Mechanisms with network pharmacology approach of Ginsenosides in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss, cognitive disorder, language dysfunction, and mental disability. The main neuropathological changes in AD mainly include amyloid plaque deposition, neurofibrillary tangles, synapse loss, and neuron reduction. However, the current anti-AD drugs do not demonstrate a favorable effect in altering the pathological course of AD. Moreover, long-term use of these drugs is usually accompanied with various side effects. Ginsenosides are the major active constituents of ginseng and have protective effects on AD through various mechanisms in both in vivo and in vitro studies. In this review, we focused on discussing the therapeutic potential effects and the mechanisms of pharmacological activities of ginsenosides in AD, to provide new insight for further research and clinical application of ginsenosides in the future. Recent studies on the pharmacological effects and mechanisms of ginsenosides were retrieved from Chinese National Knowledge Infrastructure, National Science and Technology Library, Wanfang Data, Elsevier, ScienceDirect, PubMed, SpringerLink, and the Web of Science database up to April 2023 using relevant keywords. Network pharmacology and bioinformatics analysis were used to predict the therapeutic effects and mechanisms of ginsenosides against AD. Ginsenosides presented a wide range of therapeutic and biological activities, including alleviating Aβ deposition, decreasing tau hyperphosphorylation, regulating the cholinergic system, resisting oxidative stress, modulating Ca²⁺ homeostasis, as well as anti-inflammation and anti-apoptosis in neurons, respectively. For further developing the therapeutic potential as well as clinical applications, the network pharmacology approach was combined with a summary of published studies.

Graphical abstract

1. Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss, cognitive disorder, language dysfunction, and mental disability [1,2]. The main neuropathological changes in AD mainly include amyloid plaque deposition, neurofibrillary tangles, synapse loss, and neuron reduction. The pathogenesis of AD has been attributed to intracellular neurofibrillary tangles in cortical areas of the brain caused by extracellular aggregates of amyloid β (Aβ) plaques and abnormally phosphorylated tau protein [3]. The epidemiological studies showed that the prevalence of AD has increased recently with an estimated morbidity of 50 million by 2020 and expected to reach 150 million by 2050 worldwide [[4], [5], [6]]. Although numbers of anti-AD drugs have been developed in the last decades, including anti-Aβ monoclonal antibodies, Aβ aggregation inhibitors, acetylcholinesterase (AChE) inhibitors, and N-methyl-d-aspartate (NMDA) receptor inhibitors, and these drugs can alleviate AD symptoms; however, none of them demonstrated a favorable effect in alleviating the pathological course of AD [6]. Moreover, long-term use of these drugs is often accompanied with various side effects, including nausea, vomiting, diarrhea, allergic reactions, loss of appetite, etc. [[7], [8], [9]].

Recent studies have shown that herbal medicines and their active compounds can improve memory and cognitive function in AD patients with few adverse effects [10,11]. The Panax genus belongs to the Araliaceae family, which consists of at least nine species, including Panax ginseng, Panax quinquefolium, Panax notoginseng, and Panax japonicus. Panax genus has been considered one of the most important herbal medicines for thousands of years in China and east Asian, and its use as a nutritional supplement is rapidly increasing in North America and Europe. The bioactive components of Panax genus include saponins (ginsenosides), flavonoids, polysaccharides, volatile oils, polyenes, fatty acids, and polyacetylenic alcohols [[12], [13], [14]]. Among them, the major active constituents of Panax are ginsenosides, of which more than 40 have been identified so far [15]. Based on the carbon skeletons of the aglycones structure, ginsenosides were classified into protopanaxadiols (such as Rb1, Rb2, Rc, Rd, Rg3 and Rh2) and protopanaxatriols (such as Re, Rf, Rg1, Rg2 and Rh1). The steamed ginseng also has a unique saponin profile, such as Rg5, which is likely the result of heat transformation and deglycosylation [16](Table .1).

Table 1.

Chemical structural formula of ginsenoside.

Ginsenosides have been proven to be beneficial in the prevention and treatment of AD and other nervous system diseases [14,17,18]. Accumulating studies have suggested that ginsenosides have a protective effect on AD through various mechanisms both in vivo and in vitro [18,19]. In this review, we focus on discussing the therapeutic potential effects and the mechanisms of pharmacological activities of ginsenosides in AD, to provide new insight for further research and clinical application of ginsenosides in the future.

The following databases were consulted this review: Chinese National Knowledge Infrastructure, National Science and Technology Library, Wanfang Data, Elsevier, ScienceDirect, PubMed, SpringerLink, and the Web of Science database. More than 200 publications were consulted from the databases above from inception to April 2023. To retrieve the literatures, the following words or phrases were used alone or in various combinations in the titles and/or abstracts: “Ginseng”, “Ginsenosides”, “Renshen”, “Alzheimer”, “Alzheimer's disease”, “Anti-Alzheimer”, “Mechanism”, “Pharmacology” and “Neuroprotective effect”, “Panax ginseng”, “Neuroprotective effect”, and “Pharmacological mechanisms”, total 98 articles within two decades were used in this review.

2. Pharmacological mechanisms about anti-AD effects of ginsenosides

2.1. Ginsenosides inhibit Aβ deposition

The accumulation of Aβ is a pivotal contributor to the pathogenesis of AD [20]. Two main pathways involved in amyloid precursor protein (APP) metabolization, nonamyloidogenic and amyloidogenic. APP is cleaved by α-secretase via the nonamyloidogenic pathway, producing transmembrane fragment C83 and N-terminal fragment sAPPα. C83 is subsequently hydrolyzed by γ-secretase to generate the non-toxic fragment P3 and APP intracellular domain (AICD). In the amyloidogenic pathway, APP is cut by β-secretase to generate transmembrane fragment C99 and N-terminal fragment sAPPβ. C99 is then cleaved by γ-secretase to produce neurotoxic Aβ polypeptide fragments and AICD [21]. Aβ polypeptide fragments, as non-fibrillar oligomers, aggregate to amyloid fibrils in the cerebral cortex, inducing neurotoxicity and contributing to AD. Therefore, inhibiting β-secretase pathway and activating the nonamyloidogenic pathway are the potential targets for AD treatment.

A molecular docking study showed that ginsenosides Rb1, Rb2, F1, Rh1, Rh2 and compound K (CK) have high binding affinities for the beta-site amyloid precursor protein, which cleave the enzyme beta-secretase-1 (BACE1) and exhibits potential BACE1 inhibitory activity [22]. In APP/PS1 mice, 10 mg/kg intraperitoneal (i.p.) injection of ginsenoside Rg1 increased the expression of brain-derived neurotrophic factor (BDNF), a synaptic plasticity regulator in the central nervous system that improves memory and cognitive functions, and its receptor tyrosine kinase B (TrkB) in turn lowers the synthesis of Aβ1-42, improves synaptic plasticity and decreases neurofibrillary tangles [23,24]. In streptozotocin (STZ)-induced memory impaired rats, intracerebroventricular (i.c.v.) injection of Rg5 (5, 10, 20 mg/kg) alleviated Aβ deposition by enhancing the expressions of insulin-like growth factors 1 (IGF-1) and BDNF in the hippocampus and cerebral cortex [16]. Ginsenoside Rd (10 mg/kg for 2 moths) improved cognitive and memory function in ovariectomy (OVX) rats. In the same study, ginsenoside Rd increased the expression of α-secretase and the synthesis of soluble APPα (sAPPα) via the nonamyloidogenic pathway, which ameliorates the activity of amyloidogenic pathway and consequently decrease the Aβ level in neuronal cells from HT-22 mouse hippocampus [25]. Ginsenoside Rd was also found to activate the mitogen-activated protein kinase/phosphoinositide-3 kinase (MAPK/PI3K) pathway mediated by estrogen receptor (ER), increase the production of sAPPα through the nonamyloidogenic pathway, decrease extracellular Aβ level, and subsequently alleviate the impairment of cognitive and memory in ovariectomy rats [25]. In mouse neuroma cell N2a/APP695, ginsenoside Re (100 μM) inhibited the activity of BACE1 by activating peroxisome proliferator-activated receptor-γ (PPARγ) and reduced the generation of Aβ1-40 and Aβ1-42 [26]. In tg2576 AD model mice, ginsenoside Rh2 (10 mg/kg i.p. injection for 8 weeks) increased sAPPα levels and CTFα/β ratio, decreased Aβ1-40 and Aβ1-42 concentrations, and inhibited APP endocytosis by lowering levels of cholesterol and lipid raft, which resulted in a reduced number of senile plaques in brain tissue and improved cognitive functions [27].

2.2. Ginsenosides inhibit tau phosphorylation

Tau protein, a microtubule-associated protein expressed in neurons, plays an important role in AD pathogenesis [28]. Tau is switched from non-toxic to toxic when triggered by the upstream protein Aβ, and toxic tau protein in turn increases Aβ toxicity via a feedback loop [29,30]. The neurodegeneration in AD patients is positively associated with Aβ and tau protein [[31], [32], [33]]. The activation of p38 mitogen-activated protein kinase (p38 MAPKs) promotes tau phosphorylation, which impairs the ability of neurons and glial cells to eliminate Tau and Aβ aggregates [34]. P38 phosphorates tau and promotes the release of inflammatory cytokines in astrocytes and microglia, resulting in chronic neuroinflammation and neurodegeneration [35]. In Aβ1-40-induced SK-N-SH neuroblastoma cells, ginsenoside Rg1 (50, 100 and 150 μM) mitigated Aβ neurotoxicity to SK-N-SH neuroblastoma, decreased IL-1β release, and inhibited tau phosphorylation via inhibiting the activation of p38 MAPK [36].

Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase and can induce nerve cell death and neurodegeneration by altering mitochondrial morphology or transmembrane potential [37]. CDK5 becomes hyperactive when neurons are exposed to pathogenic stimuli, leading to aberrant hyperphosphorylation of CDK5 substrates, such as APP, tau, and neurofilament, and neurodegenerative disorders [38]. When intracellular Ca²⁺ levels rise, calpain cleaves CDK5 activators p35 and p39 into p25 and p29, and the binding of CDK5 and p25 causes aberrant hyperphosphorylation of several CDK5 substrates [[38], [39], [40], [41]]. Additionally, the increased Aβ level leads to hyperphosphorylation of tau by increasing the expression of p25 [42]. Inhibiting CDK5 decreases tau hyperphosphorylation and neurofibrillary tangles, thereby delaying the process of neurodegeneration [43]. Ginsenoside Rb1 (40 μM) down-regulated p25 expression and lowered CDK5 activity via the Ca²⁺/calpain/CDK5 signaling pathway in Aβ25-35-induced rat primary cortical neurons, efficiently maintaining intracellular Ca²⁺ homeostasis, inhibiting calpain activation and minimizing tau hyperphosphorylation [44].

Glycogen synthase kinase 3 (GSK-3) is another proline-directed serine/threonine kinase, its isoform, GSK-3β promotes Aβ formation and tau hyperphosphorylation through modulating the Wnt (Wingless/Integrated) and PI3K/AKT signaling pathways [45]. In Aβ1-40-injected SD rats APP transgenic mice, pretreated with ginsenoside Rd (30 mg/kg i.p. injection) not only reduced the expression of GSK-3β, but increased the activity of protein phosphatase 2A (PP2A), a key phosphatase involved in tau dephosphorylation [46]. In the aluminum-induced tau hyperphosphorylation ICR mice model, ginsenoside Rb1 (20 mg/kg orally administration) reduced the phosphorylation of GSK-3β and the level of PP2A by activating the PI3K/AKT pathway [47].

2.3. Ginsenosides reduce neuroinflammation

Neuroinflammation is an active immune response in the central nervous system mediated by astrocytes and microglia. Microglia act as macrophages that secrete cytokines and chemokines in response to stimuli phagocytosis and subsequently regulate the internalization and degradation of Aβ [48]. In Aβ1-42-induced rat primary microglial cells, ginsenoside Rg3 (25 μg/mL) increased microglial phagocytosis of Aβ by up-regulating the expression of macrophage scavenger receptor type A (MSR-A) that promoted Aβ uptake, internalization, and digestion [49].

Toll-like receptor 4 (TLR4) is a transmembrane protein that belongs to the toll-like receptor family and activates the innate immune system. Excessive activation of TLR4 triggers the generation of various inflammatory factors such as nuclear factor-κB (NF-κB), interferon beta (IFN-β) and tumor necrosis factor alpha (TNF-α) [50]. TLR4 is the primary receptor that specifically recognizes lipopolysaccharide (LPS). The combination of TLR4 and LPS activates NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways by regulating myeloid differentiation primary response protein 88 (Myd88), which increases pro-inflammatory factors secretion and triggers the infiltration of inflammatory cells into the brain [51,52]. In the Aβ 25-35-induced NG108-15 murine neuroglial cells, ginsenoside Rg1 (2, 4, 8, 16 and 32 μg/mL) reduced neuroinflammation by decreasing the level of NF-κB, TLR3 and TLR4, and lowering the production of TNF-α, IFN-β in cell supernatant [53]. In the APP/Tg mouse model, ginsenoside Rd (10 mg/kg i.p. injection for 6 months) decreased the expression of pro-inflammatory factors IL-1β, IL-6, and TNF-α, increased the expression of anti-inflammatory factor IL-10, and consequentially improved learning and memory function [54].

NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome contains NLRP3 core protein, apoptosis-associated speck-like protein (ASC) and pro-caspase-1, which are essential components of the innate immune system [55]. A decrease in Aβ deposition was observed in cases of insufficient NLRP3 expression mice [56]. Stimulating microglia cells to release IL-1 and inducing inflammatory response via activating the NLRP3 inflammasome contributes to Aβ clearance [57]. Therefore, decreasing NLRP3 inflammasome activation would be an effective therapeutic approach for reducing inflammatory injury in brain nerves [58]. Ginsenoside Rg3 (20 mM) was demonstrated to lower Nitric oxide (NO) generation and inhibiting the activation of NLRP3 inflammasome in murine macrophage Raw 264.7 cells by regulating the expression of inducible nitric oxide synthase (iNOS) [59]. CK (10 mg/kg orally administration for 10 weeks) inhibited the activation of NLRP3 inflammasome, declined the expression of cleaved caspase-1, and mature IL-1β in the hippocampus of diabetic model mice, improving memory and cognitive dysfunction of diabetic model mice [60].

2.4. Ginsenosides regulate the cholinergic nervous system

The cholinergic metabolism was closely associated with AD, indicated by decreased activity of Choline acetyl transferase (ChaT) and presynaptic cholinergic transmitter in the brain of AD patients [61,62]. Acetylcholine (Ach) is an important central neurotransmitter in neuromuscular junctions and associated with learning and memory behavior. Acetylcholinesterase (AChE) has a strong hydrolytic activity, which blocks signal transmission by hydrolyzing Ach. The disorder of synthesis, storage, and release of Ach increases AChE accumulation and combination of AChE with Presenilin-1 (PS1), which result in cognitive impairment [63,64].

Ginsenoside Rb1 (5 mg/kg i.p. injection for 4 days) was proved to enhance ACh release in the hippocampus of SD rats after anticholinergic drug treatment, and choline absorption was boosted by promoting ACh metabolism in the central nervous system, thus alleviating the memory loss induced by anticholinergic medication [65]. Rg5 (5, 10, 20 mg/kg i.c.v. injection for 28 days) was found to improve cognitive dysfunction in streptozotocin-induced memory impaired rats by reducing AChE activity [16]. In mouse neuroblastoma N2a cells, ginsenosides Rd and Re (5 μg/mL for 48 h) increased the expression of ChAT by up-regulating microtubule-associated protein (MAP2), nerve growth factor receptors p75, p21 and tropomyosin receptor kinase A (Trka)，which resulted in an increase in the level of ACh and vesicular acetylcholine transporters for cholinergic neurotransmission [66]. In a high-fat diet induced C57BL/6 mouse model, ginsenoside Re (5, 10, 20 mg/kg orally administration for 4 weeks) increased ACh concentration in brain tissue by blocking the c-Jun NH2-terminal kinase (JNK) pathway, and decreased AChE activity, showing a beneficial effect in cholinergic neurons and mouse cognitive function [67].

2.5. Ginsenosides resist oxidative stress

Oxidative stress (OS) is one of the main causes of neurodegenerative disorders [[68], [69], [70]]. Aβ-40 and Aβ-42 induced Ca²⁺ signaling in astrocytes drives the oxidation process of decreased coenzyme II NADPH, resulting in excessive ROS and neurotoxicity [71]. On the other hand, excessive Aβ deposition aggravates OS and increases ROS production, which impairs permeability of the cell membrane, disrupts intracellular Ca²⁺ homeostasis, and causes the loss of mitochondrial function and neuronal cell death [[72], [73], [74], [75]].

In Aβ25-35-induced PC12 cells and primary cultured cortical neurons, ginsenosides Rb1 (1 mM for 24 h) and Rg1 (20 μM for 24 h) not only reduced excessive ROS and lipid peroxidation, but also lowered lactate dehydrogenase release, malondialdehyde (MDA) production, and superoxide dismutase (SOD) activity, subsequently enhancing cell survival [76,77]. Ginsenoside Rg2 (0.2 mmol/L for 24 h) demonstrated a neuroprotective effect in glutamate-induced PC12 cells by lowering excessive MDA and NO generation as well as intracellular free Ca²⁺ ions [78]. In d-galactosamine-induced rats, Rg3 (20 mg/kg/day i.p. injection for 60 days) increased the level of SOD and decreased the level of MDA, indicating that Rg3 alleviated the oxidative stress of AD rats by scavenging ROS. Rg3 improved the mitochondrial dysfunction of AD rats by regulating the abnormal energy metabolism and recovering the turbulent mitochondrial electron transport chain [79]. In Aβ-induced SH-SY5Y cells, Re (25 μM for 24 h) attenuated Aβ-induced ROS production and inhibited Aβ-triggered mitochondrial apoptosis by elevating the Bcl-2/Bax ratio, reducing the release of cytochrome c, and decreasing the caspase-3/9 ratio. Besides, Re activated Nrf2 (Nuclear factor erythroid 2–related factor 2), a regulator of cellular resistance to oxidants, indicated a beneficial effect on anti-oxidative and neuroprotection [80].

2.6. Ginsenosides regulate Ca²⁺ homeostasis

Metal ion homeostasis is critical for sustaining the function of the central nervous system. Cellular Ca²⁺ homeostasis regulates neural growth, differentiation, and synaptic plasticity, which are crucial for action potential and memory function. N-methyl-d-aspartate (NMDA) receptor locates in excitatory synapses and plays an important role in learning and memory. Aβ triggers NMDA-mediated Ca²⁺ influx and excitotoxicity in neurons, resulting in mitochondrial dysfunction, disruption of synaptic neurotransmission and cognitive impairment [81].

Ginsenoside Rg3 (10 μM for 2 days) regulates Ca²⁺ homeostasis by inhibiting NMDA receptors and shows a neuroprotective effect in rat primary hippocampal neurons [82]. In rat brain slices exposed to Aβ 25–35, ginsenoside Rg1 (20 μmol/L for 6 h) decreased hippocampal neuronal Ca²⁺ channel currents recorded by a whole-cell patch clamp by inhibiting the expression of mitogen-activated protein kinase (MAPK), thereby decreasing the neurotoxicity of Aβ induced by Ca²⁺ over influx [83]. What's more, ginsenoside Rg1 (1, 5 and 10 mg/kg orally administration for 8 weeks) improved diabetes-associated cognitive dysfunction by ameliorating neuronal Ca²⁺ overload and reducing Aβ generation in type 2 diabetic mice [84].

2.7. Ginsenosides inhibit cell apoptosis

Caspase-3 is a member of the caspase family and plays an essential role in cell apoptosis [85]. Caspase-3 immunoreactivity was increased remarkably in neurons and astrocytes of AD patients, as well as in neurofibrillary tangles and senile plaques [86]. Aβ accumulation was shown to activate caspase-3, trigger neuronal cell apoptosis, and increase neurofibrillary tangles, which are contributors to neuronal cell death and Aβ plaque formation in AD brain [87].

Ginsenoside Rg1 (12.5, 25, and 50 μmol/L for 24 h) was showed to attenuate isoflurane-induced caspase-3 activation in H4 human neuroglioma cells that express full-length APP (H4-APP cells) via inhibiting mitochondrial dysfunction, demonstrating a favor effect in neurotoxicity induced by APP [88]. In Aβ25-35 induced PC12 cells, ginsenoside Rg2 (20 mg/mL for 24 h) increased the level of B cell lymphoma protein 2 (Bcl-2) and down-regulated the level of Bcl-2 associated X protein (Bax) through the PI3K/AKT signaling pathway, thus inhibiting the activity of caspase-3 and decreasing the cells apoptosis [89]. Ginsenoside Rb1 (12.5, 25, and 50 mg/kg i.p. injection for 14 days) was proven to enhance cognitive and memory functions in Aβ1-40-induced rats by decreasing cleaved caspase-3 expression and increasing Bcl-2 expression in the hippocampus [90].

3. Network pharmacology of ginsenosides

Network pharmacology were used to explore the mechanisms of ginsenosides in AD. Combining traditional Chinese medicine (TCM) and network pharmacology should reveal the relationship between drugs, genes, and diseases. In this study, we identified and analyzed the core targets of the nine ginsenosides (Rb1, Rd, Re, Rg1, Rg2, Rg3, Rg5, Rh2 and CK) by using network pharmacology methods and found that their main pharmacological actions are anti-AD and neuroprotective effects.

The target genes of nine ginsenosides were searched by the pharmacologic database and analysis platform of TCM System (TCMSP, https://tcmspw.com/tcmsp.php/). GeneCards human genome database and OMIM database (https://omim.org/) were used to identify the target genes of AD (Fig. 1A). Subsequently, a protein-protein interaction (PPI) network was produced for all target genes using STRING (https://cn.string-db.org/), and the PPI network cores were screened by Cytoscape software (Fig. 1B and C).

Fig. 1.

Network pharmacology revealed the targets characteristics of ginsenosides. (A) Venn diagram of targets in nine ginsenosides for AD. (B) The protein–protein interaction (PPI) network based on targets of nine ginsenosides for AD. (C) Nine ginsenosides-targets-AD network.

As shown in the core of the PPI network, AKT1, EGFR, VEGFA, MTOR HSP90AA1 and STAT3 possessed high scores. AKT serine/threonine kinase 1 (AKT1)-mediated signaling is crucial for the normal development and function of the nervous system. ROS-mediated oxidative modification of AKT1 contributes to synaptic dysfunction in AD, which is necessary for synaptic plasticity and maintenance [91]. Mammalian target of rapamycin (mTOR) signaling is critical to long lasting forms of synaptic plasticity, which is important to neurodegeneration [92]. Promoting AKT1-mTOR signaling at synapses could be a potential therapeutic strategy for AD. Vascular endothelial growth factors (VEGFs) are important in the growth and maintenance of both vascular and neural cells. VEGFA appears to protect against cognitive impairment in AD pathology, whose levels are reduced in both serum and CSF among AD cases [93]. Epidermal Growth Factor receptor (EGFR) regulates the morphology and function of astrocytes. Overexpressed EGFR is found in AD astrocytes [94]. Blocking EGFR activation in astrocytes could be beneficial for the treatment of neuroinflammation, such as AD. Heat shock protein 90 kDa alpha family class A member 1 (HSP90AA1) is decreased in AD and is negatively correlated with the activities of β-secretases [95]. Signal transducer and activator of transcription 3 (STAT3) is a transcriptional factor that involved in regulation of synaptic plasticity and cognition [96]. Abnormal activation of STAT3 in the hippocampus of AD mouse model can induce astrogliosis, which could be an important therapeutic target in AD [97].

4. Conclusion and perspectives

AD is a chronic neurodegenerative disorder, and a main cause of mortality and disability in the aging population. There are no ideal treatments for patients with AD to date, even though a lot of new drugs have been developed and applied in clinical practice recently. In this article, we reviewed studies on the neuroprotective effects of ginsenosides in different pathogenic processes of AD and have shown that ginsenosides, the main active component of ginseng, have a broad spectrum of pharmacological effects on the different AD pathogenic processes, including alleviation of Aβ deposition, tau hyperphosphorylation, neuroinflammation and neuron cell apoptosis, regulation of the cholinergic system, resistance to oxidative stress, and modulation of Ca²⁺ homeostasis (Table 2 and Fig. 2).

Table 2.

Neuroprotective mechanisms of ginsenosides in AD treatments.

Ginsenoside	Subject	Dose	Duration	Targets	Actions	Reference
Rg1	MaleAPP/PS1 mice	i.p. injection (10 mg/kg)	30 days	BDNF, TrkB, BACE1, Aβ	Aβ deposition	[23,24]
	SK-N-SH neuroblastoma cells	50, 100 and 150 μM	30 min	IL-1β, tau, p38 MAPK	tau phosphorylation	[36]
	NG108-15 mouse glial cells	2, 4, 8, 16 and 32 μg/mL	24 h	TLR3, TLR4, TNF-α, IFN-β	Neuroinflammation	[53]
	PC-12 cells	20 μM	24 h	ROS, MDA, LDH, SOD	Anti-oxidation	[77]
	T2DM mice	orally administration (1, 5 and 10 mg/kg)	8 weeks	Ca2+, IP3, DAG, Aβ	Ca2+, IP3, DAG, Aβ	[84]
	SD rats	20 μmol/L	6 h	Ca2⁺, MAPK	Ca2⁺ homeostasis	[83]
	H4-APP cells	12.5, 25, and 50 μmol/L	24 h	Caspase-3	Anti-apoptosis	[88]
Rg2	PC12 cells	0.2 mmol/L	24 h	MDA, NO	Anti-oxidation	[78]
	PC12 cells	20 mg/mL	24 h	Caspase-3, Bcl2, BAX	Anti-apoptosis	[89]
Rg3	Rat primary microglial cells	25 μg/mL	8 h	Aβ, MSR-A	Neuroinflammation	[49]
	D-Gal induced rat	i.p. injection (20 mg/kg/day)	60 days	SOD, MDA	Anti-oxidation	[79]
	Murine macrophage cells	20 mM	16 h	NO, iNOS, NLRP3	Neuroinflammation	[59]
	primary hippocampal neurons	10 μM	2 days	Ca2⁺, NMDAR	Ca2⁺ homeostasis	[82]
Rg5	STZ-induced rats	i.c.v. injection (5, 10 and 20 mg/kg)	28 days	IGF-1, BDNF, Aβ	Aβ deposition	[16]
	STZ-induced rats	i.c.v. injection (5, 10 and 20 mg/kg)	28 days	AChE	Anticholinergic	[16]
Rb1	Rat primary cortical neurons	40 μM	24 h	p25, CDK5	tau phosphorylation	[44]
	Male SD rats	i.p. injection (5 mg/kg)	4 days	ACh	Anticholinergic	[65]
	Female ICR mice	orally administation (20 mg/kg/day)	4 months	GSK-3β, PP2A	tau phosphorylation	[47]
	PC-12 cells	1 mM	24 h	ROS, MDA, LDH, SOD	Anti-oxidation	[76]
	SD-rats	i.p. injection (12.5, 25, and 50 mg/kg)	14 days	Caspase-3, Bcl2	Anti-apoptosis	[90]
Rd	OVX rats	i.p. injection (10 mg/kg)	2 months	α-secretase, sAPPα, Aβ, MAPK/PI3K, ER	Aβ deposition	[25]
	APP/Tg mouse	i.p. injection (10 mg/kg)	6 months	IL-1β, IL-6, TNF-α, NF-κB, IL-10	Neuroinflammation	[54]
	Male SD rats APP transgenic mice	i.p. injection (30 mg/kg) i.p. injection (30 mg/kg)	7 days 6 months	GSK-3β, PP2A	tau phosphorylation	[46]
	N2a cells	5 μg/mL	48 h	ChAT, MAP2, p75, p21, Trka, VAChT, ACh	Anticholinergic	[66]
Re	N2a/APP695 cells	100 μM	24 h	BACE1, PPARγ, Aβ	Aβ deposition	[26]
	N2a cells	5 μg/mL	48 h	ChAT, MAP2, p75, p21, Trka, VAChT, ACh	Anticholinergic	[66]
	C57BL/6 mouse	orally administration (5, 10, 20 mg/kg)	4 weeks	ACh, AChE, JNK	Anticholinergic	[67]
	SH-SY5Y cells	25 uM	24 h	Cytochrome c, caspase 3/9, Bal 2/BAX, Nrf2	Anti-oxidation	[80]
Rh2	Tg2576 mice	i.p. injection (10 mg/kg)	8 weeks	sAPPα, CTFα/β, APP, Aβ	Aβ deposition	[27]
CK	db/db mice	orally administration (10 mg/kg)	10 weeks	NLRP3, caspase-1, IL-1β	Neuroinflammation	[60]

Fig. 2.

Protective mechanisms of ginsenosides in AD. Ginsenosides presented a wide range of therapeutic and biological activities, including alleviating Aβ deposition, decreasing tau hyperphosphorylation, regulating the cholinergic system, resisting oxidative stress, modulating Ca²⁺ homeostasis, as well as anti-inflammation and anti-apoptosis in neurons, respectively.

Ginsenosides Rb1 and Rg1 are considered the main ginsenosides of ginseng, which exhibit neuroprotective properties in anti-oxidative stress and anti-apoptosis [88,90]. Rb1 and Rg1 were also found to protect against Aβ-induced neurotoxicity and decrease the level of Aβ deposition [76,77]. Nevertheless, there are somewhat differences between the ginsenoside Rb1 and Rg1: ginsenoside Rb1 is prone to inhibit tau phosphorylation and reinforce the cholinergic systems, while ginsenoside Rg1 decreases neuronal Ca²⁺ influx and reduces neuroinflammatory responses [36,44].

The specific mechanism of ginsenosides in the treatment of AD has been unable to be clearly elucidated so far. Recent research on AD with ginsenosides is basically based on in vitro and in vivo studies. There was a clinical study to investigate the effect of total ginsenosides extracted from ginseng, which showed that treatment with ginsenosides complex for 12 weeks significantly improved the Mini-Mental State Examination (MMSE) and Alzheimer's Disease Assessment Scale (ADAS) [98]. However, the clinical significance of this study was limited by its small sample size and open-label study design. Therefore, it is necessary to conduct clinical trials with a scientific design method to validate the effects of ginsenosides in the future.

Network pharmacology results revealed multiple potential therapeutic targets of ginsenosides in AD such as AKT1, mTOR, EGFR and STAT3. Previous studies already demonstrated that ginsenosides act on these targets and exert beneficial effects in various diseases. For instance, with regards to AKT and mTOR, one study showed that ginsenoside RK1 significantly alleviated radiation-induced intestinal epithelial cell apoptosis by suppressing the PI3K/AKT/mTOR pathway in the irradiated rats model [99]. In terms of targeting EGFR, ginsenoside Re and Rf inhibited the production of pro-inflammatory factors and the expression of important cytokines in periodontitis by inducing the expression of heme oxygenase 1 via EGFR signaling pathway in human periodontal ligament cells [100]. And ginsenoside Rg1 demonstrated an anti-fatigue effect by impacting the metabolism of taurine and mannose 6-phosphate through downregulating EGFR expression in chronic fatigue syndrome rat model [101]. As for targeting STAT3, protopanaxadiol could inhibit the phosphorylation of STAT3 and its translocation from the cytosol to the nucleus in hepatocellular carcinoma cell lines [102]. Ginsenoside F2 could suppress the Y705 phosphorylation of STAT3, decrease its nuclear translocation and inhibit the growth of human hepatocellular carcinoma Hep G2 cells [103]. Additionally, in human pancreatic cancer cell lines BxPC-3 and AsPC-1, ginsenoside Rg3 enhanced the efficacy of erlotinib to inhibit the proliferation of pancreatic cancer cells via induction of apoptosis and downregulation of the EGFR/PI3K/AKT pathway [104]. Therefore, future research should focus on the PPI networks of AKT1, EGFR, VEGFA, MTOR, HSP90AA1 and STAT3 to obtain more comprehensive understanding of ginsenosides in AD.

Ginsenosides can be transformed into smaller deglycosylated through bioconversion methods by intestinal bacteria. But oral administration always accompanied with low bioavailability which may be due to the poor solubility of deglycosylated metabolites of ginsenosides [[105], [106], [107]]. Different ginsenosides displayed various bioavailability in human when administrating orally. Previous pharmacokinetic study found that ginsenoside Rb1, Rb2, Rc, and Rd were identified in human plasma after oral administration, whereas ginsenosides Re, Rh1, and Rg1 could not be found after repeated oral administration [108]. Another study showed that ginsenoside Rg1, Re, Rb1, and Rd had high bioavailability in rats. The inconsistence results from previous studies may be due to the different techniques (steaming, drying, and extracting) used in processing ginseng. Currently, limited pharmacokinetic studies also restrict the development of drug containing ginsenosides. Besides, the existence of the blood-brain barrier (BBB) is another limitation for ginsenosides in AD treatment. However, the encapsulation and nanoparticle technology showed a considerable effect in vivo, which can be developed extensively in future clinical research [109,110].

In this review, we summarized and discussed the mechanisms of ginsenosides by using database and network pharmacology tools to build target-disease networks and to analyze the PPI core network and pathways. Further investigations on the interaction mechanism of ginsenosides in AD using molecular biological techniques such as proteomics, genomics, metabonomics, and bioinformatics analysis are needed to provide more therapeutic evidence of ginsenosides.

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CRediT authorship contribution statement

Shan He: Writing – review & editing, Writing – original draft, Validation, Conceptualization. Junhe Shi: Writing – review & editing, Validation. Hua Chai: Validation. Lina Ma: Validation. Hui Pei: Validation. Ping Zhang: Validation. Dazhuo Shi: Validation, Supervision. Hao Li: Writing – review & editing, Validation, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.