Cistanche flavonoids activate the Keap1-Nrf2-ARE signaling pathway in improving cognitive dysfunction in Alzheimer's disease: A review

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder closely associated with oxidative stress, which plays a pivotal role in neuronal damage and disease progression. The Keap1-Nrf2-ARE signaling pathway plays a crucial role in regulating cellular responses to oxidative stress. Keap1 inhibits Nrf2 by maintaining its low expression, thus controlling antioxidant gene expression. Cistanche flavonoids, natural polyphenolic compounds, have been shown to activate this pathway. They suppress Keap1, preventing Nrf2 degradation and promoting its translocation to the nucleus, where it activates the antioxidant response element (ARE). This process significantly increases the production of antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase. Elevated enzyme levels enhance cellular antioxidant defenses, reduce oxidative damage at the cellular and neuronal levels, and improve cognitive function in AD mouse models. The study examined the molecular composition of Cistanche flavonoids and their impact on the Keap1-Nrf2-ARE pathway, revealing their potential in mitigating AD-related changes. By neutralizing free radicals and enhancing antioxidant defenses, Cistanche flavonoids may offer a promising approach to counteract AD pathology. This comprehensive analysis underscores their therapeutic potential in alleviating AD through oxidative stress reduction and antioxidant activation.

Keywords

Alzheimer's disease antioxidant response element Cistanche flavonoids Keap1 Nrf2

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β (Aβ) plaque deposition, tau hyperphosphorylation, synaptic dysfunction, neuroinflammation, and oxidative stress, all of which contribute to neuronal loss and cognitive decline.¹ Current therapeutic strategies, including acetylcholinesterase inhibitors (e.g., donepezil) and NMDA receptor antagonists (e.g., memantine), provide only symptomatic relief without altering disease progression.² Given the limitations of existing treatments, the search for alternative therapeutic strategies targeting oxidative stress, neuroinflammation, and protein aggregation has intensified.

Flavonoids, a class of natural polyphenolic compounds found in various plants, have emerged as promising neuroprotective agents in AD treatment.³ Their therapeutic effects stem from their potent antioxidant properties, which neutralize reactive oxygen species (ROS) and enhance endogenous cellular defense mechanisms.⁴ In addition, flavonoids exhibit anti-inflammatory activity by modulating microglial activation and inhibiting the release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Furthermore, certain flavonoids interfere with Aβ aggregation and tau hyperphosphorylation, two key pathological processes in AD.⁵ Studies have demonstrated that flavonoids such as quercetin, luteolin, and epigallocatechin gallate (EGCG) attenuate Aβ-induced neurotoxicity, reduce tau phosphorylation, and enhance synaptic plasticity, thereby preserving cognitive function in AD models.⁶

Among the diverse sources of flavonoids, Cistanche deserticola, a traditional medicinal herb, is particularly rich in bioactive flavonoids with demonstrated neuroprotective properties.⁷ Cistanche flavonoids are a specific subset of flavonoids derived from Cistanche deserticola, distinguished by their unique glycosylation patterns and structural diversity.⁸ While flavonoids as a broader class are widely found in fruits, vegetables, tea, and medicinal plants, Cistanche deserticola flavonoids include luteolin glycosides, apigenin derivatives, and other conjugated polyphenols, which differ in their metabolic stability, bioavailability, and pharmacokinetics.⁹ These structural differences influence their interaction with cellular targets, particularly in their ability to cross the blood-brain barrier and exert neuroprotective effects in AD.¹⁰

Functionally, both general flavonoids and Cistanche flavonoids share the ability to activate the Keap1-Nrf2-ARE pathway, but Cistanche flavonoids are proposed to have a more targeted and sustained effect on Keap1 inhibition, leading to prolonged Nrf2 activation.¹¹ While flavonoids from Ginkgo biloba, green tea, and Scutellaria baicalensis act through multiple signaling pathways such as PI3 K/Akt, MAPK, and NF-κB in addition to Keap1-Nrf2-ARE, Cistanche flavonoids are primarily studied for their potent effects on oxidative stress regulation through direct Keap1 interaction.¹² This pathway is a crucial cellular defense mechanism that upregulates the expression of antioxidant enzymes such as SOD, heme oxygenase-1 (HO-1), and GPx, protecting neurons from oxidative damage and apoptosis.¹³

Our study specifically examines whether Cistanche flavonoids can modulate the Keap1-Nrf2-ARE pathway to mitigate oxidative stress and improve cognitive function in AD models. The extract used in this study was derived from Cistanche deserticola and Cistanche tubulosa, including Cistanche Flavonoid Extract (CFE) as a crude flavonoid extract and specific isolated flavonoid compounds, allowing for a comparative evaluation of their neuroprotective efficacy. By investigating the molecular mechanisms through which CFE exerts neuroprotective effects, this study aims to provide new insights into its potential as a therapeutic agent for AD.

Structure and biological functions of Cistanche

Cistanche is a parasitic plant belonging to the Orobanchaceae family, predominantly distributed in arid and semi-arid regions of China, Xinjiang, Inner Mongolia, Gansu, Qinghai, Mongolia, and Kazakhstan.¹⁴ The commonly studied species include Cistanche deserticola and Cistanche tubulosa.¹⁵ Cistanche tubulosa primarily parasitizes Tamarix spp. (red tamarisk) and is distributed in Xinjiang and other regions.¹⁶ Due to its high flavonoid content, it has been extensively researched. Different species of Cistanche exhibit variations in chemical composition and biological activity, particularly in the composition and concentration of flavonoid compounds Tables 1 and 2.¹⁷

Table 1.

Flavonoid content and bioactive components of different Cistanche species.

Species	Host Plant	Main Bioactive Components	Flavonoid Content (%)
Cistanche deserticola	Haloxylon ammodendron (white saxaul)	Flavonoids (luteolin, quercetin), phenylethanoid glycosides (acteoside)	1.2–2.8%
Cistanche tubulosa	Tamarix spp. (red tamarisk)	Flavonoids (rutin, isorhamnetin), phenylethanoid glycosides, polysaccharides	0.9–2.5%
Cistanche salsa	Tamarix spp. (red tamarisk)	Steroidal saponins, iridoids	0.6–1.8%
Cistanche sinensis	Tamarix spp. (red tamarisk)	Flavonoids (luteolin, quercetin)	1.0–2.3%

Table 2.

Flavonoid composition and content in Cistanche extract.

Major Flavonoid	Molecular Formula	Content (mg/g)
Luteolin	C₁₅H₁₀O₆	5.2–12.8
Quercetin	C₁₅H₁₀O₅	4.5–10.2
Baicalein	C₁₅H₁₀O₅	3.8–9.5
Rutin	C₂₇H₃₀O₁₆	2.3–8.7
Isorhamnetin	C₁₆H₁₂O₇	2.0–7.1

Cistanche is rich in various bioactive compounds, including flavonoids, phenylethanoid glycosides, polysaccharides, and iridoids. Compared to flavonoids from other plant sources (such as Ginkgo biloba and Scutellaria baicalensis), Cistanche flavonoids exhibit unique glycosylation modifications, which enhance their solubility and bioavailability.¹⁸ Consequently, they can more easily penetrate the blood-brain barrier, thereby exerting their effects within the central nervous system.¹⁹

Cistanche flavonoid extract and its composition

In this review, most studies referenced utilized CFE. CFE is a standardized extract obtained from the stems of Cistanche deserticola and Cistanche tubulosa through ultrasound-assisted extraction (UAE) or supercritical fluid extraction (SFE). The plant material is dried, ground into powder, and subjected to high-performance liquid chromatography (HPLC) purification before being concentrated into a standardized extract.

CFE primarily consists of the following flavonoids, with their respective concentrations (per dry weight).^20–22

Among these, luteolin and quercetin are the predominant flavonoids in CFE, accounting for approximately 40–50% of the total flavonoid content. These compounds play a critical role in regulating the Keap1-Nrf2-ARE signaling pathway. Additionally, CFE contains minor amounts of other flavonoids, such as myricetin and naringenin, although their concentrations are relatively low.²³ Since this study focuses on the neuroprotective effects of Cistanche flavonoids, all references to “Cistanche Flavonoids” in this paper specifically refer to CFE, which consists of a complex mixture of multiple flavonoids rather than isolated single flavonoid compounds.

The biological roles of Cistanche flavonoids

Antioxidant activity

Cistanche flavonoids exert antioxidant effects through multiple mechanisms, including direct ROS scavenging, enhancement of endogenous antioxidant enzyme activity (such as SOD, GPx, and CAT), and regulation of the Nrf2 signaling pathway.²⁴ Their antioxidant capacity is closely linked to their molecular structure, where specific substituents enhance free radical scavenging ability and mitigate oxidative damage.²⁵ At the cellular level, Cistanche flavonoids promote Nrf2 nuclear translocation, leading to the activation of phase II detoxifying enzymes and strengthening antioxidant defenses, thereby reducing ROS-induced neuronal damage.¹² Additionally, these compounds chelate metal ions (such as Fe²⁺ and Cu²⁺), inhibiting the Fenton reaction and further suppressing oxidative stress.²⁶ Collectively, these mechanisms underscore the potential of Cistanche flavonoids in the prevention and treatment of neurodegenerative diseases.

Anti-inflammatory activity

The role of inflammatory reactions is pivotal in the progression of several neurodegenerative conditions, AD included.²⁷ The flavonoids found in Cistanche are acknowledged for their notable anti-inflammatory properties in various manners.²⁸ Importantly, these flavonoids decreased the amounts of NADPH oxidase and NLRP3 inflammasomes in the hippocampus of AD model rats induced by Aβ_1–42, leading to a significant reduction in the pro-inflammatory cytokines FL-1β and TNF-α. Furthermore, Cistanche flavonoids impede the activation of microglia and astrocytes, essential for neuroinflammatory reactions, thus diminishing the neuroinflammatory effect.²⁹ Diverse anti-inflammatory properties of Cistanche flavonoids underscore their efficiency in combating neurodegenerative conditions Figures 1–4.³⁰

Figure 1.

This case shows that flavonoids play a pivotal role in the regulation of numerous biological activities, showcasing their effects across various pathways, including anti-inflammatory and anti-apoptotic properties, and their role as strong antioxidants in mitigating oxidative stress during AD.

Figure 2.

Domains of the Keap1 protein.

Figure 3.

Domains of the Nrf2 protein.

Figure 4.

Cistanche flavonoids, including phenylethanoid glycosides, luteolin and rutin, and polysaccharides, collectively activate the Keap1-Nrf2-ARE signaling pathway. Activation of this pathway enhances neuronal antioxidant defense, modulates neuroinflammation, and attenuates oxidative stress and aging phenotypes, ultimately contributing to the amelioration of Alzheimer's disease.

Neuroprotective activity

The Cistanche-manufactured flavonoids are greatly advantageous for neural health, particularly in protecting against neurodegenerative diseases like AD and Parkinson's disease.³¹ It is recognized that these substances trigger the production of brain-derived neurotrophic factor (BDNF) along with its receptor, tyrosine kinase receptor B (TrkB), by activating the brain-derived neurotrophic factor (BDNF)/TrkB signaling route.³² This kind of stimulation improves the longevity of neurons and augments their synaptic adaptability, both critical components for maintaining cognitive functions. Furthermore, Cistanche flavonoids demonstrate brain protection by reducing levels of pro-apoptotic proteins like Bax, Caspase-9, and Caspase-3, and increasing levels of the non-apoptotic protein Bcl-2.³³ Via these systems, flavonoids play a crucial role in diminishing Aβ_1–42-triggered apoptosis in neurons, underscoring their medical promise in lessening neurodegenerative conditions.³⁴

Other biological activities

Beyond their protective effects on the brain, Cistanche flavonoids perform numerous biological functions, encompassing those for diabetes, heart, and cognition.³³ The primary reasons behind their effectiveness against diabetes are linked to preventing the creation of advanced glycation end products and enhancing insulin sensitivity.³⁵ Regarding protecting the cardiovascular system, these flavonoids aid in lessening atherosclerosis through obstructing the growth of vascular smooth muscle cells and diminishing platelet clustering.³⁶ Furthermore, Cistanche flavonoids possess the potential to improve cognitive abilities, positioning them as potential options for both preventing and treating a range of chronic illnesses.³⁷

The Keap1-Nrf2-ARE signaling pathway

The Keap1-Nrf2-ARE pathway is a crucial cellular pathway for coping with oxidative stress and is essential in preserving redox balance and in the control of antioxidant gene expression.³⁸

Keap1 (Kelch-like ECH-associated protein 1) serves as a negative regulator of Nrf2 (nuclear factor erythroid 2-related factor 2) by binding to Nrf2 in the cytoplasm, thus promoting its ubiquitination and subsequent degradation via the proteasome system, keeping Nrf2 levels low under normal physiological conditions. Keap1 achieves this by interacting with the Neh2 domain of Nrf2 through its Kelch domain, forming a Keap1-Nrf2 complex. This complex recruits the Cullin 3 (Cul3) E3 ubiquitin ligase, facilitating the ubiquitination of Nrf2 and leading to its proteasomal degradation.³⁹

However, under conditions of oxidative stress, specific cysteine residues inKeap1, such as Cys151, Cys273, and Cys288, undergo oxidative modifications. These modifications result in conformational changes in Keap1, which disrupt its interaction with Nrf2. Consequently, Nrf2 escapes ubiquitination, accumulates in the cytoplasm, and translocates to the nucleus. Inside the nucleus, Nrf2 forms a heterodimer with small Maf proteins and binds to the ARE located in the promoter regions of various antioxidant and detoxifying genes.⁴⁰ This interaction induces the expression of several critical genes, such as NAD(P)H quinone dehydrogenase 1 (NQO1), HO-1, and thioredoxin reductase 1 (TrxR1).⁴¹ The proteins encoded by these genes strengthen the cellular antioxidant defense system, effectively reducing cellular damage caused by oxidative stress. The pathway between Keap1 and Nrf2-ARE is instrumental in preventing and treating numerous diseases, such as neurodegenerative conditions, cancer, and cardiovascular diseases.⁴² Altering this route augments cell antioxidant protection, lessens harm from oxidative stress, and guards against numerous disease conditions.⁴¹

The structure and composition of the Keap1-Nrf2-ARE signaling pathway

Structure and function of Keap1

Keap1, rich in cysteine, is composed of 624 amino acids segmented into five separate structural sections. The protein's structural integrity is reinforced by the N-terminal region (NTR, 1–60) and the BTB structural domain (61–178), which facilitates the homodimerization of Keap1 and its interaction with theCul3 E3 ubiquitin ligase complex，thus aiding in the formation of the operational E3 ligase complex, crucial for Nrf2 ubiquitination and ensuing breakdown; focusing on the intermediate zone (IVR, 179–321), characterized by a dense presence of cysteine residues, which are extremely reactive to oxidative changes, making them crucial for detecting oxidative stress and for the controlled release of Nrf2; the domain for double-glycine repetition (DGR, 322–608), termed the Kelch structural domain, engages with the Nrf2 Neh2 structural domain, This entity firmly attaches to the ETGE and DLG motifs within the Neh2 structural domain; in addition to the C-terminal area (CTR, 609–624), a factor that bolsters and improves the operational soundness of the Keap1 protein.^43,44

Keap1 interacts with the Neh2 domain of Nrf2 via its Kelch domain, resulting in the formation of the Keap1-Nrf2 complex. Under physiological conditions, Keap1 facilitates the ubiquitination and subsequent proteasomal degradation of Nrf2, ensuring that Nrf2 levels remain low.

Structure and function of Nrf2

The transcription factor Nrf2, comprising 605 amino acids, is a member the basic leucine zipper (bZIP) transcription factor family. This entity encompasses seven Nrf2-ECH homology domains (Neh1-Neh7), each playing a distinct role in the function of Nrf2. Within the Neh1 domain lies the bZIP matrix, which aids in binding DNA and creating a heterodimer with the diminutive Maf protein.⁴⁵ The Neh2 domain, equipped with DLGE and ETGE patterns, engages with Keap1's Kelch domain in the process of ubiquitination and breakdown of Nrf2.Situated at the C-terminal, the Neh3 structural domain significantly contributes to activating transcription.⁴⁶ Structural areas of Neh4 and Neh5 engage with CREB-binding protein (CBP), aiding in the movement to the nucleus and the transcription function of Nrf2.

Several serine residues in the Neh6 structural domain are identified by β-TrCP, which results in the negative regulation of Nrf2. Conversely, the Neh7 domain's interaction with retinoic X receptor alpha (RXRα) significantly restricts the biological function of Nrf2.⁴⁷

Under the influence of oxidative stress, the cysteine residues of Keap1 undergo a series of modifications, leading to the dissociation of Keap1 from Nrf2. Consequently, Nrf2 translocates to the nucleus, where it triggers the activation of antioxidant genes.

Structure and function of antioxidant response element

The ARE is a cis-acting element located upstream of the promoter regions of certain cytoprotective genes. It is responsible for triggering oxidative stress responses by inducing the expression of phase II detoxifying enzymes and antioxidant enzymes. The core sequence of ARE typically is 5′- (G/A)TGA(G/C)XXXGC(G/A)-3′, with slight variations observed in different cell types.⁴⁸

Biological functions of the Keap1-Nrf2-ARE signaling pathway

Antioxidative stress response

Under normal physiological conditions, Keap1 binds to the Neh2 domain of Nrf2 through its Kelch domain, forming the Keap1-Nrf2 complex.⁴⁹ This complex facilitates the ubiquitination and degradation of Nrf2 via the Cul3 E3 ubiquitin ligase, maintaining low intracellular levels of Nrf2.⁵⁰ However, under oxidative stress, critical cysteine residues in Keap1, including Cys151, Cys273, and Cys288, undergo oxidative modification. This modification disrupts the binding between Keap1 and Nrf2, allowing Nrf2 to escape degradation and accumulate in the nucleus.⁵¹

Activation of antioxidant gene expression

Within the nucleus, Nrf2 forms a heterodimer with small Maf proteins and binds to the ARE, thereby triggering the expression of a series of antioxidant and detoxifying genes, such as NAD(P)H,NQO1, HO-1, and TrxR1.⁵² Proteins produced by these genes bolster cellular antioxidant protections and lessen the damage from oxidative stress, thereby offering a protective role.⁵³

Neuroprotective effect

The role of the Keap1-Nrf2ARE pathway is vital in the prevention and management of neurodegenerative disorders. Studies show that activating Nrf2 substantially reduces apoptosis in neurons activated by oxidative stress.⁵⁴ Case analyses of Parkinson's disease revealed that activating Nrf2 helps lessen brain injuries caused by α-synuclein (α-SYN) toxicity.⁵⁵ Additionally, Nrf2 plays avital role in reducing cerebrovascular issues by modifying the blood-brain barrier, protecting the brain from trauma.⁵⁶

Clinical applications of the Keap1-Nrf2-ARE signaling pathway

The Keap1-Nrf2-ARE pathway serves as a crucial regulatory mechanism in cellular defense, significantly contributing to safeguarding against oxidative harm and electrophilic toxicity.⁵⁷ Triggering the ARE boosts antioxidant enzymes, thus safeguarding cells against harm.⁵⁸ Research indicates that for neurodegenerative diseases like AD and Parkinson's disease, stimulating this process can diminish disease severity and improve mental capacities by lessening oxidative stress, curtailing inflammation, and averting the death of neuronal cells.

In the context of heart diseases, this approach demonstrates medical value by reducing damage from heart muscle ischemia-reperfusion and preventing atherosclerotic plaque development.⁵⁹ When cancer is being treated, controlling Nrf2 activation plays a crucial role in shielding regular cells from the oxidative damage caused by chemotherapy.⁶⁰ The Keap1-Nrf2-ARE pathway is crucial in managing a range of diseases, encompassing diabetes, COPD, autoimmune diseases, and liver-related ailments.³⁸ Diabetes serves as a case study, where triggering Nrf2 boosts antioxidant activity and lessens oxidative harm to pancreatic cells; similarly, in COPD, activating Nrf2 lessens oxidative stress and inflammation; Furthermore, in autoimmune and hepatic disorders, triggering Nrf2 plays a role in reducing inflammation and shielding liver cells against harm.⁶¹ Studies highlight the complex function of the Keap1-Nrf2ARE pathway in medical settings, especially in managing and preventing diseases linked to oxidative stress.³⁸ Subsequent research ought to concentrate on creating methods to specifically alter this route and enhance its efficiency in regulating illnesses.

Active compounds from Cistanche improve AD via activation of the Keap1-Nrf2-ARE pathway

In recent years, as our understanding of the pathogenesis of AD has deepened, signaling pathways that regulate oxidative stress and neuroinflammation have become key targets in natural product-based therapeutic research.⁶² Among them, the Keap1-Nrf2-ARE signaling pathway has emerged as a central regulatory axis for cellular defense against oxidative damage and inflammatory insults, and its role in neurodegenerative diseases is receiving increasing attention. Numerous studies have confirmed that various active compounds from Cistanche species can activate this pathway through different molecular targets, thereby exhibiting neuroprotective effects in a variety of experimental models.

Mechanistically, these compounds modulate the interaction between Keap1 and Nrf2, facilitating the release of Nrf2 from the cytoplasm and promoting its nuclear translocation. Once in the nucleus, Nrf2 binds to AREs within the promoter regions of target genes, thereby initiating the transcription of protective enzymes such as HO-1, NQO1, and GCLC.^43–45 This cascade collectively enhances cellular resilience against oxidative stress, inflammation, and apoptosis, and has become a key mechanistic basis for the pharmacological actions of Cistanche.

Accordingly, the following sections will focus on representative phenylethanoid glycosides, flavonoids, and polysaccharides isolated from Cistanche, examining their regulatory effects on the Keap1-Nrf2-ARE pathway and elucidating the underlying molecular mechanisms by which they exert neuroprotective actions in AD–related pathological models.

Phenylethanoid glycosides from Cistanche enhance neuronal antioxidant defense via activation of the Keap1-Nrf2-ARE pathway

Species of the genus Cistanche, particularly Cistanche deserticola and Cistanche tubulosa, are rich in bioactive PhGs, including echinacoside and acteoside, which have demonstrated significant neuroprotective effects in multiple experimental models.⁶³ PhGs are among the primary active constituents of Cistanche, predominantly concentrated in the succulent stems of the plant. Structurally, these compounds are composed of hydroxyphenylethanol moieties conjugated with multiple sugar residues, conferring high water solubility and biological activity.⁶⁴ Echinacoside and acteoside, as representative PhGs, are abundant in Cistanche and exhibit potent antioxidative and neuroprotective properties under oxidative stress conditions.

Yao et al. demonstrated that PhG-rich extracts significantly upregulated Nrf2 protein expression in the hippocampus and substantia nigra of MPTP-induced Parkinsonian mice. Furthermore, transcriptional upregulation of canonical Nrf2 downstream antioxidant enzymes such as HO-1 and NQO1 was observed. Since the promoter regions of HO-1 and NQO1 contain conserved AREs, their enhanced expression implies successful Nrf2 nuclear translocation and binding to ARE, a hallmark of Keap1-Nrf2-ARE pathway activation. The concomitant upregulation of Nrf2 and its transcriptional targets reflects the functional engagement of the entire signaling cascade from Keap1-mediated stress sensing to transcriptional response.⁶⁵

In another in vitro study, Jia et al. (2019) reported that echinacoside treatment protected PC12 neuronal cells from H₂O₂-induced oxidative stress in an in vitro model. This intervention significantly promoted the nuclear translocation of Nrf2 and increased the expression of HO-1 and GCLC, both of which are classic ARE-regulated genes. The transcriptional activation of these genes is contingent upon Nrf2’s dissociation from Keap1 and subsequent nuclear localization. These findings corroborate echinacoside's role in activating Nrf2 and, by extension, the Keap1-Nrf2-ARE axis.⁶⁶

Acteoside, another major PhG in Cistanche, has also been reported to activate this pathway. According to a study, Zhang et al. showed that acteoside can bind directly to the Kelch domain of Keap1, competitively disrupting the Keap1–Nrf2 interaction. This inhibition impedes Keap1-mediated ubiquitination and proteasomal degradation of Nrf2, thereby facilitating its cytoplasmic accumulation and nuclear translocation. In the nucleus, Nrf2 heterodimerizes with small Maf proteins and binds to ARE regions, initiating transcription of antioxidative genes such as HO-1 and NQO1. This molecular mechanism provides direct evidence of Keap1-Nrf2-ARE pathway activation by acteoside at the protein–protein interaction level. Intriguingly, this study also revealed that the number of glycoside moieties in PhG structures positively correlates with their Keap1 binding affinity and Nrf2 activation potency. This structure–activity relationship offers a theoretical foundation for the future rational design of optimized PhG-based neuroprotectants.⁶⁷

In summary, PhGs from Cistanche, particularly echinacoside and acteoside, exert neuroprotective effects by activating the Keap1-Nrf2-ARE signaling pathway, enhancing neuronal antioxidant capacity and mitigating oxidative stress-induced damage. While current evidence is largely derived from Parkinson's disease models, the mechanistic insights strongly support further exploration of this pathway in AD and related neurodegenerative disorders.

Flavonoids from Cistanche modulate neuroinflammation through the Keap1-Nrf2-ARE pathway

Cistanche species are known to contain flavonoids such as luteolin and rutin, which have demonstrated neuroprotective effects through well-characterized antioxidant and anti-inflammatory mechanisms. Increasing evidence suggests that these effects are mediated via the Keap1-Nrf2-ARE signaling pathway.

Kempuraj et al. found that luteolin exerted potent anti-inflammatory effects in both neurotrauma and neuroinflammation models by targeting the Nrf2 axis. Specifically, luteolin stabilized Nrf2 in the cytoplasm, promoted its nuclear translocation, and enhanced its binding to ARE. This cascade subsequently induced the transcription of key cytoprotective genes such as HO-1 and NAD(P)H quinone dehydrogenase 1 (NQO1), which play critical roles in cellular antioxidant and anti-inflammatory defense. These molecular changes led to the suppression of activated microglia and a significant reduction in pro-inflammatory cytokines including TNF-α and IL-1β, thereby mitigating inflammatory damage within the central nervous system.⁶⁸

Similarly, Li et al. demonstrated that rutin exerted neuroprotective effects against lead-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by activating the Nrf2/ARE pathway. Upon treatment with rutin, Keap1 expression was significantly reduced, while Nrf2 protein levels were upregulated. This led to enhanced nuclear translocation of Nrf2 and subsequent activation of ARE-driven transcription of downstream antioxidant enzymes, including GCLC, HO-1, and NQO1. These molecular events suppressed oxidative stress and downregulated the expression of inflammatory mediators, ultimately leading to reduced apoptosis and improved neuronal viability.⁶⁹

These findings provide compelling mechanistic evidence that flavonoid compounds found in Cistanche, such as luteolin and rutin, can modulate neuroinflammation by directly engaging and activating the Keap1-Nrf2-ARE signaling pathway, thereby enhancing cellular antioxidant defenses and attenuating pro-inflammatory cascades.

Polysaccharides from Cistanche attenuate oxidative stress and aging phenotypes via the Keap1-Nrf2-ARE pathway

Cistanche deserticola, a traditional medicinal herb, contains abundant bioactive polysaccharides (CDPs) in its succulent stem tissues.⁷⁰ In recent years, CDPs have been shown to exhibit significant antioxidant, anti-aging, and anti-inflammatory effects, with increasing evidence linking these functions to the activation of the Keap1-Nrf2-ARE signaling pathway.

In a study by Hu et al. (2020), published in Journal of Cellular and Molecular Medicine, CDPs significantly promoted Nrf2 nuclear translocation and enhanced the transcription of its downstream antioxidant enzymes such as HO-1 and GCLC in melanocytes. Nrf2 stability in the cytoplasm is controlled by the inhibitory protein Keap1, which binds to the ETGE and DLG motifs of Nrf2 and facilitates its ubiquitination and proteasomal degradation. The study demonstrated that CDPs likely interact with Keap1, modifying its redox-sensitive cysteine residues, thereby weakening Keap1-mediated suppression of Nrf2. As a result, Nrf2 is released from Keap1, translocates into the nucleus, and binds to AREs, initiating transcription of antioxidant genes. These findings clearly indicate that the antioxidative actions of CDPs are mediated by the activation of the canonical Keap1-Nrf2-ARE pathway.⁷¹

Furthermore, Takaya et al. (2023) reported that CDPs markedly improved aging phenotypes in human dermal fibroblasts. The study observed that CDPs decreased the expression of aging-associated markers such as p21 and reduced SA-β-galactosidase activity. Meanwhile, levels of inflammatory cytokines IL-1β and TNF-α were suppressed. These effects coincided with enhanced Nrf2 expression and nuclear translocation, along with elevated HO-1 and NQO1 expression. To determine whether these effects were dependent on Nrf2, the authors employed siRNA-mediated knockdown of Nrf2, which significantly attenuated the CDP-induced antioxidant and anti-aging responses. The downregulation of HO-1 and NQO1 expression and resurgence of SA-β-gal activity confirmed that these protective effects were contingent upon an intact Keap1-Nrf2-ARE axis.⁷²

Together, Cistanche polysaccharides activate the Keap1-Nrf2-ARE signaling pathway by disrupting Keap1-mediated suppression of Nrf2, promoting its nuclear translocation, and inducing downstream antioxidant and anti-inflammatory gene expression. This activation strengthens cellular defense against oxidative stress, mitigates inflammatory responses, and delays cellular senescence—providing strong mechanistic support for the potential application of CDPs in the prevention or treatment of neurodegenerative diseases such as AD.

Conclusion and perspective

In this review, we have explored the neuroprotective potential of CFE, derived from Cistanche deserticola and Cistanche tubulosa, in mitigating AD pathology through modulation of the Keap1-Nrf2-ARE signaling pathway. CFE exerts its beneficial effects primarily by enhancing antioxidant defenses, suppressing neuroinflammation, and regulating amyloid and tau pathology, which are key contributors to AD progression.

CFE's antioxidant properties are attributed to its flavonoid constituents, including luteolin, quercetin, and isorhamnetin, which interact with Keap1 to promote Nrf2 nuclear translocation and activation. This process leads to the upregulation of antioxidant enzymes such as SOD, GPx, and HO-1, reducing oxidative stress and preserving neuronal integrity. CFE's anti-inflammatory effects involve the suppression of NF-κB signaling, thereby reducing the expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, which play a critical role in neuroinflammatory damage. Additionally, CFE modulates Aβ metabolism and tau pathology, preventing the formation of neurotoxic aggregates by enhancing Aβ clearance through IDE and NEP upregulation and inhibiting tau hyperphosphorylation via GSK-3β suppression.

While preclinical studies strongly support the therapeutic efficacy of CFE in AD models, several challenges remain before it can be translated into clinical applications. Future research should focus on elucidating its precise molecular mechanisms, optimizing its bioavailability, and determining its long-term safety in human trials. Additionally, clinical studies are needed to assess its pharmacokinetics, dosage, and potential synergistic effects with existing AD treatments.

By targeting multiple pathological pathways through Keap1-Nrf2-ARE activation, CFE represents a promising multi-target approach to AD therapy. Further investigations will be crucial in establishing CFE as a viable therapeutic candidate, potentially offering a natural and effective intervention for AD management.

Footnotes

Acknowledgments

I, Ruiqi Zhao, would like to express my heartfelt gratitude to my mother, Renfei Han, and my grandmother, Longzhi Wong, for their unwavering support and encouragement throughout the process of this research and writing. I also acknowledge the assistance of the AI tool ChatGPT (developed by OpenAI, San Francisco, USA) for its help in polishing the language of the manuscript. The tool was used solely to improve clarity and fluency in expression; all academic ideas, data interpretation, and final conclusions were independently formulated by the author. The content of this manuscript has been carefully reviewed by the author to ensure accuracy and complies fully with the Journal of Alzheimer's Disease and SAGE Publications’ policies regarding the ethical use of AI. Figdraw was used to generate the final figure.

Author contributions

Ruiqi Zhao: Software; Visualization; Writing – original draft; Writing – review & editing.

Wan fu Bai: Supervision; Writing – review & editing.

Zhenyu Tian: Validation; Writing – review & editing.

Xinye Li: Writing – review & editing.

Ruijun Sun: Software; Visualization; Writing – review & editing.

Ziqi Wang: Visualization.

Xusheng Yan: Funding acquisition; Writing – review & editing.

Jian-Xin Jia: Funding acquisition; Resources; Supervision.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (Grant No. 82360264, No. 82460834); the Natural Science Foundation of Inner Mongolia Autonomous Region (Grant No. 2024MS08051, No. 2025LHMS08049, No. 2024MS08025); the Inner Mongolia Public Hospital Research Joint Fund Science and Technology Project (Grant No. 2024GLLH0791, No. 2023GLLH0183); the “Hua Lei” Program of Baotou Medical College (Grant Nos. HLJH202414, HLJH202507, HLJH202525, HLJH202508); and the College Student Innovation and Entrepreneurship Training Program of Baotou Medical College (Grant Nos. S202410130019X, S202410130021X, S202410130002, S202510130022, S202510130003).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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