Nrf2 as a therapeutic target in ischemic stroke

1. Introduction

Acute ischemic stroke treatment has progressed significantly in recent decades, and in most industrialized countries, it ranks among the deadliest diseases. Stroke is essentially caused by a blockage of a cerebral artery. Currently, there are only two modes of interventions approved: injection of a thrombolytic agent or thrombectomy. Both protocols must be performed by extremely limited World Stroke Centers that have unique brain-imaging facilities and highly skilled neurosurgeons, neurologists, and intensive care teams. Moreover, success in these interventions is dependent on a very narrow window of time after the onset of stroke, and while several neuroprotective therapies have been tested, they often fail due to the time they are administered. When considering many other neurological disorders, the issue of reaching a target by needing to cross the blood-brain barrier (BBB) presents a significant problem. Thus, by understanding the etiopathology of stroke over time, after the acute insult, one can mitigate given neuropathological events by providing specific treatment. Though similar to other effective treatments for complex diseases (e.g., breast tumor, HIV), targeting a single pathway is not sufficient. Restoration of the loss of brain homeostasis after stroke should be a priority. Therefore, targeting a transcription factor that can restore this overall imbalance is a logical step in acute treatment, and it can also build brain resistance against further attack. Nrf2 is now recognized as one of the foremost transcription factors with the required pleiotropic actions [1]. Monomodal “neuroprotective„ drugs with a single mechanism of action have failed to support long-term results. Activation of a multipronged approach by small Nrf2 activators is likely what is necessary to add to current interventions to limit acute and secondary ischemic damage and prevent further brain attack, overall enhancing the brain’s resilience to stroke to improve patient-level health outcomes.

2. Role of Nrf2 in the central nervous system

When exposed to stress, Nrf2 activation enables its translocation to the nucleus and subsequent activation of transcription factors, including cytoprotective and antioxidant genes through the antioxidant-response element (ARE) sequence [2]. Nguyen et al. [3] were the first to propose the use of the Nrf2/ARE pathway to induce antioxidant enzymes, and since, this pathway has been thoroughly studied as an effective tool for its wide range of downstream targets, that will be later discussed. Nrf2 is a transcription factor that plays a crucial role in the upregulation of pathways specific to alleviating cellular oxidative stress and the protection of cells from oxidative stress damage through its pleiotropic action. Pleiotropism is defined as the ability of one gene to influence several downstream pathways. The Nrf2 protein upregulates several pathways, including heme/iron metabolism, metal ion regulation, antioxidant regeneration, glutathione regeneration, thioredoxin, and intracellular protein recycling [1]. Recent preclinical studies have investigated the Nrf2/ARE pathway in permanent cerebral ischemia models and found that the upregulation of Nrf2 led to higher expression levels of antioxidant proteins such as heme oxygenase-1 (HO1), manganese superoxide dismutase (SOD2), and NADPH quinone reductase 1 (NQO1) [2,4,5]. Interestingly, it has been reported that in humans, the HO1 promoter has one of the most responsive ARE elements within its promoter, making it a unique targeted heat shock protein and also a useful tool for drug screening. Additionally, numerous preclinical studies have revealed the cytoprotective effects of Nrf2. For instance, different ischemia models involving Nrf2-deficient mice provide evidence regarding the functional benefit of Nrf2 on infarct volume, brain edema, and neurobehavioral deficits [2]. Consequently, it is well studied that Nrf2 plays a role in regulating inflammation. Nrf2 activation inhibits various inflammatory mediators and enzymes, including proinflammatory cytokines, cell adhesion molecules, matrix metalloproteinases (MMPs), cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) [6]. The Nrf2 pathway also interacts with the protein complex NF-κB pathway, which is involved in multiple processes such as inflammation. Numerous preclinical studies involving Nrf2-deficient mice have revealed the important role Nrf2 plays in counteracting the NF-κB-driven inflammatory response through cross-talk between the two pathways [6]. NF-κB is also connected to apoptosis, and inhibition of this pathway via Nrf2 suggests that Nrf2 plays a role in protection against so-called apoptosis/cell death and necrosis [6,7].

The transcription factor Nrf2 binds to the ARE gene sequence and increases expression of antioxidant and anti-inflammatory proteins, including NQO1, cytochromes, SOD2, catalase, glutathione peroxidases, glutathione reductase, glutathione S-transferase, glutamate-cysteine ligase subunits, thioredoxin reductase, peroxiredoxin 1, HO1, haptoglobin, biliverdin reductase, ferritin light chain, metallothionein 1, proteasomal subunits, and heat shock proteins [1]. Additionally, Nrf2 reduces the expression of inflammatory proteins, including iNOS, proinflammatory cytokines, cell adhesion molecules, MMPs, COX2, the NF-kB pathway, and the apoptotic pathway [3]. By upregulating anti-inflammatory pathways and inhibiting inflammatory pathways, it is evident that Nrf2 plays a critical role in ischemic stroke pathophysiology.

3. Nrf2 as treatment in ischemic stroke

Reactive oxygen species accumulate in the brain after ischemic stroke and contribute to neuronal cell death and BBB disruption [7]. Therefore, the significance of targeting transcription factor Nrf2 lies in its inherent ability to activate numerous downstream cytoprotective proteins and pathways. The exhibited pleiotropic action of Nrf2 thus renders a significant pharmaceutical putative advantage for targeted drug design. Currently, Nrf2-activating drugs are being evaluated for their utility and competitiveness in regard to pharmacokinetics as well as pharmacodynamics. One example is the drug omaveloxolone, which has been assessed in monkeys and revealed an increase in plasma concentrations within 1 hour of administration for a total of 24 hours. It is important to note the half-life variability between single-dose and repeated administration, with single dose exhibiting extended upregulation of Nrf2 [8]. The increased effectiveness as a single dose poses a significant advantage when it comes to administering Nrf2-inducing drugs for acute treatment in ischemic stroke. When specifically evaluating omaveloxolone, reported side effects in humans include upper respiratory tract infections, nasopharyngitis, fatigue, diarrhea, and nausea, although no severe adverse events were reported [9]. Adverse side effects of Nrf2 could be mitigated by using better Nrf2-activating drugs, optimizing drug-delivery methods, and using multiple Nrf2-activating drugs through a multipronged approach in the acute phase after ischemic stroke. It is essential to bring previously studied Nrf2-activating drugs into clinical trials to evaluate the most effective therapy and reduce off-target effects. An example of another drug that has shown promising results is auranofin, which is an oral anti-arthritic medication previously modeled in zebrafish. The protective effects of auranofin against oxidative damage through the induction of Nrf2 yielded statistically significant results with no reported severe adverse effects in acute treatment [10].

Many preclinical studies have shown the promise of targeting Nrf2 in ischemic stroke, although clinical trials have yet to explore this pathway. However, there have been a handful of clinical trials in recent years studying Nrf2 activation in other diseases. Sulforaphane, a derivative of cruciferous vegetables, is a known potent stimulator of Nrf2 and has been used as a drug and a dietary supplement in clinical trials [11,12]. Broccoli sprout extracts, which contain sulforaphane, have been used to test the activation of Nrf2. Studies using broccoli sprout extracts have shown positive and inconclusive results, but no studies have shown any severe adverse effects from sulforaphane treatment [7,8]. Sulforaphane is tolerated well with participants, although some have reported gastrointestinal side effects during their treatment, such as heartburn, nausea, stomach pain, and diarrhea [11,12].

4. Significance

Many of the reported side effects of Nrf2-inducing drugs may be mitigated because they would be administered only as an acute treatment after ischemic stroke, not as a chronic treatment. Studies that have assessed the effectiveness of Nrf2 in acute treatment of stroke are uniquely summarized in a review conducted by Zhang et al. [13]. One study, for example, was done by Tanaka et al. [14], which found that Nrf2 activity in the peri-infarct region of the brain peaked 8 hours after transient middle cerebral artery occlusion (tMCAO), and Nrf2 activity decreased 24 hours after tMCAO in mice. Expression of antioxidant proteins downstream of Nrf2, such as heme oxygenase 1, glutathione, and thioredoxin, increased 24–72 hours after reperfusion after tMCAO [14]. These results indicate the effectiveness of Nrf2 induction acutely and provide evidence to further evaluate Nrf2-activating drugs after ischemic stroke. Moreover, Liu et al. [2] summarized many preclinical studies that found brain edema, neuronal death, inflammation, and neurobehavioral deficits were significantly reduced if Nrf2-inducing drugs were administered 2–4 hours after MCAO in mice and rat preclinical models. However, many of these studies were conducted on different strains of mice that received differing levels of brain damage after MCAO, and they did not assess long-term outcomes after acute treatment [2]. While the ideal time to administer Nrf2-inducing drugs after ischemic stroke and the ideal dose of Nrf2-inducing drugs has yet to be determined, there is ample preclinical evidence suggesting that Nrf2 plays numerous neuroprotective roles after ischemic stroke [2]. It is time to bring Nrf2-inducing drugs into phase 1 and 2 clinical trials to determine the ideal dosage and administration time of Nrf-inducing drugs and better understand the cytoprotective capabilities of Nrf2 in humans.

5. Expert opinion

The role of Nrf2 has been extensively studied in preclinical neurological (acute and chronic) models, including those involving larger animals, such as piglets, cattle, and monkeys. Nrf2 exhibits the potential to be an effective therapeutic target due to its pleiotropic action and subsequent upregulation of multiple cytoprotective pathways. Despite its relative safety profile as exemplified by several Nrf2 drugs that have been approved and used for a number of years (notably in the field of psoriasis and multiple sclerosis), there is limited interest for pharmaceutical companies to invest because many Nrf2 inducers are natural compounds that are already widely used. Therefore, small drugs that are derivatives of phytochemical Nrf2 inducers are being designed that could be patented to further increase pharmaceutical interests. As most of the so-called neuroprotective drugs have failed in clinical trials, rigorous effort should be devoted to ensure they cross the BBB and have sufficient pharmacodynamics to reach the brain region and cell target and be maximally effective. Evaluating upstream regulators of Nrf2, such as protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and phosphoinositide 3-kinases (PI3K), as pharmacological targets should be considered in addition to small derivatives of Nrf2 to increase the potential for pharmaceutical investments [15,16]. Targeting various upstream regulators of Nrf2, instead of Nrf2 itself, may prove beneficial in maintaining the integrity of Nrf2 without damaging any molecular processes of the cell. Moreover, efforts are underway to screen approved drugs using well-established screening tools, with the ultimate goal of repurposing drugs with an increased safety profile and efficacy for a given condition.

Additionally, it is not entirely known how the Nrf2 pathways are involved in cross-talk with other signaling pathways and how much control the Nrf2 pathway has on the expression of approximately 200 downstream proteins in humans to restore hemostasis. Most of these induced proteins work together in the xenobiotic transformation and restore cell and organ homeostasis. However, another reason for the reticence of pharmaceutical companies is the wide and various metabolic paths being activated, not only the antioxidant or anti-inflammatory pathways. The combination of these direct and indirect biological effects may raise concerns about unwanted side effects. Ideally, to minimize off-target effects, Nrf2-inducing drugs could be inactive when first administered and activated when the drug crosses the BBB or when the drug is exposed to oxidative stress, such that the bioactive component would be released when and where it is most needed. Other pharmaceutically promoted means use a slow and sustained release of a bioactive Nrf2 activator metabolite. While some have suggested that chronic stimulation of the Nrf2 pathway may lose its power over time, acute conditions, such as stroke, are timely diseases that should be rigorously investigated in the clinical setting.

Overall, Nrf2 should be considered as a prime target for novel drug design because of its multi-target cytoprotective action as a transcription factor. Such a multipronged approach is what is needed to target complex acute and chronic neurological diseases, with stroke being a relevant example of a common etiopathology. This is further exemplified by the fact that essentially all single neuroprotective drug therapies have failed in stroke. Stroke is also a prime example because there is a brief opening of the BBB where with thrombectomy drugs can potentially be delivered at the site of the clot removal or intravenously in combination while infusing the clot-buster agent rt-PA. Therefore, the Nrf2-activated pathways could act on many of the known cascades of events after stroke, notably in cases of ischemic-reperfusion injury following rt-PA clot lysis or clot removal by thrombectomy. Additionally, Nrf2-inducing molecules could be combined with other drugs to have bifunctional properties, for example with CO-donors, MMP inhibitors, NADPH oxidase (NOX) inhibitors, other anti-inflammatory drugs, and anti-cell death medication to minimize the deleterious outcomes of acute and chronic stroke [17]. Overall, we believe that Nrf2 is a key target to boost brain resistance against oxidative stress and other inflicted stress as well as the resistance of the brain's various cell types, cells of the blood vessels, neurons, astrocytes, and microglia. Nrf2 could extend the therapeutic window of conventional/tailored neuro intensive care unit interventions.

Abbreviations

ARE:	=	antioxidant-response element
BBB:	=	blood-brain barrier
COX2:	=	cyclooxygenase-2
HO1:	=	heme oxygenase-1
iNOS:	=	inducible nitric oxide synthase
MAPKs:	=	mitogen-activated protein kinases
MMP:	=	matrix metalloproteinase
NOX:	=	NADPH oxidase
NQO1:	=	NADPH quinone reductase 1
PI3K:	=	phosphoinositide 3-kinase
PKC:	=	protein kinase C
SOD2:	=	manganese superoxide dismutase
tMCAO:	=	transient middle cerebral artery occlusion

Declaration of Interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose

Additional information

Funding

This work was supported in part by grants from the Brain Aneurysm Foundation, the American Heart Association, the National Institutes of Health (R21NS095166, NS103036, NS110008, NS116076), and the Department of Defense (AZ180127).