi powder exerts multi-ingredient, multi-target synergistic effects in the treatment of AD. Furthermore, in this network, due to the uneven distribution of the node between network nodes, the node degrees of certain ingredients/targets were far higher than the averages of their direct neighbors. In the degree centrality algorithm, the higher the degree of a node, the greater its impact on the overall network. Such a node is often considered a key node of a network known as a hub.
|Argument||Ingredient-Target Network||Argument||Ingredient-Target Network|
|Clustering coefficient||0.0||Number of nodes||191|
|Connected components||1||Number of edges||355|
|Network diameter||6||Network density||0.019|
|Network centralization||0.758||Isolated nodes||0|
|Characteristic path length||2.533||Multi-edge node pairs||15|
|Average number of neigbors||3.539||-||-|
3.3. Analysis of key targets
The algorithms for closeness centrality and betweenness centrality were included in this study. The closeness centrality algorithm focuses on the average shortest path between a node and other nodes in the network. The betweenness centrality algorithm measures the number of times a node lies on the shortest path of all other nodes, i.e., the number of times the shortest paths of other nodes pass through this node. In other words, if the shortest paths of other nodes have to pass through this node, then this node is of greater importance or capacity. It is a hub that interconnects the other nodes and controls the transmission of information. These three algorithms were used to compute the overall network, and the full results of the algorithms for the action of Yuan Zhi powder on AD targets are shown in Table 3.
|Degree Centrality||Closeness Centrality||Betweenness Centrality|
|Prostaglandin G/H synthase 2||154||Prostaglandin G/H synthase 2||0.71161049||Prostaglandin G/H synthase 2||0.82134224|
|Muscarinic acetylcholine receptor M1||76||Muscarinic acetylcholine receptor M1||0.43879908||Muscarinic acetylcholine receptor M1||0.18090792|
|Muscarinic acetylcholine receptor M2||61||Muscarinic acetylcholine receptor M2||0.41036717||Muscarinic acetylcholine receptor M2||0.11678123|
|Gamma-aminobutyric-acid receptor alpha-5 subunit||15||Neuronal acetylcholine receptor subunit alpha-7||0.34358047||Cholinesterase||0.04179894|
|Neuronal acetylcholine receptor subunit alpha-7||13||Acetylcholinesterase||0.34358047||Glycogen synthase kinase-3 beta||0.02119287|
|Acetylcholinesterase||13||Gamma-aminobutyric-acid receptor alpha-5 subunit||0.34234234||Acetylcholinesterase||0.01363547|
|Cholinesterase||6||Glycogen synthase kinase-3 beta||0.33158813||Cathepsin D||0.01068412|
|Cathepsin D||6||Cholinesterase||0.304||Gamma-aminobutyric-acid receptor alpha-5 subunit||0.00551507|
|Glycogen synthase kinase-3 beta||5||Cathepsin D||0.30206677||Neuronal acetylcholine receptor subunit alpha-7||0.00428248|
|Mitogen-activated protein kinase 14||3||Mitogen-activated protein kinase 14||0.30015798||Mitogen-activated protein kinase 14||1.4109E-4|
The topological parameters in Table 2 indicate that, in the ingredient-AD related targets network, each gene has an average of 3.539 direct neighbors, i.e., the average degree centrality is 3.539. Only six targets in this network had a degree centrality that was two-times higher than the average value (Table 3). Prostaglandin G/H synthase 2, muscarinic acetylcholine receptor M1, and muscarinic acetylcholine receptor M2 were ranked highly by all three algorithms, which implies that they are key targets in this network. Acetylcholinesterase (AchE) received a fairly high ranking by the three algorithms, and it may also be an important target in AD treatment with Yuan Zhi powder. Although gamma-aminobutyric-acid receptor alpha-5 subunit and neuronal acetylcholine receptor subunit alpha-7 were ranked highly for degree centrality and closeness centrality, their rankings were very low for betweenness centrality, which may imply that they are important targets in local networks for AD treatment.
Studies have shown15 that the expression of inflammatory factors and their related enzymes in the central nervous system are closely related to neuronal damage. Prostaglandins (PGs) are crucial inflammatory factors, and cyclooxygenases (COX) are rate-limiting enzymes that catalyze the synthesis of PGs from arachidonic acid. The expression of COX-2 is significantly increased in the brains of AD patients. Muscarinic acetylcholine receptor (M1) plays an important role in the pathological process of AD. Agonists of M1 receptors can effectively alleviate a number of typical symptoms of AD, including amyloid β (Aβ) deposition in the brain due to the incorrect cleavage of the amyloid precursor protein (APP), Tau hyperphosphorylation, and damage to the cholinergic system following the loss of cholinergic neurons16. Studies have also shown that M1 receptors mainly participate in motor and memory regulation, whereas M2 receptors are involved in the negative feedback regulation of acetylcholine (Ach) release. Antagonistic effects on M2 receptors lead to further deterioration of memory17. In addition, several studies have shown that suppressing the AchE level in the brain can improve learning and memory abilities. At present, the first-line drug for the treatment of AD is the cholinesterase inhibitor donepezil, which mainly inhibits Ach degradation by inhibiting AchE activity. It increases the concentration of Ach in the brain, thereby improving neurotransmitter delivery18.
3.4. Literature validation
Literature related to the efficacy, pharmacological effects, and targets of the five Chinese herbs was identified from multiple databases to establish a database of information on the relationship between the active ingredients and targets (see Table 4). Targets with effects were labelled with +1, giving a total of 61 targets; targets without effects were labelled with −1, giving a total of 12 targets; and targets with no recorded effect intensity were labelled with 0, giving a total of 8 targets.
|Yuan Zhi (Radix Polygalae)||Tenuifoliside A||PI3K/AKT||+1||Huang Lian (Rhizoma Coptidis)||Limonin||Cytochrome P450 3A4||−1|
|MEK/ERK/CREB||+1||Berberine||Nitric oxide synthase, inducible||−1|
|Tenuifoliside C||JNK MAPK||+1||Estrogen receptor||−1|
|NF-κB||+1||Prostaglandin G/H synthase 2||+1|
|Onjisaponin B||NGF||+1||Epiberberine||Nitric oxide synthase, inducible||−1|
|huntingtin||−1||Prostaglandin G/H synthase 2||+1|
|Onjisaponin A||NGF||+1||Coptisine||Nitric oxide synthase, inducible||−1|
|Onjisaponin E||NGF||+1||Estrogen receptor||−1|
|Ren Shen (Radix Ginseng)||Panaxynol||Prostaglandin G/H synthase 2||+1||Prostaglandin G/H synthase 2||+1|
|Muscarinic acetylcholine receptor M1||+1||Columbamine||Nitric oxide synthase, inducible||−1|
|Ginsenoside Rh4||Mineralocorticoid receptor||0||Prostaglandin G/H synthase 2||+1|
|Nuclear receptor coactivator 2||0||Beta-secretase||0|
|β-Patchoulene||Prostaglandin G/H synthase 2||0||Shi Chang Pu (Rhizoma Acori Tatarinowii)||Asarone||Dopamine D1 receptor||+1|
|Ginsenoside Rf||Tumor necrosis factor||+1||Muscarinic acetylcholine receptor M1||+1|
|Prostaglandin G/H synthase 2||+1||Prostaglandin G/H synthase 2||+1|
|Aposcopolamine||Muscarinic acetylcholine receptor M1||+1||Alpha-2C adrenergic receptor||+1|
|Sodium channel protein type 5 subunit alpha||+1||Muscarinic acetylcholine receptor M2||+1|
|5-hydroxytryptamine 2A receptor||+1||Sodium-dependent dopamine transporter||+1|
|Alpha-1A adrenergic receptor||+1||Asarylaldehyde||Muscarinic acetylcholine receptor M1||+1|
|Muscarinic acetylcholine receptor M2||+1||Prostaglandin G/H synthase 2||+1|
|Sodium-dependent dopamine transporter||+1||Alpha-2C adrenergic receptor||+1|
|Beta-2 adrenergic receptor||+1||Sodium-dependent dopamine transporter||+1|
|D(2) dopamine receptor||+1||Amine oxidase [flavin-containing] B||0|
|Neuronal acetylcholine receptor protein, alpha-7 chain||+1||β-Asarone||Dopamine D1 receptor||0|
|β-Panasinsene||Prostaglandin G/H synthase 2||+1||Muscarinic acetylcholine receptor M1||+1|
|Muscarinic acetylcholine receptor M2||+1||Prostaglandin G/H synthase 2||+1|
|α-Cedrol||Muscarinic acetylcholine receptor M1||+1||Alpha-2C adrenergic receptor||+1|
|Muscarinic acetylcholine receptor M2||+1||Muscarinic acetylcholine receptor M2||+1|
|Neuronal acetylcholine receptor protein, alpha-7 chain||+1||Sodium-dependent dopamine transporter||+1|
|Ginsenoside Rh2||JAK-STAT||-1||Methylisoeugenol||Dopamine D1 receptor||0|
|Riboflavine||Prostaglandin G/H synthase 2||+1||Muscarinic acetylcholine receptor M1||+1|
|β-Panasinsene||Prostaglandin G/H synthase 2||+1||Prostaglandin G/H synthase 2||+1|
|Muscarinic acetylcholine receptor M2||+1||Alpha-2C adrenergic receptor||+1|
|Uridine||Interferon alpha/beta receptor 2||+1||Muscarinic acetylcholine receptor M2||+1|
|Phospholipase A2, membrane associated||+1||Sodium-dependent dopamine transporter||+1|
|Bai Fu Ling (Sclerotium Poriae Cocos)||Trametenolic acid||Mineralocorticoid receptor||0||Amine oxidase [flavin-containing] B||0|
|Shi Chang Pu (Rhizoma Acori Tatarinowii)||Caryophyllene||Muscarinic acetylcholine receptor M1||+1||Bisasaricin||Nitric oxide synthase, inducible||−1|
|Prostaglandin G/H synthase 2||+1||Estrogen receptor||−1|
|Muscarinic acetylcholine receptor M2||+1||Prostaglandin G/H synthase 2||+1|
|Sodium-dependent dopamine transporter||+1||Estrogen receptor beta||−1|
3.5. Analysis of gene function and metabolic pathway
Bioinformatics enrichment included gene function and metabolic pathway analyses, which were preformed using ClueGO, as shown in Figures 2 and 3. Figure 2 indicates that the gene functions Yuan Zhi powder important to the treatment of AD were mainly associated with acetylcholine receptor activity and beta-amyloid binding. Ach is the main neurotransmitter of the central cholinergic nervous system is critical to normal learning and memory. Yuan zhi powder may promote the activity of Ach receptors (AchR), which facilitate Ach binding to AchR, thereby exciting the cholinergic system. Specific modulation of the septo-hippocampal-limbic and the cerebral cortex pathways then lead to the transfer of first-level memory to second-level memory19. Clinically, this is manifested as improvements in learning and memory. Furthermore, studies have shown that Aβ deposits can cause a decrease in acetylcholine transferase activity, reducing the number of surrounding cholinergic neurons and impairing memory. In addition, Aβ can act on an endplate of cholinergic neurons, which take up choline and reduce the synthesis of Ach, thereby decreasing Ach release in the hippocampus and cortex20. Components of Yuan Zhi powder can bind with Aβ, reducing subsequent damage to human memory functions.
The biological processes underlying AD treatment with Yuan Zhi powder were mainly concentrated in the acetylcholine receptor signaling pathway and in negative regulation of synaptic transmission, cholinergic synaptic transmission, and regulation of smooth muscle contraction. Studies have shown that Aβ can inhibit and suppress the function and expression of AchR. AchR is proposed to be the main target protein of Aβ and to play an important role in memory and cognitive functions 21The treatment of dementia may be inseparable from the AchR pathway. Changes in the intracellular signaling mechanisms of AchR during the onset and development of disease is one of the key topics for research on the pathological mechanisms of central nervous system diseases. After the synthesis of Ach molecules by cholinergic neurons, these receptors participate in the cognitive functions of the brain via the muscarinic and nicotinic signal transduction pathways. AchR exerts its function through its action at the pre-synaptic membrane, post-synaptic membrane, peri-synaptic sites, or extra-synaptic sites. The depolarization of the postsynaptic membrane not only directly excites the neurons but also regulates the release of γ-aminobutyric acid 22 Synaptic plasticity is the neurobiological basis of learning and memory. Hence, regulating the synaptic structure and function of the hippocampus can affect cognitive function, and a reduction in the synaptic plasticity in the hippocampal structure may be the cause of spatial memory impairment23
Based on network pharmacology, this study analyzed the main ingredients, key targets, gene functions, and biological processes involved in the action of Yuan Zhi powder on AD as the target disease. Three key targets (COX-2, muscarinic acetylcholine receptor M1, and muscarinic acetylcholine receptor M2) and one important target (acetylcholinesterase) were identified. By comparison with the targets reported in the literature, we know that important targets identified in our network analysis accounted for a large proportion of targets indicated in the literature, confirming the rationality of predicting these targets. In addition, our network analysis indicated that Yuan Zhi powder may treat AD by regulating AchR activity and binding to Aβ. Its biological effects may affect the acetylcholine receptor signaling pathway to negatively regulate synaptic transmission. Network pharmacology can help us elucidate the underlying mechanisms of TCM formulas in the treatment of disease, providing ideas for further research and facilitating the development of related drugs and modernization of TCM.
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阿兹海默症（Alzheimer’s disease, AD）等慢性进行性神经退行性疾病严重威胁人类健康。远志散是《圣济总录》中补虚强力益志的方剂，对学习记忆障碍类疾病具有很好的疗效，但其确切的分子作用机制尚不明确。本文通过数据库及文献构建远志散的化学成分库，并通过反向分子对接预测潜在靶点，进而构建成分-靶标网络；在此基础上又通过与AD的靶标网络进行映射、分析，获得“有效成分-AD靶标”网络，然后通过网络分析筛选出关键靶点。通过与已知文献报道的靶点进行对比，分析预测靶点的合理性。通过对其基因功能和生物过程进行分析，发现远志散对AD的治疗作用主要是通过调节乙酰胆碱水平和刺激突触传递等实现的。