Analysis of the potential and mechanism of Ginkgo biloba in the treatment of Alzheimer's disease based on network pharmacology

Introduction

Alzheimer's disease (AD), whose key symptoms are memory dysfunction and progressive cognitive dysfunction, is the most common neurodegenerative disease. Amyloid-beta (Aβ) deposition and tau protein hyperphosphorylation are pathological characteristics of AD and also the main therapeutic targets (Jeong, et al., 2020). The prevalence of AD increases progressively with age. It is estimated that there will be approximately 50 million AD patients worldwide (Armstrong RA, 2019) with approximately one in three to four people over the age of 85 having AD. More female than male suffer from dementia (27 million vs 16.8 million) which is currently the fifth leading cause of death in the world. Risk factors for AD include high body mass index (BMI), high blood sugar, smoking, and high-sugar beverage intake (GBD 2016 Dementia Collaborator, 2019). However, there are still multiple opinions on the pathogenesis of AD, including the causes of amyloid hypothesis, tau protein transmission hypothesis, cholinergic hypothesis, inflammation hypothesis, mitochondrial cascade hypothesis, calcium homeostasis hypothesis, neurovascular hypothesis, metal ion hypothesis, and lymphatic system hypothesis. At present, four drugs approved by the U.S. Food and Drug Administration (FDA) for AD are applied in the treatment of clinical AD, the main purpose of which is to restore physiological acetylcholine (Ach) levels. Acetylcholine esterase (AChE) inhibitors are to inhibit the hydrolysis of acetylcholine, including Donepezil, Rivastigmin, and Galanthamine base, all of which belong to this category of drugs. Memantine, through non-competitive antagonism of N-Methyl-D-aspartic acid (NMDA) receptors, current flow (special calcium) is prevented and the excitotoxicity effect of glutamate is reduced. However, there is no clear and effective treatment for patients with advanced AD (Zagórska and Jaromin, 2020). Therefore, the AD multiple pathogenesis hypothesis and the imperfect single-target drug therapy require further exploration of multi-target comprehensive treatment.

Ginkgo is an ancient perennial tree of the genus Ginkgo, which survived after the Fourth Glacier. Ginkgo biloba, seed kernel and outer seed coat have medicinal value and have been used as medicine for more than 600 years, the earliest record of which could be traced in Sheng Nong's Herbal Classic. China is the birthplace of ginkgo which accounts for about 70% of the world's resources. At present, more than 160 compounds have been found in Ginkgo biloba leaves and the main effective ingredients are Ginkgo flavonoids, Ginkgolide, and Ginkgolic acid (Yang HP and Gao R, 2017; Yang Y, et al. 2016).

The medicinal value of Ginkgo biloba was confirmed in Song Dynasty (960-1279), and was recorded in many kinds of herbal medicine books, such as Chinese Materia Medicain in Yuan Dynasty, Compendium of Materia Medicain in Ming Dynasty, and Mu Cao Feng Yuan in Qing Dynasty. Moreover, traditional Chinese medicine believes that Ginkgo biloba has effects of anti-oxidation, reducing blood viscosity, improving microcirculation, improving memory, increasing cerebral blood flow, protecting microvascular smooth muscle cells, reducing brain damage, improving neuroplasticity, improving neurodegenerative diseases, anti-radiation (Wang Y and Yang YF, 2001; Zhang PF, et al. 2017).

At present, there are a variety of ginkgo biloba prescriptions widely used in clinical practice, suggesting that it has an effect on improving neurodegeneration and cognitive impairment, while the mechanism remains unclear. We used network pharmacology methods to explore the drug-target-disease rationale and find new approaches for the treatment of AD in this research.

Materials and methods

Results

The target of Ginkgo biloba and AD

A total of 27 active ingredients and 191 corresponding targets were selected by the TCMSP platform. The basic information of the active ingredients of Ginkgo biloba is shown in Table 1. The target was standardized using the UniProt platform, and 191 target genes were obtained (Figure 1). Through the DisGeNET database, AD genes were obtained and a total of 99 target genes were screened out (Table 2).

Table 1. Basic Information of active ingredients in Ginkgo biloba

Syringetin	Diosmetin
Luteolin-4′-glucoside	Flavoxanthin
Isogoycyrol	sesamin
Ginkgolide M	Mandenol
ginkgolide J	bis[(2S)-2-ethylhexyl] benzene-1,2-dicarboxylate
ginkgolide C	(+)-catechin
ginkgolide B	Stigmasterol
Bilobalide	kaempferol
Laricitrin	beta-sitosterol
Linolenic acid ethyl ester	isorhamnetin
Genkwanin	quercetin
campest-5-en-3beta-ol	(-)-catechin
Chryseriol	luteolin
Ethyl oleate (NF)

Table 2. The target gene of AD

ABCA7	EPHA1	INPP5D	SLC2A4	LEP
ACE	CST3	SOD2	CHRNB2	INSR
TREM2	BDNF	MS4A4A	SNAR-I	HMOX1
BCHE	TNF	PLCG2	CASP3	INS
CLU	CR1	PRNP	MIR3622B	IGF1R
MTHFR	PCDH11X	RELN	CALM1	IGF2
BIN1	NOS3	DHCR24	SLC30A4	HFE
MAPT	CYP46A1	TF	MIR766	MPO
PSEN1	TFAM	CHRNA7	MIR4467	AMFR
CD33	CD2AP	HLA-DRB5	VEGFA	SLC30A6
PICALM	IGF1	IQCK	TPI1	ENO1
APOE	NECTIN2	NCSTN	PGRMC1	MIR708
IDE	PLAU	ARC	TPP1	F2
APP	APOC1	BAX	ADAMTS1	WWOX
TOMM40	ADAM10	CRH	DPYSL2	ABI3
BACE1	ESR1	MIR505	EIF2S1	ATP5F1A
PPARG	GSK3B	BCL2	MAOB	MIR375
PSEN2	ACHE	CASS4	MIR296	NPY
A2M	IL1B	PYY	MIR146A	GAPDHS

Construction of Venn graph

The genes of AD and Ginkgo biloba were introduced into Venn to obtain an intersection (Figure 2). The blue circle on the left represents the target gene of AD, the red circle on the right represents the target gene of Ginkgo biloba, and the overlapping part is the common target of the two. The overlapping target genes contain 18 such as plasminogen activator, urokinase (PLAU), heme oxygenase 1 (HMOX1), tumor necrosis factor (TNF), insulin receptor (INSR), myeloperoxidase (MPO), monoamine oxidase B (MAOB), insulin like growth factor 2 (IGF2), interleukin 1 beta (IL1B), estrogen receptor 1 (ESR1), B-cell CLL/lymphoma 2 (BCL2), acetylcholinesterase (AChE), BCL2-associated X (BAX), glycogen synthase kinase 3 beta (GSK3B), peroxisome proliferator activated receptor gamma (PPARG), solute carrier family 2 member 4 (SLC2A4), nitric oxide synthase 3 (NOS3), caspase 3 (CASP3) and vascular endothelial growth factor A (VEGFA).

PPI interaction network

The PPI network of Ginkgo biloba core gene for AD treatment was established by string database, including 18 points and 72 edges (Figure 3). The relevant data of core targets were imported into Cytoscape, and further filter the degree value and the Betweenness Centrality value. Using the screening results, the PPI network was constructed, and a total of 11 central targets were screened (Figure 4). The size of the target and the thickness of the line can clearly show the tightness of the important target and the line. From the visual analysis of PPI network, VEGFA, TNF, CASP3, PPARG, IL1B, NOS3, HMOX1, ESR1, MPO, AChE, play a pivotal role in Ginkgo biloba intervention in AD, link other core targets together, and play a synergistic effect.

KEGG and GO enrichment analysis

Perform analysis of GO function (Figure 5) and KEGG pathway (Figure 7) analysis using Metascape database.GO analysis includes three parts: biological process (BP), cellular component (CC) and molecular function (MF), enriching AD related genes, and setting a threshold P <0.05. A total of 640 GO items were collected, including 609 BP items, accounting for 95.1%, 16 MF items, accounting for 2.5%, and 15 CC items, accounting for 2.4% (Figure 6). Among the BP, developmental process involved in reproduction, reproductive structure development, reproductive system development, response to toxic substance, extrinsic apoptotic signaling pathway, positive regulation of cell migration, positive regulation of cell motility, positive regulation of cellular component movement, apoptotic signaling pathway, and positive regulation of locomotion are in the front position (Figure 5). Among the CC, membrane raft, membrane microdomain, membrane region, caveola, plasma membrane raft, external side of plasma membrane, side of membrane, mitochondrial outer membrane, organelle outer membrane, outer membrane are in the front position (Figure 5). Among the MF, protein homodimerization activity, protein domain specific binding, protease binding, cytokine receptor binding, receptor ligand activity, receptor regulator activity, cofactor binding, oxidoreductase activity, transcription factor binding, cytokine activity are in the front position (Figure 5).

A total of 44 pathways were obtained after KEGG enrichment. Advanced glycation end (AGE)-Receptor of Advanced Glycation End products (RAGE) signaling pathway in diabetic complications, fluid shear stress and atherosclerosis, non-alcoholic fatty liver disease (NAFLD), proteoglycans in cancer, pathways in cancer, microRNAs in cancer, hypoxia-inducible factor-1 (HIF-1) signaling pathway, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, insulin resistance, tuberculosis, are in the front position (Figure 7).

Discussion

AD with a variety of pathophysiological mechanisms is a chronic degenerative disease. Neural plaques and neurofibrillary tangles (NTF) are the pathological features of AD, which is related to the accumulation of amyloid-beta peptide (Aβ) in brain tissue and the cytoskeletal changes caused by the hyperphosphorylation of microtubule-associated tau protein in neurons (De-Paula, et al., 2012). Neural plaques are cleaved from amyloid precursor protein (APP) by β-amyloid and amyloid secretase. At present, the primary treatment of AD still revolves around the relief of symptoms, with cutting-edge therapy of the inhibition of acetylcholinesterase (AChEI), such as Donepezil, Galantamine, etc., which mainly improve cognitive symptoms and weaken the disease. AChEI approved for treatment in the mild and moderate stages of AD (MMSE 26-10), and Memantine (N-Methyl-D-aspartic acid receptor inhibitor), which can effectively bind to AChE, was used in the middle and late stages of AD (Kovács, 2009). However, these drugs can only treat the symptoms of AD. There is no evidence that they can abrogate or reverse the progression of the disease. In addition, it is impossible to predict whether it will be effective for an individual. In the world, there is an AD patient approximately every 3 seconds. By 2019, there were more than 10 million AD patients in China, which is the country with the largest number of patients in the world. It is estimated that China will have 28 million patients in 2050 (Armstrong RA, 2019). According to the theory of traditional Chinese medicine and clinical practice, it is found that Ginkgo biloba has advantages in improving memory and alleviating neurodegenerative diseases, which is consistent with the pathogenesis of AD.

Through the TCMSP database, a total of 27 effective active ingredients of Ginkgo biloba including kaempferol, ginkgolide, syringaresin, luteolin, bilobalide, and quercetin were screened for target information. Through UniProt query, 191 gene abbreviations including VEGFA, MPO, MAOB, AChE, IL6, NOS3, and TNF were obtained. NOS is a nitric oxide synthase, an isoenzyme, which exists in endothelial cells, macrophages, phagocytes and nerve cells. At present, oxidative stress induced by nitric oxide is suggested to be one of the pathogenesis of AD. Furthermore, the abnormal expression of NOS gene is also believed to induce the degeneration of brain neurons and glia. MPO is a myeloperoxidized protein, which is rich in neutrophils. It can catalyze and oxidize chloride ions to generate hypochlorous acid, kill microorganisms in phagocytes, and produce and regulate inflammation in the body. Studies have shown that there is a significant increase in MPO in the peripheral blood of AD patients, which is considered to be related to the progression of AD disease. Besides, MPO plaques and neurofibrillary tangles were found in the frontal cortex and hippocampus of AD patients (Glass, et al. 2010; Wu CY, et al. 2020).

Through DisGeNET database, with AD as the key word, 99 core targets including ATP binding cassette subfamily A member 7 (ABCA7), Angiotensin Converting Enzyme (ACE), triggering receptor expressed on myeloid cells 2 (TREM2), butyrylcholinesterase (BCHE), clusterin (CLU), methylenetetrahydrofolate reductase (MTHFR), bridging integrator 1 (BIN1), microtubule associated protein tau (MAPT), presenilin 1 (PSEN1), CD33 molecule (CD33), phosphatidylinositol binding clathrin assembly protein (PICALM), apolipoprotein E (APOE), insulin degrading enzyme (IDE), APP were screened out. Currently the pathogenesis of AD has not been fully elucidated, and is considered to be related to the cholinergic hypothesis (Bateman, et al., 2012), amyloid hypothesis (Hardy and Higgins, 1992), tau protein transmission hypothesis (Frost, et al., 2009), mitochondrial cascade hypothesis, and Ca²⁺ homeostasis hypothesis (Kim, et al., 2000), inflammatory hypothesis (Liu, et al., 2019), neurovascular hypothesis, metal ion hypothesis (Abelein, et al., 2015), lymphatic hypothesis.

The acetylcholinergic hypothesis is mainly due to choline deficiency, with AChE and butyrylcholinesterase (BuChE) as the main targets. AChE can degrade acetylcholine between cholinergic synapses, terminate the excitatory effect of neurotransmitters on the postsynaptic membrane, and ensure the correct transmission of nerve signals in the organism. At present, the most commonly used drugs are acetylcholinesterase inhibitors, such as donepezil, galanthamine and so on. However, there are also many reports that the accumulation of anticholinergic drugs is related to poor cognitive ability and functional outcome (Salahudeen, et al., 2015). The characteristic histology of AD is neurofibrillary tangle, which is mainly elicited by phosphorylated tau protein. These lesions can lead to activation of microglia and astrocytes, leading to synaptic loss and neuronal death. GSK3 β is a serine/threonine kinase, which widely exists in mammalian eukaryotic cells. GSK3β can regulate cell apoptosis by affecting signal protein structural proteins and transcription factors. Currently, GSK3 β is selected as a therapeutic target in the research of neurodegenerative diseases, neuropsychiatric diseases, cancer and other major diseases. GSK3 β regulates apoptosis by affecting glucose concentration in blood, mitochondrial permeability and cytochrome c release. It is also an important target for tau protein phosphorylation, which is also the source of tau hypothesis (Toral-Rios, et al., 2020).

MAO-B levels were found to increase in the frontal cortex and hippocampus of AD, which causing reactive oxygen activity (ROS) and hydrogen peroxide to increase, leading to oxidative damage and neurodegeneration. Based on the amyloid hypothesis of AD, the overproduction of Aβ is the result of the destruction of the steady-state process, which regulates the proteolytic cleavage of amyloid precursor protein (APP). The core of the amyloid plaque in the brain of AD patients is composed of fibrils formed by Aβ, which plays a key role in the pathogenesis of AD. It is suggested that MAO-B can regulate the level of a β in the nerve, which may be mediated by γ - secretase, which is also the reason why MAO-B is considered as the target of AD (Schedin-Weiss, et al., 2017). The “component-target-pathway” network prompts the degree of targets such as VEGFA, TNF, CASP3, IL1B, PPARG, NOS3, ESR1, HMOX1, SLC2A4, MPO, GSK3β, IGF2, BAX, ACHE, INSR, PLAU, BCL2, MAOB targets are ranked front position. Combining the above content, it is found that Ginkgo biloba can achieve the purpose of treating AD through core targets such as ACHE, MAOB, GSK3β, NOS3, and MPO. The key targets were analyzed by GO function analysis. The biological functions of 18 main targets were mainly reflected in protein binding, receptor binding, receptor regulation, redox and other processes. Enrichment analysis of KEGG pathway showed that the main biological processes involved in core targets include HIF-1 signaling pathway, NF-kappa B signaling pathway, PI3K-Akt signaling pathway, Apoptosis pathway, etc. The ATP efficiency of the thread body in AD decreases, leading to the increase of ROS and oxidation levels, and the lack of Ca⁺ buffering capacity, which promotes the release of apoptotic factors and induces apoptosis (Gadhave, et al., 2020). HIF-1 signaling pathway can increase the response of cells to hypoxia, hypoglycemia and oxidative stress by increasing glucose uptake, glycolysis and pyruvate to lactic acid transformation, so as to ensure the supply of intracellular ATP and reduce cell apoptosis, so as to achieve the effect of treating AD (Yu and Blair, 2019).The neuropathological feature of AD is the formation of senile plaques (SP) composed of small peptide Aβ and intracellular neurofibrillary tangles (NFT) (De Strooper, 2010). NF-κB pathway plays an important role in neuronal damage and synaptic plasticity in the central nervous system (Zhang, et al., 2017). In addition, the up-regulation of NF-κB activity induced by Aβ not only promotes the expression of pro-inflammatory factors, but also increases the production of Aβ, thereby participating in the development of AD (Chami, et al., 2012). Therefore, this is one of the reasons why the NF-κB pathway has become a new direction for AD treatment. PI3K-Akt signaling pathway is a downstream signaling pathway of brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB), which is widely present in cells. Glycogen synthase kinase-3 (GSK-3) is an important downstream target of PI3K-Akt signaling pathway. GSK-3 activation can lead to abnormal phosphorylation of tau protein and accumulation of neurofibrillary tangles, which ultimately leads to learning and memory impairment. These pathways are all related to the cause of AD. Ginkgo biloba affects oxidative and inflammatory responses through multiple core targets including GSK3β, TNF, and IL-1B through multiple pathways, and act on AD with multiple targets to compensate for the lack of single-target drug therapy.

In summary, the network system of Ginkgo biloba-component-target-AD was constructed by using the method of network pharmacology, public database and related software. KEGG pathway and GO biological function analysis of core targets were carried out, and the multi-target mechanism of Ginkgo biloba in the treatment of AD was explored, which provided new ideas for clinical drug research.

Ethical statement

Not applicable.

Conflict of interest

No conflicts of interest were declared.

Funding

This study was supported by the Science and Technology Fund of Guizhou Provincial Health Commission No. gzwjkj2020-1-014.

Transparency statement

All the authors affirm that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

Authors' contribution

Xue-Yan Huang, Chang-Yin Yu and Liu-Lin Xiong contributed the central idea, analysed most of the data, made figures & tables and wrote the initial draft of the paper. Ting-Ting Li, Lin Zhou and Tao Liu contributed to refining the ideas, carrying out additional analyses and finalizing this paper.