Modulation of NRF2/KEAP1 Signaling in Preeclampsia

Placentation is a key and tightly regulated process that ensures the normal development of the placenta and fetal growth. Preeclampsia (PE) is a hypertensive pregnancy-related disorder involving about 5–8% of all pregnancies and clinically characterized by de novo maternal hypertension and proteinuria. In addition, PE pregnancies are also characterized by increased oxidative stress and inflammation. The NRF2/KEAP1 signaling pathway plays an important role in protecting cells against oxidative damage due to increased reactive oxygen species (ROS) levels. ROS activate NRF2, allowing its binding to the antioxidant response element (ARE) region present in the promoter of several antioxidant genes such as heme oxygenase, catalase, glutathione peroxidase and superoxide dismutase that neutralize ROS, protecting cells against oxidative stress damages. In this review, we analyze the current literature regarding the role of the NRF2/KEAP1 pathway in preeclamptic pregnancies, discussing the main cellular modulators of this pathway. Moreover, we also discuss the main natural and synthetic compounds that can regulate this pathway in in vivo and in vitro models.


Introduction
The placenta is an essential organ that deeply changes in morphology and function during pregnancy, performing important functions during pregnancy and ensuring the normal fetal development [1,2]. Preeclampsia (PE) is a hypertensive pregnancy-related disorder involving about 5-8% of all pregnancies. It generally appears from the second trimester of gestation and it is clinically characterized by de novo maternal hypertension (a diastolic blood pressure of ≥90 mmHg and/or systolic blood pressure of ≥140 mmHg) and proteinuria (>300 mg/24 h). A high BMI, previous preeclamptic pregnancy, advanced maternal age, nulliparity and severe COVID-19 are important risk factors of PE [3][4][5]. In the case of severe PE, there is also the risk for the mother to develop comorbidities such as eclampsia, hepatic alterations (hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome), renal failure and disseminated vascular coagulation (DIC). PE may also lead to further complications for the fetus, causing fetal growth restriction (FGR), prematurity and fetal death [6,7].
Although there have been many progresses in understanding the pathophysiology of this disease over the past decade, many of the key mechanisms characterizing PE are still unknown. In particular, it has been shown that an impaired invasion of the muscle [45]. The importance of NRF2/KEAP1 signaling in PE is further highlighted by the fact that many downstream genes of this pathway (e.g., SOD3, NQO1, catalase and HO-1) have showed protective effects against hypertension [46][47][48][49]. However, it deserves to be pointed out that no difference in blood pressure has been found between NRF2-knockout and wild-type mice, suggesting that hypertension is modulated by a more complex mechanism, where NRF2 plays a minor role [50].
An interesting study showed that knocking down NRF2 in pregnancy-associated hypertension (PAH) mice (NRF2 −/− PAH mice), a mouse model of PE, improved maternal and fetal survival, ameliorated intra-uterine growth retardation and increased oxidative DNA damage. Furthermore, placentas of these mice showed many characteristics of human preeclampsia including an increased endothelial cell proliferation and a dense vascular network. The authors also found that NRF2 deficiency in these mice induced the mRNA expression of angiogenic chemokines such as Ccl2 (encoding C-C motif chemokine ligand 2), Ccl5 (encoding C-C motif chemokine ligand 5), Cxcl9 (encoding C-X-C motif chemokine ligand 9) and Cxcl10 (encoding C-X-C motif chemokine ligand 10). Moreover, there was a significant increase in mRNAs encoding inflammation-related cytokines such as interleukin (IL)-1α, IL-1β, tumor necrosis factor (TNF)-α and C-X-C motif chemokine ligand 1. The authors also reported an increase in ROS levels in labyrinth placentas of NRF2 −/− PAH mice accompanied by a low NAD(P)H dehydrogenase quinone 1 (NQO1) and sulfiredoxin 1 (SRXN1) expression, two NRF2 downstream genes, in these placentas. Thus, ROS-mediated signaling plays a key role in maintaining placental angiogenesis in PE [51].
Utero-placental interaction plays a key role in the maternal-fetal crosstalk during pregnancy and abnormal uterine vascular remodeling has been associated with PE and FGR increasing the risk of a preterm birth [52,53]. It has been found that the oxidative stress level was significantly increased but no changes were found in decidual NRF2 and KEAP1 protein expression in the decidua of PE pregnancies complicated by FGR. Interestingly, PE pregnancies with normal fetal growth also showed increased decidual oxidative stress but NRF2 expression was reduced and KEAP1 expression was increased in uterine areas of high trophoblast density, suggesting that decidual oxidative stress is modulated by trophoblast cells. This is an important study that underlines the different pathophysiology of PE with and without FGR [54]. Impaired alteration of the NRF2 pathway in PE compared to normal pregnancies was also confirmed by using genome-wide transcriptional profiling of the decidua basalis. In fact, Løset and colleagues found an increased expression of genes belonging to the NRF2/KEAP1 signaling pathway among the genes differently expressed in the decidua from PE pregnancies [55]. Furthermore, Kweider and colleagues found that NRF2 immunostaining was significantly increased in endovascular and interstitial trophoblastic cells of the placental bed and in the extravillous trophoblast of pregnancies complicated by early-onset PE-FGR compared to normal ones [56].
In addition, it has been reported that NRF2 expression significantly decreases in the syncytiotrophoblast, villous stromal cells and vascular endothelium in placentas of PE pregnancies [57]. In contrast to this study, Wruck and colleagues found that NRF2 expression was exclusively expressed in the villous cytotrophoblast. Moreover, they found that NRF2 expression in cytotrophoblastic nuclei of PE placentas was significantly higher than normal gestation-matched controls, suggesting an increased activation of this signaling in PE while the syncytiotrophoblast was negative in both controls and PE placentas. Thus, the expression of NRF2 within cytotrophoblastic cells strongly suggests that these cells are involved in the expression of NRF2-dependent antioxidant genes under oxidative stress [58].
It is known that ATP-binding cassette (ABC) transporters regulate substrate flow between maternal and fetal circulation [59], playing a key role in the antioxidant defense of the placenta by pumping out toxic oxidative metabolites [60]. Moreover, it has been reported that NRF2 can stimulate the expression of these transporters by binding the ARE regions present in their gene promoter [22,24,61]. Yu and colleagues demonstrated that the levels of NRF2, heme oxygenase-1 (HO-1), ABC transporter multidrug resistance-associated protein (MRP) 1, -2, breast cancer resistance protein (BCRP) and P-glycoprotein (Pgp) were significantly lower both in PE placentas compared to normal pregnancies and in the placentas of early-onset PE compared to late-onset PE. NRF2 silencing in JEG3 cells and NRF2 knockdown in mice significantly downregulated the expression of HO-1, MRP1, -2, BCRP and Pgp [62]. Thus, the impaired NRF2 expression in PE pregnancies can favor the increase in oxidative stress by altering the expression of ABC transporters.

NRF2 Cellular Modulators
Ferroptosis is a programmed cell death process involved in the alteration of iron metabolism, amino acids, GSH, ROS and lipid peroxides (LPOs) in the plasma membrane [63]. Thus, ferroptosis plays a key role in regulating cell fate under oxidative stimuli. DJ-1 is a small binding protein located on cell membranes that acts as a sensor for the redox state, causing NRF2 dissociation from KEAP1 and its translocation into the nucleus to activate the NRF2-responding genes [64]. It has been reported that the inhibition of DJ-1 can enhance the sensitivity of tumor cells to ferroptosis inducers and that DJ-1 depletion plays a role in promoting ferroptosis [65]. DJ-1 expression has been found to be significantly higher in PE placentas than normal pregnancies, suggesting that the overexpression of DJ-1 in PE placentas could act as a compensatory mechanism for hypoxia [66]. Interestingly, it has been found that the concentration of malondialdehyde (MDA), a ferroptosis marker, was significantly higher in the PE pregnancies compared to normal pregnancies. Moreover, NRF2, DJ-1 and glutathione peroxidase 4 (GPX4) expressions were significantly higher in PE pregnancies. Importantly, authors found the NRF2 and GPX4 expressions were significantly reduced when DJ-1 was knocked down in BeWo cells and when these cells were treated with RSL3, a ferroptosis inducer. Lactate dehydrogenase (LDH) release was significantly increased in DJ-1 knockdown compared to wild-type BeWo cells, indicating increased cell death. In this study, the authors showed that ferroptosis is involved in the pathogenesis of PE and that DJ-1 plays an important protective role in this pathology by mediating ferroptosis in trophoblast cells through the regulation of the NRF2/GPX4 signaling pathway [67].
Vascular endothelial growth factor (VEGF) has been reported to be decreased in PE and may be involved in the activation of the NRF2 pathway. In fact, an interesting study found that VEGF activated NRF2 in a BeWo cell line, leading to an increase in antioxidative enzymes such as thioredoxin (Trx), thioredoxin reductase (TXNRD1) and heme oxygenase-1 (HO-1). HO-1 metabolizes heme to generate biliverdin, iron and carbon monoxide (CO). The latter enhances VEGF synthesis in vascular smooth muscle, promoting its relaxation and vasodilatation [68]. Importantly, authors found that NRF2 activation by VEGF was ERK1/2 dependent since NRF2 activation was inhibited by ERK inhibitors. Interestingly, they found an antioxidant effect of VEGF via a positive feedback loop. In fact, VEGF activated NRF2 in an ERK1/2-dependent manner, increasing HO-1 expression and the production of carbon monoxide (due to the HO-1 activity), which in turn up-regulated VEGF expression. Thus, decreased VEGF bioavailability during PE may result in higher vulnerability to oxidative damage [69].
It has been found that serum concentration of oxidized low-density lipoprotein (oxLDL) is higher in women with PE [70,71]. oxLDL is removed from circulation by lectin-like oxLDL receptor-1 (LOX-1), which acts as a scavenger receptor for oxLDL [72]. It has been reported that oxLDL activates the NRF2/KEAP1 signaling pathway [73]. Interestingly, Chigusa and colleagues found that LOX-1, NRF2 and HO-1 expression was significantly decreased in PE placentas compared to normal pregnancies. Moreover, oxLDL treatment of JAR cells significantly increased NRF2 and HO-1 expression while the blockade of LOX-1 by TS92, an anti-human LOX-1 antibody, significantly inhibited the increase in HO-1 expression induced by oxLDL treatment. Thus, the decreased LOX-1 expression in PE may contribute to the high oxLDL concentration, low NRF2 activation and low HO-1 expression found in this pathology [74].
It has been reported that increased oxidative stress and decreased antioxidant capacity are among the main factors involved in endothelial cell hyperpermeability, a key event in the pathogenesis of PE [75]. Lipoxins are endogenous mediators derived from arachidonic acid that play an important role as anti-inflammatory compounds [76]. It has been found that lipoxin A4 (LXA4) deficiency is associated with PE-like symptom onset [77,78]. Moreover, Pang and colleagues found that LXA4 strongly attenuated lipopolysaccharide (LPS)-induced hyperpermeability in human umbilical vein endothelial cells (HUVEC) through maintaining the normal expression of VE-cadherin and β-catenin. Interestingly, LXA4 inhibited LPS-triggered ROS production, promoting the expression of NRF2 and demonstrating a key role of the LXA4/NRF2 axis in regulating vascular permeability under oxidant stimuli [79].
It is known that PE pregnancies are exposed to chronic hypoxia due to the shallow invasion of trophoblasts into the uterine wall that fails in maternal spiral artery remodeling [80,81]. It has been reported that hypoxia promoted the translocation of NRF2 into the nucleus in HTR-8/SVneo cells, leading to NRF2/HO-1 signaling activation. Moreover, hypoxia reduced the invasion of HTR-8/SVneo cells and induced oxidative stress, increasing malondialdehyde (MDA), ROS and induced ferroptosis [82]. Interestingly, NRF2 overexpression in hypoxia-induced HTR-8/SVneo cells reduced the levels of MDA and ROS, and decreased ferroptosis, proving that NRF2 signaling activation plays a protective role in PE [83]. Another study found that under hypoxic conditions, the activity of catalase (CAT), GSH-Px and SOD enzymes in HTR8/SVneo cells was significantly lower. Moreover, hypoxic conditions increased NRF2 and HO-1 expression while decreasing KEAP1 expression. In addition, the enzymatic activity of SOD, GSH-Px and CAT in placental tissues of patients with PE was significantly lower compared to normal placental tissues. Interestingly, NRF2 and HO-1 expression was significantly higher while KEAP1 expression was lower in PE placentas compared to normal placentas. NRF2 silencing significantly reduced the activities of CAT, GSH-Px and SOD in HTR8/SVneo cells under hypoxic conditions, proving an important antioxidant effect of the NRF2/KEAP1 signaling pathway in these cells [84]. The previous two studies were further confirmed by Feng and colleagues, reporting lower CAT, GSH-Px and SOD activity in HTR8/SVneo cells under hypoxic conditions and in PE placentas. Furthermore, the authors reported increased NRF2 and HO-1 expressions and reduced KEAP1 expression under hypoxic conditions and in PE placentas [85].
Advanced glycation end products (AGEs), advanced oxidation protein products (AOPPs) and advanced lipid peroxidation products (ALEs) are the three most common biomarkers used to evaluate protein modifications caused by oxidative stress [86,87]. AOPPs are a family of oxidized, dityrosine-containing products formed by plasma proteins and chlorinated oxidants' interaction under oxidative stress commonly present in several diseases [88][89][90][91] including PE [92]. In fact, it has been found that AOPPs can increase oxidative stress, leading to trophoblast dysfunction and apoptosis, showing an important involvement of AOPPs in PE pathogenesis [92]. Interestingly, it has been found that AOPPs directly increased apoptotic protein expressions and significantly inhibited the expression of the NRF2/ARE/HO-1 pathway in HTR-8/SVneo cells. The authors found that NRF2 silencing significantly aggravated the AOPP-induced cell apoptosis, activating p53 and the caspase cascade while NRF2 overexpression had the opposite effect, leading to cytoprotective effects by increasing HO-1 expression in HTR-8/SVneo cells. Hence, NRF2/ARE/HO-1 pathway activation plays an important role in AOPP-induced cell apoptosis, suggesting this pathway as a therapeutic target against PE [93].
MicroRNAs (miRNAs) are short (around 20 nucleotides) non-coding RNA sequences involved in the regulations of several cellular processes in cancerous [94][95][96][97] and noncancerous [98][99][100][101] diseases. MiR-133a has been reported to be involved in inhibiting cancer cell proliferation in various cancer types [102,103]. However, miR-133a also showed important protective functions in non-cancerous diseases such as hypoxia-induced oxidative stress in cardiomyocytes, reducing apoptosis [104]. Oxidative stress is also a characteristic feature of PE pregnancies and is the cause of many complications found in this pathology [31]. An interesting study found that transfecting HTR-8/SVneo cells with miR-133a-3p under an oxidative stress condition (induced by H 2 O 2 ) relieved the oxidative stress induced by H 2 O 2 through the reduction in ROS and MDA levels. Moreover, the authors found reduced oxidative stress-induced apoptosis. The authors proved that this antioxidant effect of miR-133a-3p was due to the inhibition of its target gene BTB domain and CNC homolog 1 (BACH1), a transcriptional repressor that competes with NRF2 for binding to ARE (antioxidant response element) sites on target genes. BACH1 can inhibit NRF2 activity by preventing its binding to ARE sites [105]. In fact, miR-133a-3p overexpression decreased BACH1 expression and increased NRF2 activation, leading to an increase in HO-1 expression [106]. Thus, miR-133a-3p can relieve the oxidative stress-induced apoptosis of trophoblast cells through the BACH1/NRF2/HO-1 signaling pathway. MiR-1246 is known to target Axin-2 and glycogen synthase kinase-3 β (GSK3β), two inhibitors of the Wnt/β-catenin pathway [107], which is involved in trophoblast proliferation and differentiation [108]. Another protein involved in trophoblast differentiation is the aromatase CYP19A1, which synthetizes estrogens, key hormones that regulate syncytiotrophoblast differentiation [109]. An interesting study by Muralimanoharan and colleagues found that NRF2, CYP191A mRNAs and miR-1246 levels were significantly upregulated in a primary culture of human trophoblast cells during syncytiotrophoblast differentiation and significantly reduced by hypoxia and in PE placentas. Moreover, the expression of Axin-2 and GSK3β was significantly downregulated during syncytiotrophoblast differentiation. The authors also found a downregulation of Jumonji and AT-rich interaction domain containing 2 (JARID2), a cell differentiation inhibitor [110]. NRF2 silencing in cytotrophoblast cells significantly inhibited miR-1246 and CYP19A1. Furthermore, NRF2 could bind miR-1246 and CYP191A promoters, explaining their increase during syncytiotrophoblast differentiation. Thus, NRF2 can promote syncytiotrophoblast differentiation by inducing JARID2 expression, and CYP19A1 and miR-1246, which inhibit the expression of Axin-2 and GSK3β [111].
CD151 is a member of tetraspanins [112] with antioxidant properties [113]. An interesting study by Wang and colleagues found that CD151 expression was significantly downregulated in PE pregnancies. Moreover, PE pregnancies showed a decreased expression of the antioxidant enzymes HO-1, NQO1, glutamate-cysteine ligase catalytic subunit (GCLC) and SOD-1. Overexpression of CD151 in HTR-8/SVneo cells enhanced HO-1, NQO1, GCLC and SOD-1 expression. These effects were reverted after silencing of CD151 in these cells. Interestingly, tail intravenous injection of siCD151 in pregnant mice led to a PE-like phenotype inducing hypertension and proteinuria. Moreover, the expression of NRF2, pERK1/2, HO-1, NQO-1, GCLC and SOD-1 was significantly decreased in mice and HTR8/SVneo when CD151 was silenced, suggesting an involvement of ERK signaling in CD151 antioxidant function. In fact, the beneficial effect of CD151 was significantly inhibited when ERK and NRF2 signaling were blocked with synthetic inhibitors (SCH7 72984 and ML385, respectively) in HTR8/SVneo cells. Thus, in trophoblast cells, CD151 exerts its antioxidant function trough NRF2 and ERK signaling pathways [114].
Bromodomains (BRDs) are functional domains present on the bromodomain and extraterminal (BET) family of proteins. The function of these domains is to activate gene transcription by recruiting polymerases and transcription factors. Among the BRD proteins, BRD4 protein has been studied as a regulator of oxidative stress [115]. BRD4 mRNA and protein expression increased after H 2 O 2 exposure while BRD4 inhibition attenuated H 2 O 2 -induced oxidative stress injury in an HTR-8/SVneo cell line [116]. BRD4 knockdown increased cell proliferation and invasion while decreasing apoptosis and ROS production following H 2 O 2 exposure. Moreover, suppression of BRD4 significantly decreased KEAP1 expression, but increased the nuclear expression of NRF2. The protective effect of BRD4 inhibition was reversed by KEAP1 overexpression or NRF2 inhibition. Thus, BRD4 inhibition attenuated oxidative stress injury by enhancing NRF2 activation via KEAP1 downregulation.
A schematic representation of NRF2/KEAP1 pathway regulation by cellular modulators is reported in Figure 1. Studies discussed in this section are summarized in Table 1. BRD4 HTR8/SVneo exposed to H 2 O 2 BRD4 inhibition attenuated oxidative stress injury by enhancing NRF2 activation via the downregulation of KEAP1. [116] CD151 HTR-8/SVneo cells and mice pertension and proteinuria. The expression of NRF2, pERK1/2, HO-1, NQO1, GCLC and SOD-1 was decreased in mice and HTR8/SVneo cells when CD151 was silenced. The beneficial effect of CD151 in HTR8/SVneo cells was inhibited when ERK and NRF2 signaling was blocked with synthetic inhibitors. [114] BRD4 HTR8/SVneo exposed to H2O2 BRD4 inhibition attenuated oxidative stress injury by enhancing NRF2 activation via the downregulation of KEAP1. [116]

NRF2 Modulation by Natural Compounds
Procyanidin B2, widely distributed in plants, exhibits antioxidative activity, mitigation of endoplasmic reticulum stress and anti-inflammatory effects [117]. In a rat model of PE, the increased soluble fms-like tyrosine kinase-1 (sFlt-1) levels were associated with decreased activity of peroxisome proliferator-activated receptor γ (PPARγ). PPARγ is a transcription factor involved in antioxidative stress, trophoblast differentiation, anti-inflammation and normal vascular function and it is induced by procyanidin B2 in primary placental tissues and endothelial cells. Since NRF2 has been reported to bind the PPARγ

NRF2 Modulation by Natural Compounds
Procyanidin B2, widely distributed in plants, exhibits antioxidative activity, mitigation of endoplasmic reticulum stress and anti-inflammatory effects [117]. In a rat model of PE, the increased soluble fms-like tyrosine kinase-1 (sFlt-1) levels were associated with decreased activity of peroxisome proliferator-activated receptor γ (PPARγ). PPARγ is a transcription factor involved in antioxidative stress, trophoblast differentiation, antiinflammation and normal vascular function and it is induced by procyanidin B2 in primary placental tissues and endothelial cells. Since NRF2 has been reported to bind the PPARγ promoter region, enhancing its transcriptional activity, the NRF2/PPARγ signaling pathway is involved in regulating the inhibitory effect of procyanidin B2 on sFlt-1 secretion [118].
Resveratrol is a polyphenolic compound present in a variety of fruits, mostly in red grapes. In cultured endothelial cells, resveratrol up-regulates gene expression of the antioxidant defense enzymes NQO1 and HO-1 in an NRF2-dependent manner [119]. HUVEC incubated with plasma from women with PE showed increased antioxidant response element (ARE) activity and the addition of resveratrol was able to potentiate this effect. Oxidative stress is a stimulus that leads to the NRF2 activation, which may be enhanced by the addition of resveratrol in PE [120]. Thus, resveratrol could mitigate or reverse placental and endothelial cellular oxidative stress. In particular, NRF2 can reduce activin A, and OH-1 decreases sFlt-1 levels [121,122]. Interestingly, the experiments of Gurusinghe et al. [123] showed that resveratrol decreased vascular cell adhesion molecule 1 (VCAM-1) and endothelin-1 expression in endothelial cells while decreasing sFlt-1 and activin A in NRF2 knockdown placental cells.
Recently, various studies evaluated the antioxidant activity of natural compounds such as flavonoids in the activation of NRF2. Silibinin is a flavonolignan composed of a flavonoid and a phenylpropane isolated from Silybum marianum, which has been shown to exhibit antioxidant and antineoplastic activities. A study showed that silibinin may reduce apoptosis in extravillous trophoblast cell lines while recent studies suggested that silibinin enhanced the activation of NRF2 in HTR8/SVneo cell lines treated with H 2 O 2 [124]. Among flavonoids, fisetin (3,3 ,4 ,7-tetrahydroxyflavone) is a natural flavonoid commonly found in many fruits and vegetables with a wide range of pharmacological functions including anti-inflammatory, antioxidant and anti-tumor activities [125]. The antioxidant activity has been demonstrated by increasing the transcriptional activity of NRF2 [126]. In preeclampsia-like rat models, fisetin reduced the clinical and biochemical setting of PE by activation of Toll-like receptor 4 (TLR4)/NF-κB and NRF2/HO-1 pathways [127].
Apocyanin and sulforaphane are natural compounds widely present in plant extracts with biological interest [22,128]. Apocyanin is an inhibitor of the NADPH-oxidase complex that acts on impairing membrane translocation of the cytosolic component p47phox (a member of the NADPH-oxidase complex) [129]. Combined treatment of apocyanin and aspirin ameliorated the PE symptoms by activating the phosphoinositide 3-kinase (PI3K)/NRF2/HO-1 pathway in rat models of PE [130]. Sulforaphane, an aliphatic isothiocyanate found in many cruciferous vegetables, is rapidly metabolized after catalyzation by glutathione S-transferase. Sulforaphane is believed to exert antioxidant effects by up-regulating NRF2 expression [32,131]. An interesting study evaluated the effect of sulforaphane in endothelial dysfunction, a characteristic of PE pregnancies. This study demonstrated that sulforaphane is able to protect endothelial cells and reduce placental secretion of vasoactive agents such as endothelin 1, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. In placental explants, sulforaphane reduced sFlt-1, soluble endoglin (sEnd) and activin A secretion. However, this effect was not blocked by NRF2 silencing, suggesting that the NRF2 signaling pathway is not involved in this mechanism [132].
Crocin is a hydrophilic carotenoid derived from saffron (or Crocus sativus) with beneficial effects on cardiovascular pathologies, obesity and hypertension [133]. In a PE rat model, the administration of crocin led to a significant reduction in blood pressure and placental growth factor (PlGF) and decreased sFlt-1 levels. Clinically, an increase in the fetal weight, fetal survival and fetal/placenta ratio was achieved. These phenotypic changes were accompanied by upregulation of protein levels of NRF2 and HO-1 [134]. Astragaloside IV, an extract from Astragalus membranaceus, has been studied primarily for its properties on the cardiovascular system through its action in protecting the endothelium from lipoprotein damage and hyperglycemia [135]. This compound has been studied for its anti-inflammatory action in liver, lung and cardiovascular disease [136]. In a PE rat model, administration of astragaloside IV, in a dose-dependent manner, significantly reduced sFlt-1 and PlGF levels as well as improved the systolic blood pressure, proteinuria, placental weight and overall fetal survival. These clinical improvements were accompanied by an overall reduction in placental cellular oxidative stress, inducing a partial recovery of NRF2 and HO-1 placental levels [137].
Pyrroloquinoline quinone is an essential animal nutrient with important functions on mammalian development, growth, reproduction and immune function [138]. Pyrroloquinoline quinone is an important antioxidant compound, which exerts its function through the protection of the mitochondria against oxidative stress-induced lipid peroxidation, protein carbonyl formation and inactivation of the mitochondrial respiratory chain [139]. In a PE rat model, dietary intake of pyrroloquinoline quinone significantly increased NRF2 expression [140]. Another powerful antioxidant, 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), a derivative of vitamin E, showed several positive effects on diabetes, hepatic cirrhosis, neurodegenerative diseases and cancer [141][142][143]. It has been reported that HTHQ treatment of a PE mouse model significantly induced nuclear translocation and expression of NRF2, leading to an increased HO-1 expression in placental tissues and providing antioxidant and antiapoptotic effects [144].
Coenzyme Q10 is a fat-soluble ubiquinone with intracellular antioxidant activity synthesized endogenously from phenylalanine (benzoquinone ring) and mevalonic acid (responsible for isoprenoid units) but it can also be provided (in a minor part) from the diet. In the mitochondrial respiratory chain, it is responsible for electron transport from complex I and II to complex III [145,146]. Coenzyme Q10 was found to play an important role as an antioxidant in plasma lipoproteins, where it also regenerates the active form of vitamin E. When orally administered to mice [147] and rabbits [148], coenzyme Q10 showed a significant anti-atherosclerotic effect. It has been reported that coenzyme Q10 levels were significantly decreased in PE pregnancies [149]. Coenzyme Q10 treatment induced NRF2 and HO-1 up-regulation in the placental tissues of PE rat models (both nuclear and total NRF2 protein levels) [150].
Melatonin, a lipid-soluble hormone with antioxidant activities, is primarily produced by the pineal gland but it is also produced in large quantities by the placenta and it is rapidly transferred from maternal to fetal circulation [151]. This hormone regulates the circadian rhythm but also shows direct free radical scavenging properties and can induce the expression of antioxidant enzymes [152]. The melatonin levels are significantly reduced in PE, as well as decreased levels of the enzymes responsible for melatonin synthesis and decreased levels of melatonin receptors [153]. In animal models (sows), dietary supplementation with melatonin was associated with a significant increase in placental and newborn weight and with a significant increase in placental mRNA levels of antioxidant-related genes involved in the NRF2/ARE pathway (NRF2, SOD, GPx1 and NQO1) [154]. When administrated intraperitoneally to a PE rat model, melatonin treatment decreased blood pressure and proteinuria. These effects were accompanied by an increased serum sFlt-1/PlGF ratio and increased levels of NRF2, PlGF and HO-1 in the placenta [155]. These effects have been found to be comparable to the use of aspirin, a drug that significantly reduces PE onset and mortality [156]. The properties of melatonin on PE have also been studied in humans. In explants of PE placentas, melatonin did not reduce placental production of activin A, sFlt-1 or sEnd but it reduced oxidative stress, increasing NRF2 and HO-1 expression [157].

NRF2 Modulation by Synthetic Compounds
Tert-butylhydroquinone (tBHQ) is a synthetic aromatic organic compound, which derives from hydroquinone (substituted with a tert-butyl group). In humans, tBHQ acts as a phase II detoxification enzyme inducer and a NRF2 agonist [158]. In fact, tBHQ increases the level of NRF2, inhibiting its degradation [159]. tBHQ treatment increased NRF2 protein and HO-1 mRNA expression in an HTR-8/SVneo cell line. In addition, these authors found that serum levels of HO-1 protein decreased while placental HO-1 mRNA levels, HO-1 and NRF2 proteins increased in severe PE [160].
Simvastatin is a well-known statin extensively used to reduce the morbidity associated with cardiovascular disease and the risk of developing Alzheimer's disease. Many studies showed that statins activate HO-1, decrease ROS levels and have antioxidant effects in some cancer cells, fibroblasts and neuronal cells [161]. In trophoblast cells, under experimental hypoxia conditions, simvastatin has been shown to counteract oxidative stress through activation of NRF2 signaling. This activation is dependent on KEAP1 inhibition while NRF2 knockdown resulted in insufficient augmentation of HO-1, GCLC and glutamate-cysteine ligase modifier subunit (GCLM) mRNA expression under oxidative stress [162].
Angiogenesis, inflammation and oxidative stress are among the most important processes involved in PE pathogenesis. An interesting study evaluated the role of NRF2 in modulating these three key processes involved in PE onset. To this aim, Zhang and colleagues found that ML385 (a synthetic NRF2 inhibitor) treatment slightly reduced systolic blood pressure increases and proteinuria in a PE rat model while treatment with CDDO-Im (a NRF2 activator) significantly increased placental HO-1 expression (showing a protective effect) but also increased proteinuria and blood pressure, worsening PE. These authors also found no morphological changes in placentas of rats treated with ML385 and CDDO-Im. However, treatment with ML385 did not lead to significant alteration in HO-1 expression in placental tissues. Moreover, inhibition of NRF2 significantly increased the levels of chemokine 2 (CCL2), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), angiotensin II receptor type 1 (AT1R) and ROS in the embryonic tissues. Important results were also obtained in in vitro studies using HTR8/SVneo and hESC cells. In fact, NRF2 knockdown significantly suppressed cell proliferation, improved cell apoptosis and invasion and increased ROS and HO-1 expression. This study showed that inhibition of NRF2 increased oxidative stress, apoptosis and inflammation in the placental tissues but also increased placental angiogenesis and improved fetal and maternal outcomes, suggesting that the relationship between NRF2 and HO-1 expression in PE might not be a simple upstream and downstream relationship but a more complex pathway where other factors play an important role [163]. A schematic representation of NRF2/KEAP1 pathway modulation by natural and synthetic compounds is reported in Figure 2. Studies discussed in this section are summarized in Table 2.

Conclusions
The NRF2/KEAP1 pathway could be a promising target for treatment of PE, improving or avoiding the onset of this disease. In this review, we discussed several studies focusing on the role of the NRF2/KEAP1 signaling pathway in regulating the oxidative stress response in placental cell lines and in in vivo models. We found that NRF2 can be regulated by important cell modulators such as DJ-1, VEGF, oxLDL, LXA4, AOPPs, miR-133a-3p, CD151 and BRD4 (see Table 1 and Figure 1), favoring the cell response to oxidative stress, avoiding apoptosis and ferroptosis and promoting cell proliferation. Moreover, this pathway plays a key role in important processes for placental development such as hypoxia and syncytiotrophoblast differentiation. These processes are deeply impaired in PE placentas. In addition, we showed that natural and synthetic compounds can act as potent antioxidants, activating the NRF2/KEAP1 signaling pathway, reducing apoptosis and inflammation and improving the clinical signs of PE. In particular, some compounds can   NRF2 knockdown abolished some of the protective effects of resveratrol on endothelial cells, but not in primary trophoblast cells. [123] Silibinin HTR8/SVneo exposed to H 2 O 2 Silibinin protects the trophoblast from apoptosis, enhancing the activation of NRF2. [124] Fisetin PE rat model Reduction in hypertension and proteinuria; reduction in TNF-α, IL-6, IL-1β, MDA and the sFlt-1/PlGF ratio; and promoting the NRF2/HO-1 pathway in placental tissues. [127] Apocyanin PE rat model Reduction in preeclampsia symptoms with combined treatment of apocyanin and aspirin by activating the PI3K/NRF2/HO-1 pathway. [130] Sulforaphane HUVEC and placental explants In HUVEC, reduction in endothelin-1, VCAM1, ICAM1 and E-selectin. In placental explants, reduction in sFlt-1, endoglin and activin A. In HUVEC, induction of activation and nuclear translocation of NRF2, and induction of HO-1. NRF2 silencing blocked some but not all of sulforaphane's effects and did not prevent inhibition of trophoblast secretion of sFlt-1 or activin A. [132] Crocin PE rat model Crocin upregulated protein levels of NRF2 and HO-1. [134] Astragaloside IV PE rat model Improvements in clinical signs of preeclampsia, reduction in placental cellular oxidative stress and strengthening of the NRF2/HO-1 signaling pathway in placental tissues. [137] Pyrroloquinoline quinone PE rat model Pyrroloquinoline quinone improved the antioxidation effect in preeclampsia models, activating the NRF2 pathway. [140] 1-O-hexyl-2,3,5trimethylhydroquinone (HTHQ) PE mouse model HTHQ treatment induced NRF2 expression and nuclear translocation, increasing HO-1 expression in placentas. [144] Coenzyme Q10 PE rat model Coenzyme Q10 protected the rats from preeclampsia through activating the NRF2/HO-1 pathway. [150] Melatonin Pregnant sows Increase in mRNA levels of antioxidant-related genes involved in the NRF2/ARE pathway (NRF2, SOD, GPx1 and NQO1). [154] Melatonin PE rat model Increased NRF2, PlGF and HO-1 placental levels with reduction in blood pressure and urine protein content, and recovery in the fetus alive ratio, fetal weight and fetal weight/placental weight ratio. [155] Melatonin Placental explants Improved oxidative stress, presumably due to the potentiation of NRF2 and HO-1. [157] Tertbutylhydroquinone (tBHQ)

HTR-8/SVneo
Increased NRF2 protein and HO-1 mRNA expression after stimulation with tBHQ. HO-1 was located in the cytoplasm and NRF2 was located in both the nucleus and cytoplasm. [160] Simvastatin JAR cells exposed to hypoxia and treated with diethyl maleate (DEM) In hypoxia conditions, activation of NRF2 signaling depending on KEAP1 inhibition. [162] ML385 and CDDO-Im PE rat model, HTR-8/SVneo and hESC cells ML385 treatment reduced SBP and proteinuria in PE rats while treatment with CDDO-Im increased proteinuria and systolic blood pressure, worsening PE. HO-1 expression decreased in the PE group compared with the control group while it increased after CDDO-Im treatment compared with the PE group. ML385 did not alter HO-1 expression in placental tissue. NRF2 inhibition increased CCL2, IL-1β, TNF-α, AT1R and ROS in the embryonic tissues. NRF2 knockdown in HTR-8/SVneo and hESC cells suppressed cell proliferation, improved apoptosis and invasion and increased ROS and HO-1 expression. [163]

Conclusions
The NRF2/KEAP1 pathway could be a promising target for treatment of PE, improving or avoiding the onset of this disease. In this review, we discussed several studies focusing on the role of the NRF2/KEAP1 signaling pathway in regulating the oxidative stress response in placental cell lines and in in vivo models. We found that NRF2 can be regulated by important cell modulators such as DJ-1, VEGF, oxLDL, LXA4, AOPPs, miR-133a-3p, CD151 and BRD4 (see Table 1 and Figure 1), favoring the cell response to oxidative stress, avoiding apoptosis and ferroptosis and promoting cell proliferation. Moreover, this pathway plays a key role in important processes for placental development such as hypoxia and syncytiotrophoblast differentiation. These processes are deeply impaired in PE placentas. In addition, we showed that natural and synthetic compounds can act as potent antioxidants, activating the NRF2/KEAP1 signaling pathway, reducing apoptosis and inflammation and improving the clinical signs of PE. In particular, some compounds can improve newborn weight, systolic blood pressure, proteinuria, placental weight and overall fetal survival (see Table 2 and Figure 2).
The studies reviewed above suggest that several compounds and cell modulators can regulate the NRF2/KEAP1 pathway. Thus, these compounds may be used in combination with classical therapy to improve the efficiency of the treatment ameliorating/avoiding PE onset.