Role of the transcription factor NRF2 in maintaining the integrity of the Blood-Brain Barrier

Abstract

Background

The Blood-Brain Barrier (BBB) is a complex and dynamic interface that regulates the exchange of molecules and cells between the blood and the central nervous system. It undergoes structural and functional throughout oxidative stress and inflammation, which may compromise its integrity and contribute to the pathogenesis of neurodegenerative diseases.

Main body

Maintaining BBB integrity is of utmost importance in preventing a wide range of neurological disorders. NRF2 is the main transcription factor that regulates cellular redox balance and inflammation-related gene expression. It has also demonstrated a potential role in regulating tight junction integrity and contributing to the inhibition of ECM remodeling, by reducing the expression of several metalloprotease family members involved in maintaining BBB function. Overall, we review current insights on the role of NRF2 in addressing protection against the effects of BBB dysfunction, discuss its involvement in BBB maintenance in different neuropathological diseases, as well as, some of its potential activators that have been used in vitro and in vivo animal models for preventing barrier dysfunction.

Conclusions

Thus, emerging evidence suggests that upregulation of NRF2 and its target genes could suppress oxidative stress, and neuroinflammation, restore BBB integrity, and increase its protection.

Background

The Blood-Brain Barrier (BBB) forms a multicellular vascular unit that separates the central nervous system (CNS) from the peripheral blood circulation. This structure is composed of endothelial cells, pericytes, smooth muscle cells, astrocytes, and neurons [1], which together control the selective passage of small molecules, metabolites, and cells to the brain parenchyma, maintaining an environment suitable for brain homeostasis [2, 3]. It also provides protection from pathogens, inflammation, injury and disease [4]; as well as regulating the cerebral blood flow (CBF) [1]. All these cell types constitute the neurovascular unit (NVU), a term often used to refer to the functional unit of the BBB, enabling paracrine regulations [5]. Maintenance of the CBF is key to the clearance of toxic by-products of brain activity, such as metabolic waste [6, 7].

The BBB is not an impermeable barrier, but due to the presence of several specialized and diverse transport and channels, it can mediate the transport of ions, glucose, amino acids, and well-defined substrates [5]. BBB limits the entry of plasma components and leukocytes under healthy conditions. However, upon endothelial damage, the integrity of the BBB is compromised, allowing for the entry of said cells. Therefore, it’s key to protect brain endothelial cells (BEC) to maintain barrier function [5]. The BBB also releases a series of diffusible signals (nitrogen monoxide (NO), prostanoids, adenosine, ions, etc.) to regulate cerebral blood flow [1]. Loss of these barrier properties is highly linked to neurological diseases, including stroke, multiple sclerosis, traumas, and neurodegenerative disorders [3, 4].

NRF2 (Nuclear factor erythroid-related Factor 2) is the main regulator of cellular defense against oxidative and inflammatory stress [8, 9]. It regulates the expression of ~ 250 genes that present regulatory sites termed Antioxidant Response Elements (ARE). Many of the ARE-containing genes are involved in maintaining the glutathione and thioredoxin antioxidant defense systems, iron and drug metabolism, the detoxification of oxidants that could alter the redox states, and genes implicated in the repair of damaged tissue [10, 11]. In addition, the NRF2 pathway is involved in proliferation, immune cell migration, and anti-inflammatory processes [12,13,14].

Lack of NRF2 transcriptional activity compromises BBB integrity and increases inflammatory markers, including vascular adhesion molecules and pro-inflammatory cytokines, while also disrupting the redox and metabolic functions of endothelial cells [10, 15, 16]. In vitro models, have demonstrated a BBB alteration in NRF2-knockout samples, along with reduced expression of tight junction (TJ) proteins such as Occludin and Claudin-5 [17, 18] and adherens junction proteins such as VE-Cadherin. Furthermore, vivo models also provide evidence that in Nrf2-deficient mice, there is compromised BBB integrity, leading to leakage and an increased extravasation of plasma constituents from to vessel to into the brain parenchyma. This is also associated with an exacerbated neuroinflammatory response, showing an increased number of activated microglia and upregulation of inflammatory mediators (e.g., IL-1β) [19].

Conversely, the activation of the NRF2/ARE system can potentially prevent/reduce BBB impairment and, consequently, decrease some types of brain injury in astrocytes and BEC, which resulted in moderate peroxynitrite generation, protecting against BBB disruption [20]. In this manner, NRF2 can also serve as a potent therapeutic agent to diminish CNS injury-induced brain damage upon increasing BBB integrity. Thus, in this review, we discuss the role of NRF2 in protecting and maintaining against BBB disruption for the prevention of neurological disorders.

Features of the Blood-Brain Barrier

BBB blood vessels are comprised of several cell types, endothelial cells (EC) which make up the walls of blood vessels, mural cells, which sit on the abluminal surface of the EC layer, astrocytes and neurons (Fig. 1). The properties of BBB are mainly derived from the ECs but are induced and maintained by the interaction with astrocytes, smooth muscle cells, pericytes, and neural cells [4].

One of the distinguishing features of the BBB is the unique properties of its endothelial cells [21] which form a tightly sealed monolayer [5] and exhibit characteristics that differ from endothelial cells in other tissues, ensuring proper substance movement and maintaining barrier function [4]. Key differences include the presence of continuous intercellular TJ and adherens junctions (AJs), the absence of fenestrations, and low rates of transcytosis [22, 23]. These features limit both paracellular and transcellular movement of molecules [2, 4], while selective transporters and receptors facilitate regulated transport.(Fig. 1) [24,25,26]. Additionally, endothelial cells at the BBB secrete vasodilators and vasoconstrictors to regulate vascular tone [27].

BBB TJs allow for a high-resistance paracellular barrier to molecules and ions, polarizing the luminal and abluminal sides [4]. These TJs are present in the apical lateral membrane [28] and are linked to the cytoskeleton through interactions with cytoplasmic adaptors, creating paracellular barriers that, depending on local transport requirements, differ on the level of permeability (Fig. 1) [29]. TJ are protein complexes composed of integral membrane proteins, cytoplasmic plaque proteins, and cytoskeletal proteins, among these TJ-associated transmembrane proteins, claudins, occludins, JAMS, and ZO proteins are directly involved in the formation of TJ strands in endothelial cells [3, 30]. Claudin-5 is highly expressed and plays a key role in vertebrate BBB [31], tightening the barrier for many physiological active substances [25], which includes most physiologically active substances [28]. Occludin, a TJ-associated MARVEL protein (TAMP), is found ubiquitously in the TJ [32] and interacts with claudins to establish the integrity of the endothelium [33]. JAMs, part of the immunoglobulin superfamily [34], help to establish the correct localization of other junctional protections [35].

TJs also interact with basal AJs, connecting all ECs, and are composed of vascular endothelial (VE)-Cadherin and platelet EC adhesion molecules (PECAM1), linked to the cytoskeleton by catenins [5, 21, 23]. VE-Cadherin is essential for endothelial integrity in quiescent vessels and may regulate endothelial functions by activating directly signaling pathways related to survival and actin cytoskeleton as Phosphoinositide 3-Kinases (PI3K) [36].

For the establishment of the BBB, the transport of materials through transcellular pathways must be suppressed completely, therefore needing specialized transport systems in the plasma membranes of endothelial cells for the uptake of even small molecules such as glucose and amino acids by the CNS [37]. Moreover, the paracellular route is tightly sealed thanks to the barrier formed by the TJs [25]. So, by limiting the passive passage of molecules through the BBB, its exchange needs to be strictly regulated using particular transporters. For instance, Slc2a1, also called Glucose transporter 1 (GLUT1), has been largely studied for its crucial in supplying glucose to the CNS, with nutrient concentration gradients regulated according to the CNS’s metabolic needs. Other transporters are involved in adaptive responses for nutrient uptake and removing waste from the CNS to the blood [4].

BBB damage is a consequence of several pathological processes of the brain [28], and can contribute to the progression of the disease [38]. Most common brain disorders can lead to BBB disruption, which shares an impaired TJ network, imbalance of transporters and fluxes, and increased extravasation of blood cells [39].

Role of NRF2 in Blood-Brain Barrier integrity

NRF2 is a key transcription factor in the antioxidant and anti-inflammatory response [8]. It mediates the induction of phase II antioxidant defense enzymes, activating transcription through the ARE sequence present in the promoter region of target genes [40]. NRF2 possesses a bZIP-containing C-terminal domain, capable of heterodimerizing with small Musculoaponeurotic Fibrosarcoma (sMAF) proteins [41]. The heterodimers can recognize AREs located in the upstream regulatory regions of the target genes [42], which can initiate the transcription of roughly 250 genes related to cytoprotection [43]. These genes encode a network of cooperating enzymes that participate in phase I, II, and III biotransformation reactions and antioxidant processes. These processes include the generation of NADPH, glutathione (GSH), and thioredoxin; lipid and iron catabolism; and interactions with other transcription factors, among other functions [10]. NRF2 has also been shown to regulate the expression of various proteasome subunits and autophagy-related genes, highlighting its role in the control of proteostasis [44,45,46]. Some key NRF2 target genes related to antioxidant enzymes include those encoding NAD(P)H quinone oxidoreductase-1 (NQO1)), Heme oxygenase 1 (HO-1), catalytic and modulatory subunits of γ-glutamyl synthase (GCLM, GCLC), and the glutamate-cystine transporter (SLC7A11) [8, 47] playing a key role in the cellular defense system against oxidative stress [48].

NRF2 activity control mechanisms

NRF2 levels are kept low under physiological conditions with the possibility of suddenly increase them upon oxidative or inflammatory stress and this is achieved by the constitutive synthesis and degradation of NRF2 [49]. The main proteins responsible for maintaining NRF2 levels low are Kelch-like-ECH-Associated Protein 1 (KEAP1) and β-transducin repeat-containing protein (β-TrCP) [8]. KEAP1 is an E3 ligase adapter that, in unstressed conditions, degrades NRF2 through ubiquitination by recruiting the Ringbox1-Cullin 3 complex (Fig. 2). KEAP1 presents redox-active cysteine residues that react under oxidative conditions, with several electrophiles or Reactive Oxygen Species (ROS), preventing KEAP1 from degrading NRF2, leading to the stabilization and accumulation of NRF2 [50]. The main alternative pathway to KEAP1 regulation is the GSK3-β-TrCP axis. Glycogen synthase kinase (GSK-3) is a serine-threonine protein kinase that has been demonstrated to inhibit NRF2 through the ubiquitin ligase β-TrCP by phosphorylating NRF2 and forming a phosphodegron which is recognized by β-TrCP, which recruits the Ringox1-Cullin 1 ubiquitination system (Fig. 2) [51,52,53]. Thus, the constitutive degradation of NRF2 is inhibited, which allows NRF2 to accumulate within the cell, translocate to the nucleus, and regulate cytoprotective gene expression [54].

NRF2 regulation of inflammation and oxidative stress

Multiple studies have noted that imbalanced ROS levels have been implicated in endothelial vessel integrity by acting as signaling molecules that promote angiogenesis through proliferation, cell migration, and tube formation. This phenomenon involves cells losing junctions and, thereby, losing barrier properties [55]. ROS can function as low-level signaling intermediates and regulate fundamental cell activities including growth and adaptive responses [56]. However, at higher concentrations, ROS can cause cell injury and death in endothelial cell dysfunction and loss of BBB integrity [57]. Furthermore, oxidative stress has been consistently reported to occur in most neurodegenerative conditions [58], correlating with the development of cognitive and motor disturbances [59, 60].

Neuroinflammation plays a physiological role in phagocytosis and the clearance of cellular debris, pathogens, and aberrant proteins, which is beneficial for maintaining brain homeostasis unless it becomes chronic. For instance, microglial activation promotes phagocytosis of astrocytic perivascular end-feet and reduces the expression of two TJ proteins: Occludin and Claudin-5. These changes support leukocyte infiltration across the BBB and further exacerbate neuroinflammation and neurodegeneration [61]. As such, increased BBB permeability following prolonged systemic inflammation may contribute to cognitive dysfunction. In contrast, microglia can protect the BBB from inflammatory attacks by elevating the levels of anti-inflammatory factors, such as IL-10, IL-4, IL-13, and transforming growth factor-β (TGF-β) [62, 63]. Thus, depending on the perivascular microenvironment, the highly dynamic switch of microglial phenotype between pro- and anti-inflammatory states is closely linked to their impact on BBB integrity.

In addition, other key components of BBB such as astrocytes can modulate BBB development and function under physiological conditions by secreting a series of molecules that regulate immune responses. Astrocytes are also able to release extracellular vesicles containing inflammation-related proteins and miRNAs that help support a healthy BBB integrity [64]. However, chronic inflammation can cause injury to the CNS, highlighting the importance of regulating the neuroinflammatory process [65, 66]. Triggered by pathophysiological conditions, activated astrocytes disrupt the BBB components by secreting matrix metalloproteinases 9 (MMP-9) and proinflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor-α (TNFα), which leads to BBB leakage due to exacerbated inflammation [67, 68]. Moreover, neuroinflammation contributes to BBB disruption by damaging TJs, allowing leukocytes to infiltrate the brain when the barrier is compromised [69, 70].

Persistent inflammation is a hallmark of several diseases in which NRF2 mediates, likely because inflammation increases local and systemic pathologic formation of ROS [71]. In addition, ROS stimulates inflammatory response through the activation of nuclear factor k-light-chain enhancer of activated B cells (NF-κB) p65 subunit [71]. NRF2 has been extensively studied for its complex crosstalk with the NF-κB pathway, engaging in feedforward and feedback loops [15]. NF-κB is a transcription factor that plays a crucial role in regulating inflammation and suppressing ARE-dependent gene transcription [72]. However, it can also bind to the NFE2L2 gene promoter, activating NRF2 expression and establishing a negative feedback loop [73].

Initially, NRF2’s anti-inflammatory effects were attributed to its role in modulation of redox metabolism and interactions with NF-κB. However, NRF2 can also directly suppress the transcription of proinflammatory genes like interleukin-6 (IL-6) and interleukin-1β (IL-1β) in macrophages in response to lipopolysaccharide (LPS) stimulation [74]. Upon LPS exposure or pharmacological activation, NRF2 binds to the proximal promoters of these genes, blocking the recruitment of RNA polymerase II. This mechanism is independent of NRF2’s typical binding to the ARE enhancer [74].

Thus, NRF2 exhibits a pleiotropic functional role in the endogenous defense system, protecting against oxidative stress and inflammation [18, 40]. The defense against oxidate stress in the brain also acted by modulating microglial dynamics [75], regulating inflammatory signaling [47], and protecting neurons and astrocytes [40]. Conversely, the deletion or downregulation of NRF2 exacerbates cell susceptibility to the damage [18]. Vascular endothelial dysfunction and consequent CNS damage have been connected to ROS [76,77,78] and ROS-driven inflammation [79]. NRF2 activation likely preserves the BBB by maintaining ROS homeostasis, which ultimately leads to a decrease in the risk of cerebrovascular, neurodegenerative, and CNS disorders [77, 80, 81].

Several authors have reported that treatment with different NRF2-inducing compounds results in reduced inflammation due to NRF2 having an anti-inflammatory property, as seen previously in LPS-stimulated cells [72, 82, 83]. In inflammation assays with BEC, NRF2 activation is associated with antioxidative protection conferred by NQO1, alongside its anti-inflammatory effects [84]. Furthermore, astrocyte and microglia activation of NRF2 provides protection from neurotoxicity, ameliorating the oxidative stress and inflammation surrounding neurons and blood vessels [75]. Additionally, NRF2 plays a protective role in the conservation of the NVU [85,86,87] and, in conjunction with the BBB, upregulates the expression of TJs proteins [87,88,89].

NRF2 regulates intercellular binding proteins

There is strong evidence that NRF2 may regulate the imbalance that compromises BBB integrity, preventing leakage by maintaining ROS homeostasis and controlling inflammation. Further evidence supports the idea that NRF2 can also control the expression of junction proteins, as demonstrated by promoter analysis and detection of promoter activity [17, 90]. For instance, it had been found an ARE motif on the mouse Cldn5 (Claudin-5) and two electrophile response elements (EpRE) motifs have been identified on Cdh5 (VE-Cadherin) promoters. Using promoter-luciferase activity assay, they verified that both Claudin-5 and VE-Cadherin might be NRF2 direct target genes [17, 90].

TJ protections, such as occludins, claudins, and Zonula occludens-1 (ZO-1) are vulnerable to the effects of MMPs and were found in the extracellular matrix surrounding the brain endothelium after BBB disruption [91], leading to the degradation of these junctions [92]. MMPs belong to a family of enzymes implied in tissue remodeling [93]. Astrocytes can release MMPs that degrade the basal lamina proteins, exposing the TJ proteins to be hydrolyzed by MMPs contained in the blood vessels, such as MMP-9 [91]. Immune and endothelial cells can also release MMP-9 [94], to facilitate leukocyte diapedesis [95, 96]. MMP-2, -3, and − 9 are the primary MMPs present in the brain. MMP-2 and 9, compared to MMP-3, have a narrower spectrum of substrates but include basement membrane elements. MMP-2 is also expressed in astrocytic feet, supporting cerebral endothelial cells. MMP-9 is particularly harmful to BBB integrity, as it can hydrolyze extracellular matrix and TJ proteins, such as those involved in the BBB [97]. For example, after hypoxia/reperfusion MMP-9 is released by several cell types, including infiltrating neutrophils and lymphocytes, worsening the condition of the BBB integrity. Additionally, ZO-1 degradation has been linked to increased MMP activity, causing enhanced barrier permeability [98, 99].

NRF2, as an anti-inflammatory regulator, can mitigate the release of MMPs by downregulation of NF-kB and TNF-α signaling [96]. MMPs-2 and MMP-9 have been known to present a heightened activity following oxidative stress in other tissues [100], making NRF2 a key mediator in the antioxidant response. NRF2 activation also contributes to the inhibition of extracellular matrix (ECM) degradation, among them by decreasing the expression of two members of the MMP family; MMP-9 in macrophages and MMP-7 in epithelial cells of the human intestine, improving the disease state of inflammatory bowel disease [101, 102]. Consequently, during inflammatory processes, MMP production may be controlled directly by the NRF2 pathway or by the effect of NRF2 in the activation of the NF-κB pathway [101] in BBB extracellular matrix remodeling. For instance, Nrf2-deficient mice with skin damage induced by UV radiation show higher levels of MMP-9 compared to controls [103].

NRF2 regulation of transport system

Besides regulating the expression of neuroendothelial TJs, NRF2 also influences BBB endothelial glucose uptake. This transcription factor regulates metabolism by re-directing the metabolic flux through the pentose phosphate pathway [104]. Given the high energy demand of the CNS and the presence of various glucose transporters and mitochondria in the BBB endothelium [105, 106], NRF2 regulation of glucose uptake is likely critical for BBB function and integrity [18]. Impairment in endothelial glucose uptake downregulates TJ protein expression, resulting in loss of BBB integrity [107]. Therefore, NRF2 role in TJs population and glucose uptake appears to be entwined [18].

GLUT1 is one of the targets for transcriptional regulation by Hypoxia-inducible factor-1α (HIF-1α) [108], which in turn presents ARE sequences in its promoter that NRF2 can bind to promote its expression. Activation of NRF2 by metformin in tobacco smoke-induced cerebrovascular toxicity restores GLUT-1 levels [87], which correlates with increased cellular glucose uptake occurring with increased NRF2 activity [109].

NRF2 protective effects on Blood-Brain Barrier damage concerning neurological diseases

Several studies have indicated that BBB leakage contributes to neuro-disorders, related to cerebrovascular diseases, altered glycemia, neurodegeneration diseases, cigarette smoke toxicity, and sepsis, pointing out that the BBB should be considered for therapy [99, 110,111,112]. This is supported by the fact that both BBB loss of integrity and neurodegenerative disease share oxidative stress and inflammation as a factor of worse prognosis [58]. Thus, we discuss how NRF2 may regulate the imbalance of BBB integrity homeostasis in concerns of neurological diseases, related to neurovascular pathologies, hyperglycemia diseases, cigarette smoke toxicity, sepsis, and neurodegeneration.

NRF2 protective effects on cerebrovascular diseases

Cerebrovascular diseases include a reduced or altered blood flow to the brain, most commonly as a result of hypoperfusion and thrombosis, an ischemic stroke, hypertension, or traumatic brain injury, which subsequently leads to oxidative stress, hypoxia, and neuroinflammation which can be protected by NRF2 activation [113].

Ischemic/stroke

Ischemic/Stroke (I/S) occurs when a vessel supplying blood to the brain becomes obstructed and the brain is deprived of oxygen, glucose, and other essential nutrients. The pathogenesis of stroke is complex, with oxidative stress due to the overproduction of ROS and/or reactive nitrogen species (RNS) being central [114,115,116]. When the physiological balance between ROS generation and ROS clearance is disrupted and exceeds antioxidant capacity, oxidative stress occurs and is followed by cellular injury (Fig. 3).

Following a stroke and reperfusion, the cerebral vessels are key to resupplying blood flow, cerebral vessels are crucial for restoring blood flow and minimizing further tissue damage due to reduced oxygen levels [117]. However, reperfusion can lead to excessive production of ROS in the brain, including hydrogen peroxide and peroxynitrite [118]. This results in BBB destruction, edema, and brain parenchymal damage [119, 120]. Ischemic brain injury triggers a pathological cascade causing rapid and irreversible neuronal injury within the ischemic core, while the surrounding hypoperfused brain tissue, the penumbra region, can be salvaged if the blood flow is restored. A common mechanism is reperfusion from recanalized cerebral vessels but can cause cerebral edema, brain hemorrhage, and neuronal death [121]. The responses to the ischemia result in ROS accumulation which promotes, in turn, pro-inflammatory and pro-apoptotic mediators [40].

Hypoxia is a common phenomenon during this process, causing significant oxidative stress and ROS accumulation [77, 122], activating the NRF2 signaling pathway as a key regulator in antioxidant and anti-inflammatory processes (Fig. 3). A key player involved in the hypoxia response is HIF-1α, which induces the transcription of genes related to glucose metabolism, angiogenesis, erythropoiesis and cell survival [123]. HIF-1α levels are primarily elevated in the salvageable penumbra tissue [123] promoting angiogenesis, glycolysis, apoptosis and inflammation, crucial at first for brain healing after the stroke [124]. HIF-1α is described as a mediator of inflammation, which is needed to recruit leukocytes to the injury site and activate local immune cells [125]. NRF2 has been described to regulate HIF-1α activation since NRF2 upregulation led to an increase in HIF-1α pathway activity because the HIF-1α have a conserved functional ARE was identified in the promoter region of the HIF1A gene [126]. More specific causal relationships have been reported, where HIF-1α induction repressed the NRF2 transcription of HO-1 and interleukin-8 (IL-8) in endothelial cells; the decreased NRF2 activity was found to be due to the elevation of BTB Domain and CNC Homolog 1 (BACH1), a repressive partner of NRF2 [127]. Thus, the tight balance between the NRF2/HIF-1α axis regulates BBB preservation and health.

TJs are often altered and less expressed at the BBB during hypoxia [128,129,130], which increases paracellular flux, mainly by degradation and decreased expression of Claudin-5 and ZO-1 [130, 131] which can be upregulated by NRF2 to compensate it. Moreover, in in vitro BBB models, NRF2-knockdown was associated with in vitro BBB disruption evidenced by increased dextran leakage and decreased transendothelial electrical resistance [17, 18]. This was accompanied by reduced expression of TJ proteins ZO-1, Occludin, and Claudin-5 [132], and adherens junction protein VE-Cadherin [18, 133, 134]. In vascular cognitive impairment and dementia (VCID) rats, NRF2 activation increased the expression of Claudin-5 and Occludin [17]. In the stroke mice model, NRF2 could also protect against BBB disruption [20], and it was proved critical for ischemic preconditioning-afforded BBB preservation after stroke [90]. Besides, there has been reported an increase in nuclear NRF2 levels in the hippocampus following ischemia, which facilitated those responses [122], contributing to the overall neuroprotective effect of this transcription factor in this pathology [135].

Traumatic brain injury

Traumatic Brain Injury (TBI), also known as acquired intracranial injury, is caused by an external force, such as a blow, bump, or jolt to the head, or from an object suddenly impacting or piercing the skull into the brain tissue [136]. While the primary injury of trauma can cause direct damage to neuronal structures, the mechanical tissue deformation triggers secondary injury leading to BBB damage, edema, increased intracranial pressure, inflammation, and cell death [137]. Secondary injury is the progression of TBI as a long-term neurological problem by affects physically, cognitively, and emotionally which often leads to a permanent disability of the victim [138] which is characterized by a neuroinflammatory response [139]. Secondary injury is primarily due to oxidative stress causes, which include neuroinflammation, edema formation, BBB damage, cell death, and finally leads to cognitive impairments in TBI. Immediately after a brain injury, a huge quantity of inflammatory cytokines such as IL-1β, IL-6, TNF-α and TGF-β are released into the blood circulation which further exacerbates the trauma condition of the brain with oxidative stress [140, 141].

Emerging evidence suggests that oxidative stress not only contributes to TBI pathogenesis TBI but also initiates and promotes excitotoxicity, mitochondrial dysfunction, neuroinflammation, and other risks. NRF2 plays a protective role in TBI via fighting against oxidative damage and inflammatory response in TBI (Fig. 3) [142], and modulating microglial and macrophage function [139], by blocking the expression of proinflammatory cytokines [74]. NRF2 also suppresses reactive astrocyte activation and mitigates neuroinflammation and ferroptosis by inhibiting NF-κB, providing neuroprotective effects [143, 144]. Conversely, NRF2-genetic deletion delays the recovery of motor and cognitive functions post-TBI [145]. NRF2 activation plays a crucial role in mitigating ferroptosis by decreasing intracellular iron levels by upregulating the expression of ferritin heavy chain (FTH1) for iron storage and ferroportin (FPN) for iron efflux [146, 147]. Additionally, it inhibits glia-driven neuronal ferroptosis by positively regulating the Glutathione Peroxidase-4 (GPX4), a key regulator of ferroptosis, and Ferroptosis Suppressor Protein-1 (FSP1) pathways, which are also crucial for protecting against oxidative damage [148,149,150]. NRF2 can also be activated through the Tropomyosin-related Kinase Receptor type B (TrkB) receptor, upregulating NRF2 via the PI3K/AKT pathway, reducing ferroptosis and neuroinflammation, and partially restoring neurocognitive function impaired by injury [151]. This mechanism also helps reduce endothelial cell death, TJ protein loss, and BBB permeability [152].

Hypertension

High blood pressure has been found to alter the BBB [153] reduce resting cerebral blood flow, and suppress the neurovascular coupling and endothelium-dependent responses, leading to its dysfunction [154]. Severe hypertension is associated with reduced oxygen consumption and blood flow [155]. Hypertension’s role in cognitive decline, aside from its link to stroke, affects similar areas as other causes of cerebrovascular diseases [154, 156,157,158]. A key factor that contributes to the pathogenesis of hypertension in the brain is sympathoexcitation [159], which is prevalent in the hypothalamic paraventricular nucleus (PVN). PVN is the center of artery pressure control [160] and can affect the sympathetic outflow of all the CNS [161]. ROS is one main contributor to the sympathoexcitation in this area in different kinds of hypertension [162,163,164,165].

Regarding BBB damage, a physiopathological increase in blood pressure can ultimately result in NVU dysfunction [6, 156], often correlated with a framework of oxidative stress, ischemia, and inflammation (Fig. 3). the protective role of NRF2 in maintaining BBB integrity during hypertension may be linked to mitigating endothelial damage [166], shielding this cell type against oxidative stress [14] and reducing the blood pressure and heart rate in rats after activation as seen by using the naturals compound [167]. For instance, in hypertensive rats reduced NRF2 protein expression in the rostro-ventrolateral medulla neurons impairs mitochondrial biogenesis and contributes to the development of hypertension [168]. Silencing of Nrf2 in this rat model led to higher levels of ROS and increased hypertension, showing a 20–25% increase in the mean arterial pressure suggesting that the basal level of NRF2 in the normal state is crucial for restraining sympathetic outflow [169]. Upon NRF2 activation, reduced activity of the sympathetic nerve and blood pressure was observed in hypertensive rats [159], being able to be correlated with a protective mechanism of BBB integrity and health.

NRF2 protective effects on glycemic load-related diseases

Glycemic load-related diseases involve imbalances in the glucose levels in the body, which have been associated with an increased risk of diabetes mellitus, coronary heart disease, obesity, and cancer [170]. Diabetes mellitus (DM) is a chronic metabolic disease characterized by elevated levels of blood glucose (or blood sugar), leading to complications in several organs, including the heart, blood vessels, eyes, kidneys, and nerves, and increased premature death [171]. Diabetes and other hyperglycemic-related diseases such as obesity, are linked to proliferative lesions in small brain vessels [172] which is correlated with an increase in barrier permeability, also named diabetic encephalopathy [173]. Disruption of BBB was paired with changes in glucose transport rates, efflux transporter activity, BEC expression of matrix MMPs and immune cell trafficking [174]. Excessive glucose oxidation via the Krebs cycle induced oxidative stress, which provoked BBB loss of integrity and pericyte death (Fig. 4) [175]. Hyperglycemia has been shown to alter BBB permeability, while hypoglycemia has been seen to trigger GLUT-1 overexpression to compensate for low circulating blood glucose levels and the opposite was described for hyperglycemia [172, 176]. In both cases they present an effect on the TJ expression and endothelial oxidative and inflammatory responses, correlating with higher released levels of vascular endothelial growth factor (VEGF) in the cell medium [176]. BBB leakage in diabetes and obesity with concomitant type II diabetes correlates with the downregulation of Claudin-5 and 12, Occludin and ZO-1 [177, 178].

Increased mitochondrial ROS appears to be a common mediator, linking increased glucose metabolism via the polyol pathway, accumulation of advanced glycation end products (AGEs), protein kinase C (PKC) activation, and increased activity of the hexosamine pathway with vascular damage in diabetes [179]. AGEs can increase BBB permeability, by inducing VEGF production in BEC and TFG-β in pericytes, which both then can act to induce secretion of MMP which can degrade TJ (Fig. 4) [173, 174].

Diabetic complications are associated with increased ROS generation resulting from elevated blood glucose and free fatty acids [180,181,182]. NRF2 counteracts high-glucose-induced damage and downstream markers of oxidative stress such as oxidized low-density lipoprotein (ox-LDL) [183], 4-hydroxynonenal (HNE) [184,185,186], and TGF-β [187] are elevated in diabetic patients and have all been shown to activate NRF2/ARE-linked gene transcription. Research in cultured bovine aortic endothelial cells [188] has established that high glucose and AGEs increase ROS production and NRF2-dependent HO-1 expression. As a result, certain antioxidant compounds may confer protection against AGE cytotoxicity by upregulating NRF2-linked antioxidant enzyme activity preventing oxidative stress in diabetes [188]. In human microvascular endothelial cells, although high glucose concentrations (30 mM, 6 h) do not significantly induce NRF2 translocation, activation of this pathway by NRF2-inducers significantly reduces hyperglycemia-induced ROS generation and protein glycosylation [189].

As stated before, BECs are highly vulnerable to oxidative and inflammatory stress, leading to a loss of BBB integrity through a reduced composition of TJ complexes as the main mechanism [58]. In pathologies related to altered glycemia pathologies, such as prolonged hypoglycemia, signification downregulation of NRF2 in BBB ECs has been detailed (Fig. 4) [18]. NRF2-knockdown resulted in a reduced expression of Claudin-5 and VE-Cadherin in BECs, without significant effects on Occludin expression [176]. The effects of NRF2 silencing on TJ were parallel to those observed with hypoglycemia [176], whose activation allowed for the partial restoration of TJ and prevented BBB permeability. Furthermore, VE-Cadherin was shown to positively regulate the transcription of Claudin-5 [190]. The effect on TJs may be explained by NRF2 involvement in the upregulation of RAS family member homolog A, an essential factor that promotes the reorganization of the actin cytoskeleton [191].

NRF2 protective effects on neurodegeneration diseases

Alzheimer’s disease

Alzheimer’s Disease (AD) accounts for 60–70% of all dementia cases and is generally characterized by an accumulation of amyloid-β (Aβ) in the form of senile plaques (SPs) and TAU protein in neurofibrillary tangles (NFTs) [192,193,194]. Additionally, AD involves low levels of acetylcholine, disrupted calcium regulation, oxidative stress, and neuro-inflammation [195]. The accumulation of SP and NFTs occurs in specific regions like the frontal cortex, hippocampus, and basal forebrain, which results in the decline of cognitive skills related to learning and memory [196]. Morphological changes have been observed in post-mortem brains of AD patients, such as basement membrane thickening, decreased microvessel density, capillary leakages, and accumulation of fibrinogen, albumin, prothrombin, and hemoglobin-derived peptide levels [197,198,199] indicative of BBB damage.

There is growing evidence that the BBB is compromised in the deposition of Aβ (Fig. 5) [200, 201]. Aβ causes mitochondrial dysfunction which results in the release of ROS to the cytosol, causing oxidative stress in neurons and endothelial cells, and it is seen as a causative factor for microhemorrhages within lobar regions [118, 202]. This enhanced permeability could lead to increased deposition of Aβ within AD brains, worsening the disease [203, 204]. A reduction in TJ proteins in BBB has been shown to enhance the clearance of brain Aβ into the blood [6, 205]. Angiogenic processes can be triggered by Aβ, which in turn results in the loss of the capillary TJ proteins (Claudin-1 and 5) and adherens junctions causing an increased permeability in rodent brains [206, 207]. The angiogenic process may be related to the Aβ accumulation that impairs the angiogenic signaling pathways, leading to reduced blood flow which decreases oxygen and nutrient supply in the brain [208].

Particularly, the hippocampus is especially vulnerable to BBB disruption, due to the intrinsic fragility of its vascular network, which showed that BBB permeability in the hippocampus is higher compared to several other brain regions, even in human subjects with no known brain pathology [209]. The hippocampus is a region particularly reliant on TJs for proper functioning that is enriched in this cell-cell junction for mediating the transport of ions and metabolites, which ensures a balance of neurotransmitters and signaling molecules [207, 210], which is highly affected in this neurodegenerative disorder [211]. Decreased Occludin and Claudin-5 have been detailed in AD mice [210] and postmortem AD patients [205].

Development of cerebral β-amyloidosis and cognitive decline in patients with AD, with related familial cerebrovascular disorders, may be linked to defective Aβ clearance from the brain and a lack of NRF2 protection of BBB integrity [212, 213]. Animal models suggest that NRF2 plays a crucial role in mitigating Aβ cytotoxicity and clearing Aβ. For example, the 5XFAD AD-like mice model lacking NRF2 shows an increase in astrocytes and microglia and increased levels of interferon-γ (IFN-γ) and exhibits worsened cognitive deficits [214, 215]. Bahn et al. showed that NRF2 can also negatively regulate β-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) expression, which is the main secretase for Aβ peptides, through binding to ARE sites in the BACE1 promoter. These researchers showed that an NRF2 deficiency increases BACE1 expression and exacerbates Aβ plaque loads and cognitive deficits in 5XFAD mice [216], and probably a BBB injury. On the other hand, macroautophagy is the main route involved in Aβ oligomer species and Aβ plaque clearance [217] and has been linked to NRF2. Recent studies have reported that NRF2 levels are regulated by the autophagy-related adaptor protein p62 [44]. p62 shuttles ubiquitinated proteins to the proteasomal and lysosomal degradation machinery and sequesters damaged proteins into aggregates before their degradation [44]. In this context, in Nrf2-deficient mice models, increased intraneuronal Aβ aggregates were found in the hippocampus of APP/PS1^ΔE9 mice when NRF2 was absent [44], which may present a compromised BBB.

It has also been described that the AD brain presents microvascular alterations, such as increased capillary tortuosity, rarefaction, and higher levels of atherosclerosis [218, 219]. In vitro, experimental evidence shows that these Aβ deposits induce cerebrovascular dysfunction in the rat brain [220] and that the Aβ peptide produces endothelial dysfunction in cerebral microvessels via ROS. This occurs when the ROS superoxide–scavenging enzyme, superoxide dismutase, prevents acetylcholine-induced endothelium-dependent vasodilation [220]. This excessive ROS production could be a primary mechanism explaining BBB disruption [221]. In this way, the activation of the NRF2 defense pathway can prevent oxidative stress and would be beneficial for preserving BBB disruption and neurological impairment during its progression, in a similar manner in other neuro disorders [213]. Conversely, an NRF2-deficiency condition would increase the intracellular ROS content, suggesting that it is important for maintaining ROS homeostasis [222].

Another component in AD progression is the accumulation of TAU oligomers in cerebral microvessels. That has also been reported in other human neuro-disorders as Lewy Body (LB) dementia, and progressive supranuclear palsy patients [223]. Microbleeds have been detected in the brains of patients affected by frontotemporal dementia [224] and cerebrovascular inflammation has been associated with TAU pathology. In these cases, areas with significant accumulation of neurofibrillary tangles exhibited upregulation of adhesion molecules, disruption of TJs, morphological alterations in brain microvessels, including thickening of the vessel wall, vessel lumen reduction as well as increased in collagen-type IV content per vessel [225, 226], and this damage can stimulate inflammation.

In AD, Blood-Brain Barrier (BBB) damage is not solely due to endothelial TJ disruption but also involves the loss of mural cells, particularly pericytes, which are crucial for maintaining BBB integrity. Pericyte loss is strongly associated with BBB disruption in the cortex and hippocampus [227]. Soluble TAU could be a contributing factor to the decrease of TJ [228] and, like Aβ, can lead to dysfunction of the BBB in the hippocampus (Fig. 5) [207, 229]. Additionally, TAU pathology has been linked to small vessel disease [230], and immune cell trafficking across the BBB also appears to be modulated by neurofibrillary pathology in Tauopathies [231].

In vitro models using primary rat BECs exposed to oligomeric TAU changed the endothelial properties of the BBB that promote the migration of immune cells from the blood into the brain [231]. This process is driven by glial cell activation, which leads to neuroinflammation in TAU pathology-affected regions and may accelerate BBB damage [232]. Activated glial cells activate and release proinflammatory cytokines, including IL-1β, TNF-α, and IL-6, which alter endothelial properties [232,233,234].

NRF2 gains focus on this pathology as an anti-inflammatory capacity for response through several mechanisms. It suppresses the expression of several proinflammatory cytokines secreted from microglia, macrophages, and monocytes, which can affect the neurovascular unit [74] and infiltrate into the brain parenchyma.

Several reports have suggested that misfolded or phosphorylated TAU could be degraded through the proteasome [235,236,237]. However, recent studies found that TAU may also be turned over by the autophagy-lysosome pathway [238,239,240]. Of particular interest, NRF2 activation may also reduce phosphorylated TAU protein via the autophagy-lysosome pathway through the induction of the autophagy adaptor protein NDP52 [241]. It functions similarly to p62, recognizing ubiquitinated proteins and directing them to lysosomes for degradation. Five putative ARE sequences have been identified in the promoter region of the Ndp52 gene [241], and Ndp52 mRNA levels were found to be reduced in the hippocampus of Nrf2-knockout mice [241].

Overall, these findings highlight the relevance of NRF2 in the neuronal protective process and can prevent onset and progress to neurodegenerative diseases that could go head to BBB disturbed and leaky.

Parkinson’s disease

Although previously not as extensively studied in Parkinson’s Disease (PD), evidence increasingly suggests that BBB dysfunction plays an active role in PD. PD is a neurodegenerative disease characterized by motor dysfunction and cognitive deficits, which begin in the nigrostriatal pathway and eventually spread to the cortex [242]. BBB leakage has been observed in PD patients in areas typically associated with the disease, such as the basal ganglia [243]. Furthermore, perivascular deposits of fibrinogen or fibrin, immunoglobulin G (IgG), and hemosiderin in specific regions, indicate BBB disruption [244,245,246]. Post-mortem samples of advanced cases have revealed capillary damage in the frontal cortex [247] and BBB permeability [6]. This suggests that BBB disruption might be implicated in the development of PD (Fig. 5).

Astrocytes, which are crucial for maintaining the BBB [248], may play a central role in BBB disruption when they become dysfunctional. These cells support the secretion of TJ proteins through the production of growth factors [249]. However, in PD, reactive astrocytes have been shown to significantly reduce growth factor expression [250], leading to TJ protein production and, consequently, BBB leakage. Although PD patients show augmented vascular density in the substantia nigra pars compacta (SNpc), in the proximity of neuronal damage, these new microvessels display impaired maturation processes and altered diameters [251].

Astrocyte-specific NRF2 expression exerts neuroprotection through several mechanisms. Primarily, NRF2 enhances metabolic efficiency and helps maintain neuronal metabolic homeostasis following mitochondrial complex II inhibition [252]. Additionally, astrocyte NRF2 mitigates oxidative stress and protects against cell death [253], maintains water and glutamate balance, and reduces excitotoxicity in cases of NRF2-deficiency [254]. Furthermore, VEGF production in astrocytes is dependent on NRF2, as demonstrated in a study using Nrf2-deficient mice [255].

α-Synuclein (α-SYN) has also been shown to impact ECs homeostasis differently (Fig. 5) [256]. In mouse models expressing the A53T mutant form of human α-SYN, TJ expression is reduced, leading to increased vascular permeability and accumulation of α-SYN in activated astrocytes, which then release VEGF. Toxic forms of α-SYN can further disrupt EC function by downregulating TJ expression and inflammatory cytokine release [257, 258].

Activation of the NRF2 pathway has protective effects on ECs, reducing oxidative stress-related damage and alleviating PD symptoms. A recent study reported that over-expression of HO-1 triggered intracellular proteasomal degradation of α-SYN [259]. HO-1 also stimulates autophagy in astrocytes, another mechanism leading to the clearance of aggregated α-SYN [260, 261]. HO-1, a heat shock protein (HSP), is part of a family that, when induced by stress, prevents protein aggregation through refolding or degradation mechanism, including α-SYN [262]. Another pathway through which α-SYN degradation occurs is via the NRF2/p62 axis, offering alternative routes for clearing α-SYN aggregates and mitigating BBB injury [263].

Besides, upregulating HO-1 in ECs via NRF2 activation exerts cytoprotective effects against oxidative stress. This enzyme degrades heme to release free iron, carbon monoxide (CO), and biliverdin, which is converted to the antioxidant bilirubin [264]. By activating antioxidant phase II enzymes, NRF2 diminishes inflammation caused by microglia in the hippocampus, reducing inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines [47, 265]. Moderately elevated CO and biliverdin generated by HO-1 may confer neuroprotection by inhibiting NADPH oxidase and scavenging nitrogen monoxide (NO), thereby protecting the neurovascular unit [265, 266].

Huntington’s disease

Huntington’s Disease (HD) is a dominantly inherited autosomal neurodegenerative disorder often diagnosed at the age of 40, although onset varies from under ten to over eighty years of age. While not traditionally considered an aging-related disease, research has revealed epigenetic age acceleration in specific brain regions of that HD [267]. Recently, morphological changes in blood vessels and BBB leakage in the caudate and putamen were observed in HD patients using magnetic resonance imaging. In HD patient samples from the putamen and striatal samples of the HD model R6/2 mice, Occludin and Claudin-5 protein levels were decreased and evidence of increased BBB permeability was found [268].

HD is distinguished by the neuropathic phenotypes in glial cells, which disrupt these cells regulatory roles and involvement in BBB breakdown (Fig. 5). Mutant huntingtin accumulates in glial cells, impairing their regulatory functions, which leads to BBB disruption [269]. Glial cell activation occurs through cytokines like TNF-α, IL-6, chemokines, iNOS, and cyclooxygenase-2 (COX-2) [270], along with NF-κB pathway activation in both neural and glial cells [271]. Activation of the NRF2 pathway may reduce the synthesis of these inflammatory and, by limiting ROS production, could help protect against oxidative stress-induced BBB damage in HD [272].

Studies using STHdhQ111/Q111 HD-like transgenic mice indicate reduced NRF2 activity and altered KEAP1 and p62 in striatal cells [273]. As NRF2 regulates macroautophagy pathways, including p62 expression, decreased p62 levels in STHdhQ111/Q111 models could impair autophagy, preventing huntingtin degradation and ultimately compromising BBB integrity [273].

NRF2 protective effects on cigarette smoke toxicity

Cigarette smoking (CS) is considered a major risk factor for several neurological disorders and neurovascular complications including stroke, small vessel ischemic disease (SVID), and cerebrovascular diseases. Research attributes these detrimental effects to the oxidative and inflammatory damage caused by a large and poorly identified number of high ROS contained in tobacco smoke (TS) [274]. The BBB is directly exposed to this host of harmful toxicants and ROS present in TS, making it a critical factor in TS-promoted CNS disorders (Fig. 6). When cigarette smoke is inhaled, many soluble and gaseous components bypass first-pass metabolism and enter the brain microvasculature via arterial circulation [274].

Despite substantial evidence linking smoking and vascular impairment, the impact of CS on the BBB has only been marginally addressed. Previous studies have disclosed that soluble TS extracts trigger strong pro-inflammatory responses at the BBB endothelial level [275]. Nicotine exposure, a key component of TS, has been found to downregulate the expression of TJ proteins such as ZO-1, occludins, and cadherins, leading to an increased BBB permeability [276, 277].

NRF2 pathway is activated in response to CS in resident macrophages, bronchial and alveolar epithelium, and lung fibroblasts of chronic smokers [278]. Following TS exposure, NRF2 translocates to the nucleus and promotes the expression of detoxification enzymes and antioxidants (Fig. 6) [88], which reduce inflammation and restore TJ protein expression [279]. However, chronic TS exposure can impair the NRF2/ARE antioxidant pathway, resulting in cerebrovascular damage and BBB breakdown, as alterations in the transcription and activation of this pathway were among the most significant changes observed in human BEC [18, 87]. Studies suggest that pharmacological activation of NRF2 reduces CS toxicity by stabilizing TJ proteins, upregulating GLUT1, and decreasing oxidative stress and inflammation [87].

NRF2 protective effects on sepsis-associated encephalopathy

Sepsis-associated encephalopathy (SAE) is a common and fatal disease, especially in critically ill patients due to systemic infection. The pathogenesis and progress of sepsis are life-threatening organ dysfunctions caused by an abnormal immune response to infection [280, 281]. This dysregulated immune response disrupts brain homeostasis and leads to a substantial risk of long-term cognitive impairment, resulting in sepsis-associated encephalopathy (SAE). SAE is characterized by excessive microglial activation, endothelial barrier dysfunction, and BBB impairment (Fig. 6), often accompanied by psychological disorders, including cognitive impairment and coma [282].

The classic model that is usually carried out in both in vitro and in vivo models is an LPS treatment, which leads to inflammation by NF-kB activation and can increase the production of ROS [48, 283, 284]. This oxidative stress damages cells, triggering neuroinflammation and the secretion of neurotransmitters such as ATP and nitric oxide (NO) [48, 65]. Moreover, ROS acts as a secondary messenger, sustaining immune activation in a feedback loop that leads to persistent inflammation and more oxidative stress. RNS can activate MMPs which trigger BBB disruption and neuroinflammation [65], and reduce the expression of TJ proteins such as ZO-1, Occludin, and Claudin-5 by stimulating the secretion of more MMPs [283].

In this context, NRF2 serves as a crucial protective factor against LPS-induced BBB injury. Studies in mouse brain microvascular endothelial cells and the hippocampus show that NRF2 protects the BBB from LPS-induced damage (Fig. 6) [48, 99]. NRF2 significantly alleviated LPS-induced BBB dysfunction both in vitro and in vivo, as evidenced by the improved TJs proteins (ZO-1 and Occludin) and the reduced expression levels of vascular cell adhesion molecule 1 (VCAM-1), an adhesion molecule that promotes BBB integrity (Fig. 6) [285, 286]. By reducing BBB permeability, NRF2 helps prevent inflammatory cytokines from infiltrating the brain, limiting microglial activation and subsequent neural cell damage [112].

Cecal ligation and puncture (CLP) is another sepsis mice model to evaluate the BBB integrity. It consists of the perforation of the cecum allowing the release of fecal material into the peritoneal cavity to generate an exacerbated immune response induced by polymicrobial infection. In a study by Yu et al., NRF2 activators in pretreated mice were associated with better cognitive outcomes, reduced BBB damage markers (e.g., decreased brain water content and dye leakage), and improved BBB integrity [287]. Nrf2-deficient mice showed greater expression of pro-inflammatory markers and oxidative stress indicators, compromising BBB stability. In contrast, wild-type mice with functional NRF2 maintained higher levels of TJ proteins like VE-Cadherin and ZO-1 in the cortex, protecting against BBB injury in SAE [287].

NRF2 inducers are protective of the Blood-Brain Barrier integrity

From a clinical perspective, the main asset of NRF2 is its potential as a pharmacological target for patient therapy [8]. Several NRF2 activators are plant-derived phytochemicals, including isothiocyanates, triterpenoids, curcumin, resveratrol, and lycopene [41], or synthetic sourced compounds [48, 288]. Many of these activators can prevent the KEAP1-mediated proteasomal degradation of NRF2, the main regulator of NRF2, thus enhancing the stabilization of the protein. Other NRF2 activators mediate the pathway through signaling cascades, further increasing its protective effects [289, 290].

This review highlights the potential of NRF2 activators, particularly phytochemicals, in maintaining BBB integrity across various neurological disorders (see Summary in Table 1).

Table 1 Summary of NRF2 inducers involved in BBB integrity protection

BBB protective compounds in ischemic/stroke models

Oridonin, a natural diterpenoid compound extracted from Chinese herbs, has been proven to exert anti-oxidative stress effects in various disease models [291, 292]. Li et al. (2021) demonstrated that oridonin protects BBB integrity in an ischemic stroke (I/S) mouse model by upregulating TJ protein expression, inhibiting peripheral immune cell infiltration, and reducing neuroinflammation, ultimately lowering infarct volume [293]. Mechanistically, oridonin promotes NRF2 nuclear translocation through c-Jun N-terminal kinase (JNK) phosphorylation, allowing NRF2 to bind antioxidant response elements ARE and activate gene transcription [293, 294].

Rhein is the major ingredient of several traditional Chinese medicines (TCM) which has various pharmacological effects such as anti-inflammatory, antioxidant, and anti-cancer properties [295,296,297]. In the middle cerebral artery occlusion (MCAO) model in rats, Rhein improved neurological deficits, reduced infarct size, and preserved BBB integrity. Rhein’s neuroprotective effects are attributed to its NRF2-dependent activity, which reduces ROS, suppresses ferroptosis, and mitigates oxidative stress [298]. Rhein protected against Oxygen and Glucose Deprivation/Reoxygenation (OGD/R)-induced by regulating the NRF2 signaling pathway, and its effect was abolished upon NRF2 inhibition, suggesting that Rhein’s neuroprotective action is NRF2-dependent [298]. Although the molecular pathway behind NRF2 activation remains undescribed, however, molecular docking has been revealed to be capable of binding directly to the NRF2 protein [298].

Kinsenoside (KD), a major bioactive component in Anoectochilus roxburghii, has demonstrated efficacy in treating several disorders, such as diabetes, liver disease, osteoporosis, osteoarthritis, hyperlipidemia, endotoxin shock, and acute lung injury [299, 300]. Qiao et al. (2023) found that KD reduced infarct volume, neurological deficits, and brain edema while preserving BBB integrity by upregulating TJ proteins like Occludin, Claudin-5, and ZO-1 [301]. KD improved BBB structure and function, as evidenced by a lower 18 F-fluorodeoxyglucose pass rate of the BBB and upregulation of TJ proteins such as Occludin, Claudin-5, and ZO-1. It was proposed that the activation of NRF2 happened through the binding to ERK 1/2, which could promote NRF2 phosphorylation, causing its dissociation with KEAP1 and nuclear translocation, as well as, NRF2/HO-1 axis signaling protein stimulation [301].

Nomilin (NOM) is a triterpenoid, which exists in common edible citrus fruits. NOM has anticancer properties, immunomodulatory and antiproliferative activities, among others [302, 303]. Shi et al. (2019) investigated NOM’s neuroprotective effects in a MCAO stroke model and found it reduced infarct area, brain edema, and BBB disruption while stabilizing TJ proteins ZO-1 and Occludin [304]. Further results revealed that NOM treatment effectively mitigated oxidative stress and facilitated the expressions of NRF2 and NQO1, achieved by preventing the binding of KEAP1 to NRF2 [305], which might confirm that the loss of TJ proteins in the microvasculature was likely mediated by oxidative stress [304].

Procyanidin B2 (PB) composed of two molecules of the flavan-3-ol (−)-epicatechin is one of the most common procyanidins found in plants such as cocoa, apples, or grapes. PB is considered a bioactive component due to its benefits in health promotion as well as in the restoration and maintenance of homeostasis [306]. Wu et al. (2015) demonstrated PB’s neuroprotective effects in the MCAO stroke model, where it decreased infarct volume, reduced brain edema, and preserved BBB integrity by reducing Evans blue leakage and IgG levels as well as increasing TJ expression levels such as ZO-1 [307]. The activities of antioxidant enzymes were elevated, because PB reversed the suppression of NRF2 nuclear translocation, and increased the protein expression of HO-1, Glutathione S-transferase-α (GSTα), and NQO1 in the ipsilateral ischemic area of the brain [307]. PB’s effect on NRF2 increased activated is mediated through the deacetylase sirtuin-1 (SIRT1), which induces NRF2 transcription [308]. SIRT1 has been linked to increased NRF2 transcription and improved DNA binding of NRF2 to its target genes by promoting the deacetylation of NRF2, stabilizing the transcription factor and enhancing its activity [309]. This interaction improves NRF2’s ability to bind to antioxidant response elements (ARE) in the DNA, leading to the activation of numerous genes involved in the antioxidant defense mechanism [308].

Dimethyl fumarate (DMF) is known for its anti-inflammatory and anti-oxidative activity in a variety of tissues and cell types. DMF is a well-characterized activator of NRF2, acting as an electrophile which reacts to KEAP1 redox-sensitive cysteines, promoting a conformation change which is unable to recruit the ubiquitination machinery, allowing for the free NRF2 to travel to the nucleus and promote its transcription [263]. In vitro, DMF and its primary metabolite monomethyl fumarate (MMF) increased the survival rate of astrocytes and neurons exposed to oxidative stress conditions [310,311,312]. Clinically used for treating psoriasis [313] and, more recently, for multiple sclerosis [314, 315], Kunze et al. (2015) validated the DMF effect in it stabilized the BBB by preventing disruption of endothelial TJs and gap formation, and decreased matrix metalloproteinase activity in the brain stroke model [316], as well as inhibited inflammatory cytokine expression and attenuated leukocyte transmigration. DMF activated the NRF2 pathway as shown by the upregulation of several target genes in the brain in vivo, as well as in cerebral endothelial cells and astrocytes in vitro, where DMF also increased the protein abundance of nuclear NRF2 [316].

BBB protective compounds in traumatic brain injury models

Sulforaphane (SFN) is an isothiocyanate abundant in cruciferous vegetables (e.g., broccoli), and was identified as being one of the most potent inducers of NRF2 [317, 318]. SFN acts as an electrophile, inhibiting the KEAP1 degradation pathway [290], and showcases antioxidant, antiproliferative, and anticarcinogenic properties [319]. Zhao et al. (2007) analyzed the effect of the NRF2 activation by SFN in traumatic brain injury (TBI) models using pneumatic piston-induced injury [152]. Postinjury administration of SFN reduced the loss of endothelial cell markers and TJ proteins and preserved BBB function. This effect was NRF2-dependent, as benefits were not observed in Nrf2-deficient mice or those pretreated with NRF2-decoy oligonucleotide [152].

Allyl isothiocyanate (AITC) is a member of a group of naturally occurring compounds called isothiocyanates and is found in radish, mustard, and wasabi [320]. These compounds have been shown to exhibit antioxidant, anti-inflammatory, anticancer, and antimicrobial properties by inducing NRF2 activation [321,322,323,324]. In a cryogenic TBI model in mice, Caglayan et al. (2019) analyzed its effects on NRF2 and NF-κB signaling pathways [325]. AITC which was administered immediately after the injury significantly decreased infarct volume and BBB permeability. Protein levels of pro-inflammatory cytokines IL-1β and IL6, glial fibrillary acidic protein (GFAP), and NF-κB were decreased, while NRF2 and neural cell adhesion molecule levels were increased with AITC when compared with vehicle control [325].

Fisetin is a flavonoid present in fruits and vegetables like apples, strawberries, grapes, cucumbers, and persimmon, known for its anti-carcinogenic, anti-inflammatory, and antioxidant effects [326, 327]. In addition, fisetin was also a potent scavenger of reactive oxygen species (ROS). It has been used to prevent oxidative stress-induced pathologies, such as Alzheimer’s and Parkinson’s diseases, skin damage, liver injury, and diabetic neuropathy [328,329,330,331]. Zhang et al. (2018) demonstrated that fisetin improved neurological function, reduced brain edema, and preserved BBB integrity in TBI [332]. Administration of fisetin suppressed neuron cell death and apoptosis, increased the expression of B-cell lymphoma 2 (BCL-2), and decreased the expression of Bcl-2-associated X protein (BAX) and caspase-3 after TBI. In addition, fisetin activated the NRF2/ARE pathway following TBI. However, fisetin only failed to suppress oxidative stress in Nrf2-deficient mice [332]. Activation of NRF2 by fisetin has been described to occur by activating the PI3K/AKT pathway, which, in turn, prevents NRF2 degradation by the β-TrCP1 pathway through GSK3 inhibitory phosphorylation [333].

BBB protective compounds in cigarette smoking toxicity models

Metformin (MF) widely used in diabetes management, also has possible renoprotective properties as well as neuroprotective effects on BBB integrity [334,335,336,337]. Prasad et al. (2017) found that MF, through NRF2 activation, counteracted cigarette smoke (CS) toxicity by preserving TJ protein expression, reducing inflammation and oxidative stress, and normalizing GLUT-1 and thrombomodulin levels [87] which drastically reduces CS toxicity at the cerebrovascular level. Although the mechanism by which can activate NRF2 remains to be fully elucidated, it has been known to inhibit AMPK. AMPK addresses phosphorylation to NRF2 and prevents its activation by degradation through β-TrCP2. Thus, MF can lead to the induction of a selected group of NRF2-target genes [336, 338].

Rosiglitazone (RSG), is a thiazolidinedione compound that is well known to improve insulin resistance through regulating adiponectin gene expression and is used for the treatment of type-2 diabetes mellitus [339]. It is considered to act as a transcription factor peroxisome proliferator-activated receptor (PPARγ) agonist [340, 341]. Since chronic smoking and diabetes carry similar risks for cerebrovascular diseases and stroke, it is plausible that RSG can prevent/reduce BBB impairment promoted by chronic TS and recent e-cigarette vaping exposure. Although the exact mechanism of RSG is not fully understood, previous studies have revealed that RSG can promote counteractive protective mechanisms primarily associated with the enhancement of NRF2 activity through activation of the peroxisome proliferator-activated receptor gamma [342]. as well as through the PI3K/AKT pathway [343]. Sivandzade et al. (2019) reported that RSG increased PPARγ expression and NRF2 activity, which protected the BBB in models of tobacco smoke and e-cigarette exposure [279].

BBB protective compounds in sepsis-associated encephalopathy models

Liensinine, an alkaloid extracted from lotus plumule [344], has multiple biological activities, such as anti-oxidative stress [345, 346], anti-inflammation [347], and anti-hypertension [348, 349]. Wang et al. (2023) found that liensinine preserved BBB integrity in a sepsis-associated encephalopathy (SAE) model by upregulating TJ proteins and decreasing oxidative stress through NRF2 activation [350]. Mice were pretreated with LPS, which triggered brain necrosis and disrupted the integrity and permeability of the BBB. While liensinine restored cerebrum structure and improved BBB integrity with upregulated TJs proteins, decreased Evans-Blue leakage and fibrinogen expression with decreased MMP-2/9 in serum, thereby reducing BBB permeability. Moreover, LPS triggered cerebrum oxidative stress and inflammation, whereas liensinine enhanced antioxidant enzyme activities and weakened malondialdehyde through the NRF2 pathway [350], which could be by the inhibition of KEAP1 binding to NRF2 [351].

Astragaloside IV (ASIV) is one of the active components present in Astragalus membranaceous, a plant used in traditional Chinese medicine. Many studies have shown that the administration of ASIV may facilitate the alleviation of CNS diseases, such as multiple sclerosis, traumatic brain injury, and cerebral ischemia or ischemia/reperfusion [352,353,354,355,356,357,358], through antioxidant, anti-inflammation, or anti-apoptosis. Li et al. (2017) demonstrated ASIV’s efficacy in preventing BBB disruption by increasing TJ protein expression and reducing VCAM-1 in LPS-stimulated mice [132]. ASIV was found to prevent the leakage of BBB in LPS-induced mice, which was accompanied by increased ZO-1 and Occludin but reduced VCAM-1 in brain microvessels. Moreover, in bEnd.3 cells line, ASIV mitigated the increased permeability induced by LPS. ASIV also enhanced the expression of TJ proteins such as ZO-1, Occludin, and Claudin-5 in LPS-stimulated bEnd.3 cells. Meanwhile, it inhibited inflammatory responses and prevented the monocyte adhesion onto bEnd.3 cells upon LPS stimulation. Further study disclosed that ASIV could alleviate ROS levels and activate the NRF2 antioxidant pathway in bEnd.3 cells. When Nrf2 was silenced, the protective effect of ASIV was abolished [132]. It has been proposed to activate NRF2 through different pathways, such as SIRT1 and PI3K/AKT pathway [359,360,361].

Baicalin is one of the main bioactive components in the extract of Scutellaria baicalensis. It has anti-inflammatory, anti-cancer, anti-diabetic, anti-thrombotic, cardioprotective, liver protection, and neuroprotective pharmacological properties [362]. Thus, Wang, X. et al. 2021 demonstrated that treatment with baicalin can inhibit the production of pro-inflammatory cytokines induced by LPS in mice and bEnd.3 cells, including IL-1β and TNF-α [99]. At the same time, LPS caused a decrease in TJ proteins in the BBB, but baicalin can alleviate the damage by up-regulating Claudin-5 and ZO-1 protein expression. In addition, the results showed that baicalin reduced the production of ROS and oxidative parameter malonaldehyde (MDA) in bEnd.3 cells and promoted the production of SOD, and up-regulated the expression of NRF2, HO-1, and NQO1 [99]. The mechanism of this change was mediated by activating the NRF2 signaling pathway through the KEAP1 pathway [363].

GYY4137, a new synthetic compound of hydrogen sulfide (H₂S), can release lower concentrations of H₂S over a longer period in the form of an aqueous solution, and it has extensive biological benefits. Currently, GYY4137 is widely used in studies focusing on the protective effects of H₂S [364, 365]. In septic mice, Cui et al. (2021) found that GYY4137 inhibited TJ degradation and oxidative stress by activating NRF2 through KEAP1 modification [112]. These results suggested that administrated GYY4137, in combination with LPS, significantly alleviated the clinical symptoms, improving the pathological abnormalities of septic mice. Moreover, the degradation of TJs in the BBB was considerably inhibited by GYY4137, and significantly attenuated inflammation and apoptosis in the brain.

Fenretinide (FEN), the synthetic retinoid 4-hydroxy(phenyl) retinamide, is a KEAP1-NRF2 protein-protein interaction inhibitor. It has been widely studied in cancer therapeutic because of its favorable toxicological profile and is presently undergoing phase II clinical trials for the suppression of insulin resistance in obese humans with hepatic steatosis [366,367,368].

Previous studies have shown that FEN reduced the expression of pro-inflammatory genes and the levels of oxidative stress markers after spinal cord injury (SCI) [369]. Li et al. (2020) observed that the FEN treatment markedly improved NRF2 expression and nuclear translocation in bEnd.3 cells, and promoted NRF2/ARE transcription activity, and its downstream signals, which were NRF2-dependent [48], possibly acting as an NRF2-KEAP1 Protein-Protein Interaction-inhibitor. FEN also exhibited a cytoprotective role in LPS-stimulated bEnd.3 cells through improving antioxidant capacity and inhibiting inflammation by blocking NF-κB signaling. In a mouse model with brain injury induced by LPS, FEN administration markedly attenuated the behavior impairments, BBB, and histological changes in hippocampus samples [48].

Even though other NRF2 inducers have been tested for BBB protection in various neurological conditions, studies exploring NRF2 compounds in diabetes, obesity, hypertension, and certain neurodegenerative diseases (e.g., Alzheimer’s, Parkinson’s, Huntington’s) remain limited, indicating a significant area for future research.

Conclusion remarks

There is a growing list of CNS pathologies involving BBB dysfunction, including multiple sclerosis [370,371,372]; hypoxia and ischemia [373,374,375]; edema [375, 376]; Parkinson’s disease [377,378,379,380], Alzheimer’s disease [378, 380,381,382,383]. Other well-known diseases in which a disturbed Blood-Brain Barrier (BBB) plays a recognized role include epilepsy [384, 385]; tumors [386, 387] lysosomal storage diseases [388, 389] and glaucoma [390, 391] in blood-retinal barrier (BRB) injury. The barrier dysfunction can range from mild and transient TJs opening to chronic barrier breakdown [392,393,394], and changes in transport systems and enzymes can also occur. While the causal relationship between BBB disturbance and disease initiation is not always clear, its contribution to disease progression is well documented [395, 396].

Research has identified multiple mechanisms contributing to neurological disorders during secondary injury, including inflammatory responses, oxidative stress, mitochondrial dysfunction, and Blood-Brain Barrier (BBB) disruption. NRF2 plays a crucial role in this context, as its activation helps mitigate oxidative stress and inflammation. Furthermore, NRF2 has been shown to regulate tight junctions and matrix metalloproteinases, which are vital for maintaining BBB integrity under neurophysiological conditions.

Numerous compounds, both phytochemicals derived from fruits, vegetables, and medicinal herbs, as well as synthetic agents that inhibit the KEAP1-NRF2 interaction, exhibit significant neuroprotective effects. These effects include enhancing BBB integrity, preserving neuronal viability, and curbing excessive inflammation through NRF2-mediated oxidative stress responses. Despite extensive evidence of the neuroprotective properties of various phytochemicals in vitro and in vivo, there remains a notable gap in effective clinical applications. In spite of promising preclinical data, translating these findings into effective clinical treatments remains a challenge. To date, only a few compounds, like sulforaphane and dimethyl fumarate (DMF), have progressed to clinical trials targeting CNS diseases and may offer protection against BBB damage [397, 398]. Repurposing existing drugs, such as metformin and rosiglitazone (currently used in diabetes treatment), for CNS diseases is an attractive option due to their known mechanisms involving similar pathways.

To optimize the therapeutic potential of NRF2 activators for BBB protection, careful consideration must be given to patient selection, drug dosage, delivery methods, timing of administration, and administration protocols to maximize benefits while minimizing side effects. Additionally, the safety and pharmacokinetics of these phytochemicals are still poorly understood, highlighting the need for further research to facilitate their clinical introduction.

Fig. 1

Structure of the BBB. The BBB is composed of several cell types, the smooth muscle cells are present in vessels with higher diameters, astrocytes bind to the vessel through their feet, and pericytes enclose the vessel and are surrounded by a basement membrane. The endothelial cells are tightly bound together through several cell junction proteins, which include the TJs (Claudin and Occludin) which connect to the actin cytoskeleton through ZO-1, JAM, PECAM, and adherens junctions (such as VE-Cadherin)

Fig. 2

Regulatory pathways of NRF2. NRF2 levels are kept low in physiological conditions through two main mechanisms. KEAP1 binds to NRF2 and promotes its ubiquitination and proteasomal degradation, upon ROS, oxidative stress or electrophiles these molecules are going to interact with a series of electrosensitive cysteines present in KEAP1. This would lead to a change in conformation which results in KEAP1 being unable to ubiquitinate NRF2. The other main pathway is through GSK3-β-TrCP. GSK3-β phosphorylates NRF2 when is not inhibited by AKT phosphorylation which is activated in the presence of growth or other signaling factors. Upon NRF2 phosphorylation, this site acts as a phosphodegron which is recognized by β-TrCP, which recruits an E-3 ubiquitin ligase complex for its degradation. When NRF2 is not degraded it is able to travel to the nucleus, binding to the ARE sequence of its target genes and promoting an antioxidant and anti-inflammatory response. Regarding BBB, it also projects by downregulation of MMP expression and signaling and promotes cell junctions and BBB key transporters expression

Fig. 3

The effect of cerebrovascular damage leads to brain pathologies. Cerebrovascular diseases can be caused by ischemia, traumatic head injury, or hypertension. These pathologies share an oxidative stress-inflammation feedback loop which results in a compromised BBB. There is a transport dysregulation coupled with cell death and loss of instability, thus altering brain homeostasis by the entry of toxins or antibodies. The BBB damage can accumulate and result in neuron loss, and alteration of the brain BF which ultimately can end in cognitive decline

Fig. 4

Effects of altered glucose blood levels on the BBB. A), high levels of glucose in the blood, such as in diabetes, lead to high activity in mitochondria which increases oxidative stress coupled with higher AGE levels, which can disrupt BBB integrity through the promotion of VEGF release by BEC and TFG-beta by pericytes, which, in turn, activate metalloproteinase to degrade cell junction. MMPs are also activated by the oxidative and inflammatory stress frame caused by elevated glucose levels. B), hypoglycemia also leads to BBB disruption, there is an increase of GLUT1 expression to compensate for the low levels of glucose and a reduced NRF2 expression, which in turn, is not able to downregulate MMPs and to mediate the antioxidative and anti-inflammatory response

Fig. 5

BBB landscape across different neurodegenerative diseases. A), in physiological conditions the BBB is stable, which results in an adequate transport regulation that allows the brain homeostasis. B), Alzheimer’s disease severely affects the hippocampus and cortex, in those areas we have an accumulation of amyloid β plaques and TAU neurofibrillary tangles which can be released to the bloodstream, causing inflammation and oxidative stress, which can activate microglia and astrocytes. These stresses result in a loss of the BBB integrity, where there is cell death and loss of TJ. C), Parkinson’s disease most common areas that affect in the early stages are the substancia nigra (in pink) and the basal ganglia, it is known to accumulate both in neurons and astrocytes, damaging the neurons and causing the release of growth factors that can induce an angiogenesis response from the endothelium, which needs a loss of the TJ for the cells to migrate and form novel vessels. High levels of ROS are present in the bloodstream, which contributes to the BBB damage. D), Huntington’s disease has been shown to affect the basal ganglia and the brain cortex and in the context of the BBB, it causes the release of pro-inflammatory cytokines which can damage the brain endothelium. NRF2 protects the BBB in neurodegenerative diseases through various mechanisms. Aberrant protein aggregations generate ROS and inflammation, which NRF2 can suppress while promoting autophagy pathways to clear these aggregates. NRF2 also inhibits BACE1 gene expression, reducing Aβ protein release, and activates autophagy-related genes such as p62 and NDP52 to further support the removal of harmful protein accumulations. Macroautophagy is one main pathway, which is capable of removing neurofibrillary tangles, amyloid plaques, α-synuclein and huntingtin aggregates

Fig. 6

Effects of damaging substances present in the bloodstream. A), healthy conditions of a brain vessel, with all the components of the neurovascular unit. B), BBB damage caused by smoking, which causes the entry into the bloodstream of damaging gases such as CO, toxic products, and nicotine which results in inflammation, and nicotine is known to dysregulate the BEC cell junctions. C), sepsis is characterized by the elevated levels of pro-inflammatory cytokines in the bloodstream, causing an elevated inflammatory situation in the tissue, LPS present in bacteria is known to stimulate the release of nitric oxidate that activates metalloproteinases, degrading cell junctions