Dissecting the Crosstalk Between Nrf2 and NF-κB Response Pathways in Drug-Induced Toxicity

1When our body is continuously exposed to external stresses, it’s easy to produce the excessive free radicals and reactive oxygen species (ROS) in the mitochondria, peroxisomes, and endoplasmic reticulum (ER), and downregulate endogenous antioxidants such as enzymes and antioxidant proteins (may be stress upregulated sometimes, but not enough to resist oxidative stress), thereby leading to damage of cellular components including proteins, DNA, and lipids. Antioxidant proteins are substances that can protect cells against free radical damage. However, supplementation of exogenous antioxidants can target oxidative stress by inhibiting the production of free radicals and ROS and bolstering the endogenous antioxidant capacity. Originally characterized as a master regulator of oxidative stress, the transcription factor Nrf2 continues to emerge as a critical mediator of cellular defense. Through the ongoing study on the Nrf2 pathway, more researchers find that Nrf2 is beneficial for cell survival and proliferation from redox homeostasis, drug/xenobiotic metabolism to DNA repair.

2Nrf2 is a key transcription factor with a basic-region leucine zipper (bZIP) structure in the Cap-n-Collar (CNC) family, which controls many aspects of cell homeostasis in response to oxidative stress and toxic insults in various tissues and cells. It is a modular protein composed of seven Nrf2-ECH homology domains (Neh1-7) that perform different functions. Neh2 contains two important motifs known as DLG and ETGE, which are essential for the interaction between Nrf2 and its negative regulator Kelch-like ECH-associated protein 1 (Keap1). The synthesized Nrf2 will translocate to the nucleus, and then form heterodimers with small musculoaponeurotic fibrosarcoma protein (MAF) K, G, and F. The heterodimer recognizes an enhancer sequence termed antioxidant response element (ARE) which is located in the regulatory regions of cellular defense enzyme genes. The target genes of Nrf2 encode the antioxidant proteins, metabolic enzymes, transporters, redox balancing factors, and glutathione-conjugated coenzyme.

3In the review of previous studies, the regulation of Nrf2 is considered a beneficial target for patients to release the disease. The best-characterized mechanism of Nrf2 regulation is the control of protein stabilization by the keap1-Cullin3 (Cul3)- Ring-box protein 1 (Rbx1) complex, which acts as a protein level rheostat. Keap1 binds to Nrf2 via a specific amino acid sequence on its homodimeric N-terminal domain, including siting with low (aspartate, leucine, and glycine; DLG) and high (glutamate, threonine, glycine, and glutamate; ETGE) affinity (hinge and latch hypothesis). Under the physiological state, Keap1 binds to Nrf2 and promotes ubiquitination and subsequent proteasomal degradation. Due to the short half-life of Nrf2 (10–30 min), Nrf2 remains at a very low basal level by sustained inhibition of Keap1. When stimulated by oxidative stress or Nrf2 activators, the thiol groups on cysteine residues in Keap1 are modified by electrophiles, and the function of Keap1 is diminished, ultimately resulting in the dissociation of Nrf2 from Keap1 and activation of Nrf2 and ARE genes. Studies show that products of the Nrf2/ARE pathway, including NADPH, NAD (P) H quinone oxidoreductase 1 (NQO1), glutathione peroxidase, and heme oxygenase 1 (HO-1), protect cells from various injuries and enhance body resistance through their activation (Figure 1A).

4Interestingly, Keap1-independent Nrf2 regulation is achieved through Glycogen synthase kinase 3 (GSK-3)/β-transducin repeat-containing protein (β-TrCP). β-TrCP is a substrate receptor for the S-phase kinase-associated protein 1 (Skp1)-Cul1-Rbx1/Roc1 ubiquitin ligase complex that targets Nrf2 for ubiquitination and proteasomal degradation. GSK-3 is responsible for phosphorylating a domain of Nrf2 (aspartate, serine, glycine, isoleucine, serine; DSGIS), creating a recognition motif for β-TrCP and then presenting Nrf2 to the Skp1-Cul1-Rbx1/Roc1 complex, finally leading to an alternative pathway for inhibition of Nrf2 (Figure 1B).

5Additional pieces of evidence suggest that other regulators also may activate the Nrf2 pathway. P62, for example, sequesters Keap1 to its autophagic degradation and ultimately activates Nrf2-dependent genes (Figure 1C).

6The NF-κB family, a regulator of κB light chain expression in mature B and plasma cells, regulates many genes involved in different cellular processes, such as cell differentiation, proliferation, development, and apoptosis. This family is composed of five structurally related members NF-κB1 (also called p50), NF-κB2 (also called p52), RelA (also called p65), c-Rel, and RelB, encoded by NF-κB1, NF-κB2, RELA, REL, and RELB, respectively. They share an N-terminal Rel homology domain (RHD) which is responsible for sequence-specific DNA binding, and homo- and hete-rodimerization. Based on the different mediators in the signaling cascade, the NF-κB signaling pathway can be categorized as either a canonical or noncanonical (alternative) pathway.

7Activation of the canonical NF-κB pathway relies on its ability to bind with DNA controlled by the inhibitor of κB (IκB). IκB protein, a subfamily of the large ankyrin repeat domain (ARD) containing superfamily, can bind NF-κB dimers to prevent the activation of NF-κB. The most common member of IκB in mediating the activation of the typical NF-κB pathway is IκBα, which plays a central role in subsequent transcription. Degradation of IκBα involves phosphorylation of IκB kinase (IκK) which is a trimeric complex composed of two catalytic subunits, IκKα and IκKβ, and a regulatory subunit, IκKγ (also named NF-κB essential modulator or NEMO). In response to external stimulation, NF-κB-bound IκB protein undergoes specific phosphorylation, ubiquitination, and proteasome-mediated proteolysis, allowing NF-κB to localize to the nucleus and bind to DNA (Figure 2A).

8On the contrary, activation of the non-canonical NF-κB pathway is slow and depend on a different set of protein synthesis, including CD40 ligand (CD40L), receptor activator of nuclear factor kappa-B ligand (RANKL), lymphotoxin beta (LTβR), and B cell-activating factor (BAFF). In addition, the non-canonical NF-κB pathway relies on the processing of the NF-κB2 precursor protein, p100 rather than the degradation of IκBα. The NF-κB inducing kinase (NIK) phosphorylates and activates IκKα, then synergistically regulating the phosphorylation of p100. After that, this phosphorylation in turn induces the ubiquitination and proteasome-mediated partial degradation of p100 to generate p52. Meanwhile, the degradation processing of p100 not only forms p52 but also allows the nuclear translocation of the noncanonical NF-κB complex p52/RelB and then activates the NF-κB. In the nucleus, p65 coordinates gene transcription by recruiting coactivators (e.g., CREB-binding proteins (CBP)) or corepressors (e.g., histone deacetylases (HDACs)) (Figure 2B).

9In the review of previous clinical and experimental studies, we find that tissues after external stimulation secrete cytokines including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α), of which TNF-α stimulates the activation of other inflammatory mediators (including NF-κB), resulting in the release of inflammatory genes, and then leading to the persistence and deterioration of inflammation.

10The Nrf2 and NF-κB pathways co-regulate cellular responses to oxidative stress and inflammation. The underlying complex molecular mechanisms between these two important pathways remain to be further elucidated.

11The NF-κB p65 subunit can directly inhibit the Nrf2 pathway at the transcriptional level. On the one hand, the NF-κB p65 subunit competes with Nrf2 for the CH1-KIX domain of the transcriptional co-activator CBP, leading to the inactivation of the Nrf2 pathway (Figure 3A). On the other hand, NF-κB recruits HDAC3 by promoting the interaction of HDAC3 with CBP or MafK, causing local hypoacetylation and impeding Nrf2 signaling (Figure 3B). Interestingly, a large number of anti-inflammatory agents can activate the Nrf2 pathway by interfering with NF-κB activation.

12Nrf2 deficient mice subjected to severe head injury showed higher NF-κB activity compared to wild-type Nrf2 mice (Figure 3C). A study explored the effect of Nrf2 on the activation of NF-κB and expression of inflammatory cytokine by using primary cultured astrocytes from Nrf2 wild-type (WT) or knockout (KO). The activity of NF-κB and the expression of inflammatory cytokines including TNF-α, IL-1β, interleukin-6 (IL-6), and matrix metallopeptidase9 (MMP9) were upregulated in Nrf2 KO astrocytes and WT astrocytes, but the increase of which was more apparent in Nrf2 KO astrocytes. These results suggest that loss of Nrf2 may induce more aggressive inflammation by activating NF-κB and downstream pro-inflammatory cytokines. However, NF-κB-DNA-binding activity was significantly inhibited in a diabetic mouse model with Nrf2 overexpression. These studies suggest that we should also notice the negative regulation of NF-κB by Nrf2.

13There are results indicated that the Nrf2 gene has an NF-κB binding site which p65/p50 heterodimer is recruited, and IκK-β plays a core role in NF-κB signaling, contains an ETGE motif that enables it to bind to Keap1. Thereby, the selective recognition mechanism of Keap1 with IκKβ or Nrf2 is vital to the crosstalk between NF-κB and Nrf2 signaling. Besides, the Rho GTPase protein (RAC1) through IκBα induces the increase of the transcriptional level of NF-κB, which in turn up-regulates the expression of Nrf2. However, Nrf2 deficiency induces IκBα phosphorylation and subsequent degradation, increasing NF-κB levels and inducing inflammation. Collectively, Nrf2 inhibits NF-κB as a regulatory feedback loop. Moreover, much evidence indicates that Nrf2 downstream gene HO-1, one of the centers of the crosstalk between Nrf2 and NF-κB, plays a vital role in regulating inflammatory responses (Figure 3D). HO-1 may inhibit the nuclear translocation of the NF-κB pathway to mediate inflammation via its end-products, i.e., carbon monoxide and bilirubin which have been reported to prevent the activation of NF-κB. Also, a study demonstrates that HO-1 inhibits LPS-induced TNF and IL-1β expression through suppression of NF-κB activation.

14The liver is an important central organ for metabolism and detoxification in the human body and is susceptible to many factors, such as excessive alcohol consumption, hepatoviruses, various toxins, and drugs, leading to the development of liver diseases. Among them, drug-induced liver injury (DILI) and liver failure have high morbidity and mortality worldwide and it deserves further to explore the mechanisms and new targets against drug-induced hepatotoxicity. Transcriptomic analysis of a variety of DILI compounds in primary hepatocytes showed that NF-κB inhibition coincided with a strong Nrf2 stress response, which might sensitize hepatocytes to pro-apoptotic signaling cascades induced by endogenous cytotoxic proinflammatory cytokines and play a key role in the regulation of DILI.

15Nrf2 signaling pathway is an important way to regulate cellular oxidative stress, which plays an important role in DILI. Various regulatory factors are affecting the Nrf2 pathway. A study suggests that intestinal microorganisms modulate hepatic susceptibility to oxidative injury via the Nrf2 signaling pathway. Also, anti-inflammatory agents contribute a lot against hepatotoxicity.

16Most DILI is caused by an intentional or unintentional overdose of APAP, which accounts for approximately 50% of all cases of liver injury in the United States. APAP, also known as N-acetyl-p-aminophenol, or paracetamol, is one of the most widely used over-the-counter analgesics and antipyretics and is considered safe at recommended therapeutic concentrations. However, excessive APAP can cause severe liver dysfunction, which threatens patients’ lives. Many studies have shown that Nrf2 KO mice are highly sensitive to APAP treatment compared to controls, and the cause of death is the inability to supply glutathione, a product downstream of Nrf2, promptly. Meanwhile, it has been reported that some protective agents could reduce mitochondrial oxidative stress, the inflammatory response by inhibiting signaling axes such as Keap1/Nrf2/HO-1, and thus protecting hepatocytes from APAP-induced injury. Among them, corilagin and farrerol, the common hepatoprotective preconditioners, can effectively protect liver cells from APAP-induced acute liver failure (ALF) by activating Nrf2 or upregulating the AMPK/GSK3β-Nrf2 signaling pathway, which in turn is involved in the prevention of hepatotoxicity.

17Meanwhile, the role of NF-κB in hepatoprotection cannot be ignored. Maltol, a food-flavoring agent, was found to mitigate APAP-induced inflammatory responses by restraining the phosphorylation of IκKα, IκKβ, and I-κBα in a dose-dependent manner, which exerted a significant liver protection effect. And one study demonstrated that corilagin exerted protective effects against APAP-induced hepatotoxicity by inhibiting ERK/JNK mitogen-activated protein kinases (MAPK) and NF-κB signaling pathways. At the same time, a large number of hepatoprotective reagents (e.g., rutaecarpine, tovophyllin A) have been demonstrated to have significant therapeutic effects by up-regulating Nrf2 transcript levels and inhibiting NF-κB expression. In addition, in APAP-induced hepatotoxicity models, the protein levels of Sirt1 are downregulated by IL1β/NF-κB signaling, resulting in inflammation and oxidative stress. However, the limonin can alleviate APAP-induced hepatotoxicity by activating Nrf2 antioxidative signals and inhibiting NF-κB inflammatory response via upregulating Sirt1.

18Carbamazepine is a widely used antiepileptic drug that causes acute cholestatic injury and leads to hepatotoxicity. Using the live-cell imaging of the green fluorescent protein (GFP)-based reporter models for Nrf2 and NF-κB signaling, the researchers found that treatment with carbamazepine could cause DILI through an interconnected network of temporal changes in Nrf2 and NF-kB signaling, which influence each other and bring about phenotypes (slightly higher cell death). A study on the gene regulatory networks (GRNs) tells us that in azathioprine-induced liver injury, Nrf2 is widely activated and the activity of NF-κB is inhibited. At the same time, the inferred network reveals the overlap of regulatory genes between NF-κB and Nrf2, demonstrating the relationship between NF-κB, Nrf2, and hepatotoxicity.

19Anti-tuberculosis drugs (e.g., INH)-induced hepatotoxicity (ATDH) is a serious problem in tuberculosis treatment and hepatotoxicity management. INH reduced the phosphorylation of ERK1, then prevented Nrf2 translocation into the nucleus, and raised the cytosolic Nrf2 protein levels, thereby inhibiting Hep3B cell cytoprotection produced by the ARE response. Further studies revealed that INH increased ROS levels, promoted apoptosis, and decreased Nrf2 importer Karyopherin β1 expression levels, thereby inhibiting the Nrf2 pathway. Accordingly, some protective candidates may target Nrf2 against INH-triggered hepatotoxicity or ALF. Sagittaria sagittifolia (SSP), a protectant against hepatotoxicity, increased the expression of Nrf2, glutamate-cysteine ligase, and HO-1, whereas that of keap1 was inhibited in INH-caused liver injury models. Interestingly, a new sight against hepatotoxicity may be to promote the stress activation of Nrf2. A study observed a significant increase in the mRNA expression of Nrf2 and the promotion of downstream factors such as HO-1 during INH-induced liver injury in zebrafish larvae. The Nrf2 signaling pathway was activated in the early stage of INH- triggered hepatotoxicity, as a stress compensatory mechanism, but just partially counteracting hepatotoxicity. Then when the liver injury was aggravated, the activation of the Nrf2 pathway was not enough to resist oxidative stress. Instead, a continuous accumulation of Nrf2 in the nucleus leads to more severe oxidative stress.

20Meanwhile, some results suggested that the expression of inflammatory mediators NF-κB was also upregulated in INH-induced liver injury. In rat models, pyrrolidine dithiocarbamate significantly reduced hepatic biochemical and histological damage by inhibiting the activation of NF-κB and oxidative stress caused by INH in a rat model demonstrating its hepatoprotective effect. Other hepatoprotective agents have also been shown to exert the same therapeutic effect. What’s more, other drugs like gallic acid and quercetin attenuate INH-induced liver injury by improving hepatic redox homeostasis through influence on Nrf2 and NF-κB signaling cascades in Wistar rats. So in conclusion, many drugs protect against anti-tuberculosis-induced liver injury by activating Nrf2 and blocking NF-κB, and it is of greater clinical significance to investigate the interaction between Nrf2 and NF-κB.

21TP is one of the main biological activities and active ingredients of Tripterygium wilfordii Hook F, which has excellent immunoregulatory and anti-tumor activities and can be used to treat nephrotic syndrome, cancer, rheumatoid arthritis, and other autoimmune inflammatory diseases. However, the widespread use of TP remains limited by the narrow therapeutic window and hepatotoxicity. To better understand the causes of TP-induced hepatotoxicity, we focused specifically on the role of Nrf2 and NF-κB in TP-induced hepatotoxicity, which also provides direction for future therapeutic targets. MiR155, a regulator of the Nrf2 antioxidant pathway, was significantly activated and then inhibited the activity of the Nrf2 pathway in TP-induced hepatotoxicity. TP indeed decreased the protein expressions of the Nrf2 pathway including HO-1, NQO1, and Nrf2 associated with the oxidative stress pathway. And a study found that TP-induced oxidative stress and cell damage in HepG2 cells could be aggravated by Nrf2 knockdown or counteracted by overexpression of Nrf2. In addition, treatment with sulforaphane (SFN), a typical Nrf2 agonist, attenuated TP-induced toxic effects such as liver dysfunction, structural damage, and glutathione depletion in mice by activating Nrf2 and its downstream targets. To illustrate a deeper mechanism, some scholars have found that catalpol (CAT), the main active ingredient of Rehmannia Glutinosa (RG), enhanced Nrf2 transcript expression by reversing TP-induced Nrf2 transcriptional repression. The idea further proved that CAT induced UGT1A6, a phase II detoxifying enzyme of TP, through the Nrf2 pathway, thereby increasing TP metabolic conversion and reducing TP hepatotoxic effects. Meanwhile, another agent panax notoginseng saponins (PNS) significantly increased Nrf2 phosphorylation for relieving the potential inhibition of TP-induced Nrf2/ARE binding activity. These results highlight the necessity of the Nrf2/ARE pathway in the TP-triggered hepatotoxicity.

22As an upstream regulator, Nrf2 not only regulates the oxidative stress response but also suppresses inflammation by regulating cytokine production and cross-talk with the NF-κB signaling pathway. It has been previously reported that the alleviation of TP-induced hepatotoxicity was involved in the inactivation of the NF-κB pathway. Besides, TP treatment might disrupt immune homeostasis and induce liver hypersensitivity through the regulation of NF-κB-dependent pro-survival genes. These shreds of evidence support the close correlation among unbalanced expression of Nrf2, NK-κB, and hepatotoxicity.

23PM, the root of P. multiflorum Thunb, has been used to treat various diseases, such as constipation, early graying of the hair, and hyperlipemia for several decades. The hepatotoxicity caused by PM treatment has attracted attention in recent years, which was initially considered to be non-toxic. As one of the main active components of PM, rhein could up-regulate the mRNA and protein levels of Nrf2, HO-1, and glutamate-cysteine ligase catalytic subunit (GCLC), down-regulate the protein expression of NF-κB, TNF-α and caspase-3, increase the survival rate of LO2 cells, decreasing the apoptosis rate and acting as a hepatoprotective drug. However, there is evidence to confirm that rhein has potential hepatorenal toxicity. A study clarified that immunological idiosyncratic hepatotoxicity induced by PM was associated with inhibition of the NF-κB pathway. We know that abnormal activation of the TLR4/NF-κB signaling pathway in response to external stimulation can trigger severe liver injury. Also, a large number of results support that PM regulates the Nrf2 pathway, but the specific toxicological mechanism remains to be explored in PM-induced liver injury models.

24Overall, Nrf2 and NF-κB interact with each other to coordinate antioxidant and inflammatory responses. There is evidence indicating that Nrf2/HO-1 pathway and NF-κB pathway are involved in olaquindox (OLA)-induced hepatotoxicity. Meanwhile, curcumin can efficiently alleviate OLA-induced hepatotoxicity by further activating Nrf2/HO-1 pathway and inhibiting the p53 and NF-κB pathways to inhibit oxidative stress, apoptosis, and mitochondrial dysfunction. In the previous study, teicoplanin (TIL) treatment significantly upregulated the relative hepatic expression of HSP70 and NF-κB mRNAs and blocked Nrf2/HO-1 mediated responses, indicating that TIL could induce oxidative stress and hepatotoxicity by blocking Nrf2 mediated defense responses and increasing NF-κB signaling pathways. On the other hand, the combination of astragalus polysaccharide (APS) and TIL improved the expression of the Nrf2 pathway, suggesting that the crosstalk between Nrf2 and NF-κB could be a new therapeutic target against hepatotoxicity. These pieces of evidence strongly complement our above discussion on the crosstalk of Nrf2 and NF-κB in drug-induced hepatotoxicity, demonstrating that this connection may provide an insight into the development of future hepatoprotective agents.

25Among the adverse effects, there is a great association between nephrotoxicity and the crosstalk between Nrf2 and NF-κB. Next, we will elaborate in-depth on typical drug-induced nephrotoxicity.

26Cisplatin, a potent chemotherapeutic agent used for treating various types of solid organ tumors, is limited by its serious side effects in clinical application. Cisplatin can cause an increase in the expression of NF-κB and TNF-α in the liver and kidney. And abundant evidence suggests that antioxidant agents promote the anti-inflammatory effect by inhibiting the production of NF-κB in the kidney even in the liver. Celastrol, a potent chemotherapeutic agent, could ameliorate cisplatin-induced nephrotoxicity through suppressing NF-κB and protecting mitochondrial function in vitro and in vivo. Similarly, polysulfide and H2S could afford protection against cisplatin-caused nephrotoxicity via persulfidating STAT3 and IκKβ and inhibiting NF-κB-mediated inflammatory cascade. In addition, Nrf2 is a key molecule involved in targeting specific proteins in the NF-κB pathway and may be implicated in inflammatory regulation. Cisplatin can down-regulate Nrf2 and HO-1 while upregulating inflammatory factors. And embelin, an anti-inflammatory drug, can alleviate cisplatin-induced nephrotoxicity by activating the signaling pathway of Nrf2/HO-1 and impeding NF-κB. Some studies have found that vinorelbine protects against cisplatin-induced acute kidney injury (AKI) by activating Nrf2/HO-1 signaling pathway and hindering TLR4-IFN-γ-CD44 cell inflammatory cascade when investigating the molecular mechanism of renal protection against nephrotoxicity. Accumulating evidence suggests that the antioxidant mechanism protecting against enucleation-induced nephrotoxicity involves the Nrf2/HO-1 signaling pathway.

27Some scholars have found that in the cisplatin-induced AKI mice, the C. cicadae mycelium extract regulated the inflammatory response by inhibiting renal pathological changes, inflammatory cell infiltration, and the release of a variety of inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Further data revealed that the C. cicadae mycelium extract exerted potent detoxifying effects by inhibiting TLR4/NF-κB/MAPK and regulating the HO-1/Nrf2 signaling pathway while regulating autophagy and inhibiting apoptosis to alleviate renal injury. Furthermore, thymoquinone and curcumin combination also prevented cisplatin-induced kidney injury by attenuating NF-κB, KIM-1 and ameliorating Nrf2/HO-1 signaling.

28Ingestion of Chinese herbal medicines containing AA can induce aristolochic acid nephropathy (AAN) which is a rapidly progressive tubulointerstitial disease. Earlier evidence supports that AA-induced AKI is associated with impaired activation of Nrf2 and its downstream target gene expression. Therefore, we boldly guess that nephrotoxicity can be alleviated by activating the Nrf2 signaling pathway and increasing the expression of downstream target genes. Unsurprisingly, enhancement of the Nrf2 signaling pathway ameliorated AA-induced tubular epithelial cell injury, while down-regulation of the Nrf2 signaling pathway caused the opposite results, fully demonstrating that Nrf2 plays an important role in the AA-induced nephrotoxicity. Furthermore, a study has found that methylbarbadenolone (BARD), an antioxidant modulator, could play a renoprotective role through the Nrf2 signaling pathway. It significantly ameliorated AA-induced tubular necrosis and interstitial fibrosis by up-regulating the renal expression of Nrf2 and Smad7.

29In addition, NF-κB is also significantly activated in AA-induced nephropathy. Notably, AA exhibited cytotoxicity in a dose-dependent and time-dependent manner. At a safe dose, AA could significantly inhibit the activity of NF-κB in HK2 cells and show the pharmacological effect. However, in the AA-induced renal injury model, the expression of NF-κB was abnormally elevated, suggesting that the dual effects of NF-κB may be one reason for the different properties of AA on the kidney. Demonstrating our idea from the reverse aspect, the Chinese herb Sedum sarmentosum extract (SSB) reduced the activity of the NF-κB signaling pathway when exerting renoprotective effects. Also, AA may be involved in the alteration of matrix homeostasis during renal fibrosis in vivo, including the imbalance of extracellular matrix (ECM) accumulation and matrix metalloproteinase (MMP) activation involving NF-κB. Therefore, we can improve renal fibrosis by regulating the activity of the NF-κB pathway, and providing new ideas for drug therapy of nephropathy. All the above evidence suggests that in AA-induced nephrotoxicity, NF-κB is a network core molecule that can modulate the immune and inflammatory responses that occur in the kidney.

30As the third most common cause of AKI, the use of contrast agents and contrast-induced nephropathy (CIN) have become the focus of global public health attention. This event has been reported to occur in about 30% of patients receiving invasive image examinations and is strongly associated with a high risk of death, long hospital stays, and adverse outcomes such as a high risk of chronic kidney disease. In exploring possible therapeutic options for CIN, researchers have found that a renal protective drug could increase the activation of the Nrf2 antioxidant defense pathway. Further mechanism elaboration, the protective effect against CIN could be exerted through Nrf2/Sirt3/SOD2 or Nrf2/HO-1 pathways. Moreover, recent studies have found that STC1, a conserved glycoprotein with anti-apoptosis and function, could target Nrf2 to reduce kidney injury and provide a promising preventive target for the treatment of CI-AKI.

31Similarly, NF-κB is aberrantly activated in CIN, but NF-κB expression is decreased and renal tissue hyalinization, hemorrhagic casts, and necrosis are reduced after infliximab treatment. And TLR4/Myd88/NF-κB is also involved in the protective pathway. In addition, a study has revealed that overexpression of miR429 inhibits the NF-κB signaling pathway by targeting programmed cell death 4 (PDCD4) to reduce apoptosis and improve cell viability in a CI-AKI cell model.

32GM is an aminoglycoside antibiotic commonly used to treat acute gram-negative bacterial diseases. However, at least 30% of patients who received GM for more than 7 days had an increased risk of AKI. Melatonin (MT) restores antioxidant enzyme activity and blocks NF-κB and nitric oxide synthase (iNOS) activation in rat kidneys, thereby preventing GM-induced nephrotoxicity, and these experimental results confirm that MT protects the kidney as a potent antioxidant. Besides, GM was found to promote nuclear NF-κB (p65) expression and inflammatory cytokine cytokines (TNF-α and IL-6) and upregulate NF-κB-DNA-binding activity in kidney cells. In contrast, nephroprotective drugs can attenuate the abnormity of these genes, thereby reducing the degree of histological damage and neutrophil cell infiltration in renal tubules. Also, NF-κB inhibitors have the same effect. On the other side, both Keap1/Nrf2/ARE and PKC/Nrf2 antioxidant pathways can be activated to enhance the attenuation function of the kidney. In addition, Nrf2 and NF-κB pathways can be simultaneously regulated in GM-induced nephrotoxicity models, and which provides direct evidence between the crosstalk of Nrf2 and NF-κB and drug-induced toxicity. In future therapeutic strategies, increasing the nuclear immunoreactivity of Nrf2 and decreasing that of NF-κB could be taken into account.

33MTX is an anti-proliferative folic acid antagonist widely used in clinical treatment for cancers and chronic inflammatory diseases, but its nephrotoxicity limits its use. Some researchers have found that MTX-induced nephrotoxicity could be attenuated by vincamine, playing a protective role in the already peripheral and central nervous systems of the kidney. The therapeutic effect of vincamine against MTX-induced renal injury was through inhibiting oxidative stress while decreasing the expression of the NF-κB pathway by increasing the expression of Nrf2 and HO-1. And some other nephroprotective agents, such as chicoric acid, umbelliferone and berberine, also exert their protective effects by the same mechanism. Indeed, the importance regarding the modulation of NF-κB and Nrf2 pathway activity in MTX-induced AKI has been efficiently shown, especially Nrf2. Activation of the Nrf2/HO-1 signaling pathway can effectively protect kidney structure from destruction, while various factors lead to the elevated activity of Nrf2/HO-1, such as the expression level of miR145-5p which directly targets Sirt5.

34Let us pay attention to whether, the body affected by nephropathy also causes further nephrotoxicity by breaking the balanced relationship between the two pathways, in addition to nephrotoxicity caused by the crosstalk of Nrf2 and NF-κB. When exploring whether diabetes aggravates I/R-induced AKI in rats, some results have shown that diabetes aggravated oxidative stress, inflammatory response, and apoptosis after renal I/R by enhancing TLR4/NF-κB signaling and blocking the Nrf2/HO-1 pathway. However, pretreatment with tBHQ (an Nrf2 agonist) inhibited NF-κB signaling and enhanced the function of anti-apoptosis. These results indicate that the crosstalk between Nrf2 and NF-κB is involved in the development of nephrotoxicity.

35As same as nephrotoxicity, there are lots of results that have shown a tight association between the crosstalk and drug-induced cardiotoxicity.

36DOX, an anthracycline antibiotic, has been widely used to treat both solid and hematologic malignancies. However, DOX can lead to myocardial cell loss, mitochondrial dysfunction, myelofibrosis, and congestive heart failure in the body, triggering cardiotoxicity after long-term use. DOX-induced cardiotoxicity is mediated by ROS production and the Nrf2/ARE pathway. Natural compounds (NCs), such as Asiatic acid, α-linolenic acid, apigenin, and β-LAPachone, were confirmed to ameliorate DOX-induced cardiac injury by activating Nrf2 in different pathways. For example, cardamonin (CAR), a flavone found in Alpinia plants, can reduce the NF-κB signaling pathway and improve Nrf2 signaling to suppress oxidative stress, apoptosis, and inflammatory response, ameliorating DOX-induced cardiotoxicity. And previous studies have shown that miR140-5p increases DOX-induced myocardial oxidative damage by inhibiting Nrf2 and Sirt2 signaling pathways. Conversely, miR200a protects against DOX-induced cardiotoxicity by activating the Nrf2 signaling pathway. This suggests that microRNA acts as a regulatory medium to modulate the Nrf2 pathway to aggravate or ameliorate DOX-induced cardiotoxicity.

37Notably, DOX induced the expression of NF-κB p65 and caspase-3 in myocardial nuclei in a DOX-induced cardiotoxicity model. Interestingly, NF-κB p65 and caspase-3 were significantly inhibited by trifluoperazine, a strong calmodulin antagonist, suggesting that the cardioprotective effect conferred by trifluoperazine involves inhibition of NF-κB and apoptosis. Furthermore, biochemical and histopathological examinations revealed that trifluoperazine ameliorated DOX-induced renal and hepatic injury both functionally and structurally. Similarly, another drug geraniol may function as a potential activator of Nrf2, subsequently improve Nrf2 dependent antioxidant signaling, reduce apoptosis, and attenuate inflammatory responses. Antioxidant drugs may be a potential therapeutic target to prevent the development of DOX-induced cardiotoxicity by inhibiting oxidative stress and NF-κB pathways.

38The study suggests the necessity and importance of discussing the crosstalk between Nrf2 and NF-κB in drug-induced cardiotoxicity. On top of that, among the organ injuries, the rising morbidity and mortality of highlighting cardiovascular diseases (CVDs) make cardiac injury attract people’s attention. Some studies have found that aging is a major risk factor for CVDs. Under physiological conditions, Nrf2 and NF-κB are degraded via the proteasome without cardiotoxicity. When aging disrupts the balance of Nrf2 and NF-κB, it can increase ROS/RNS and activate inflammatory signaling pathways, thereby damaging cellular components and leading to cardiac injury. Also, the ability of adhesion, proliferation, migration, and formation of capillary-like structures is compromised by dysfunction of Nrf2 signaling and it revealed Nrf2 might be a prominent target to release angiogenesis and microvascular rarefaction. It has earlier been reported that the NF-κB p65 subunit inhibits the Nrf2/ARE pathway at the transcriptional level, so there may be crosstalk between NF-κB and Nrf2 in the decrease of Nrf2 expression.

39BLM, a glycopeptide antibiotic, is an effective antineoplastic agent commonly used to treat breast, ovarian, lung, and different types of leukemia. However, the effective use of BLM in chemotherapy remains limited, as it precipitates dose-dependent interstitial pneumonia, resulting in pulmonary fibrosis in at least 10% of individuals treated with BLM, ultimately leading to irreversible lung structural damage. BLM aggravates pulmonary inflammation and apoptosis by up-regulating the expression of NF-κB signaling pathway and apoptosis regulator caspase-3, and substantially reducing the activity of antioxidant enzymes in a rat model of BLM-induced pulmonary fibrosis. Furthermore, NF-κB p105 and NF-κB p65 are involved in the progression of acute lung injury (ALI) and play important roles in tumorigenesis, inflammation, and immunity, which are important for elucidating therapeutic strategies for ALI. Juglans regia extract can inhibit NF-kB activation and reduce the expression of proinflammatory biomarkers such as cyclooxygenase 2 (COX-2) and iNOS to slow down pulmonary fibrosis. The protective effect is to reduce BLM-induced oxidative stress and pulmonary inflammation by regulating the inflammatory response of rat alveolar macrophages. Meanwhile, the involvement of the Nrf2 pathway in the process of BLM-induced lung injury has also been mentioned. It has become clear that artemisinin, a novel Nrf2 activator, activates the Nrf2 pathway by reducing Nrf2 ubiquitination and improving its stability. On the other hand, the subsequent antioxidant protection can be achieved by regulating the Nrf2/HO-1 signaling pathway.

40The hepatoprotective effects of NF-κB and Nrf2 have been demonstrated in BLM-treated animals. It is curious whether there are potential interaction mechanisms between the two genes that together regulate antioxidant levels to protect against lung injury? Earlier studies have shown that NF-κB and Nrf2 expression levels are simultaneously regulated by hepatoprotective drugs. In BLM-treated pneumonocytes, salidroside inhibits IκBα phosphorylation and the nuclear accumulation of NF-κB p65 while activating Nrf2-antioxidant signaling. In addition, hesperidin attenuates BLM-induced lung toxicity by inhibiting the IκBα/NF-κB pathway, which in turn improves the regulation of oxidative inflammatory markers (Nrf2 and HO-1) and pro-inflammatory markers (TNF-α, IL-1β, and IL-6) to reduce collagen deposition during pulmonary fibrosis. Besides, treatment of Glycyl-L-histidyl-L-lysine (GHK)-Cu complexes modulates the Nrf2 and NF-κB pathways, and Smad2/3 phosphorylation partially prevents the development of epithelial-mesenchymal transition (EMT) and has a protective effect against BLM-induced inflammation and oxidative stress. However, the specific interaction mechanism between the two remains to be elucidated. A study revealed that the activation of NF-κB and Nrf2 pathways played opposite roles in lung injury, respectively. And in the hepatoprotective mechanism, the activity of Nrf2 increases, which has a potential inhibitory effect on the NF-κB pathway, plays a better protective effect.

41Psoriasis is a skin disease mediated by immune response disorders, with a prevalence of 2–3% in the world’s population. And the patients are suffered from skin itching, pain, seriously affecting the quality of life of patients. While the etiology of psoriasis remains to be fully elucidated, studies are revealing the crosstalk between Nrf2 and NF-κB in psoriasis.

42The results from IMQ-induced psoriasis models in vitro confirm the involvement of NF-κB1 in psoriasis pathogenesis through mediated T cell (Th1 and Th17) activation. The role of Nrf2 and NF-κB pathways in psoriasis has been previously documented, but the exact interaction between the two has not been elucidated. Tussilagonone (TGN), a sesquiterpenoid isolated from Tussilagofarfara, has been used to treat IMQ-induced psoriasis-like dermatitis mice when further exploring transcription factors associated with psoriasis pathogenesis. As a potent Nrf2 activator, TGN can significantly activate the Nrf2 pathway and induce the expression of its downstream target gene HO-1 by inhibiting NF-κB and STAT3. In turn, activation of Nrf2/HO-1 by TGN also inhibits activation of NF-κB and STAT3 which are the key transcription factors in psoriasis pathogenesis and promote the skin defense system while reducing the expression of immune mediators and epidermal proliferation. Further proving our subject, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) aggravated IMQ-induced psoriasis by increasing the expression of phosphorylated NF-κB p65 and inhibiting the antioxidant marker Nrf2. These studies have provided a novel and clinically meaningful perspective on the pathogenesis and therapeutic targets of psoriasis in the world.

43Experimental results from other perspectives have also demonstrated the importance of the Nrf2 and NF-κB pathways in skin inflammation, especially psoriasis. Fumarate esters (FAEs) such as dimethyl fumarate (DMF) and its active metabolite monomethyl fumarate (MMF) can play a vital role in the treatment of psoriasis because of their immunomodulatory effects. On the one hand, the binding of DMF and MMF to the cysteine residues of Keap1 induces a conformational change that dissociates Nrf2 from Keap1 and enters the nucleus, thereby activating cytoprotective and anti-inflammatory genes. On the other hand, DMF can also directly or indirectly inhibit the nuclear activity of NF-κB, affecting cytokine production in human T cells, human breast cancer cell lines, and mouse dendritic cells.

44Interestingly, some researchers have found that synchronized circadian clocks could not only enhance the protective role of Nrf2 but also attenuate the validated response through the NF-κB signaling pathway when testing the differential responses of keratinocytes clock synchronized or desynchronized in challenged keratinocytes by O3. The synchronized circadian clocks can strengthen the cellular defense function and alleviate skin inflammation, which provides new insight into the crosstalk of Nrf2 and NF-κB.

45Chemotherapy agents such as oxaliplatin, cisplatin, paclitaxel, and bortezomib frequently cause severe peripheral neuropathy and there is currently no effective strategy to prevent drug-induced toxicity.

46Cisplatin can cause not only nephrotoxicity but also cause severe neurotoxicity, which significantly limits its clinical use. A study showed that a continuous high dose of cisplatin stimulation disrupted the blood-brain barrier, leading to brain cell edema and neuronal necrosis. Regarding the study of prevention and treatment of cisplatin-induced neurotoxicity, the neuroprotective effects exerted by many agents are closely related to the Nrf2 and NF-κB pathways. Schisandrin B (Sch B) has been reported earlier to effectively inhibit the activation of NF-κB, p53 and cleave caspase-3 expression in cisplatin-treated mice, thereby exerting the efficacy of protective drugs. Recently, a large number of articles have found that agents including edaravone and epigallocatechin-3-gallate (EGCG) prevented the development of cisplatin-related brain inflammation and oxidative damage by up-regulating the gene expression level of the Nrf2/HO-1 pathway and preventing the cisplatin-induced NF-κB activation.

47Bortezomib was the first proteasome inhibitor approved to treat multiple myeloma (MM), mantle cell lymphoma, and other solid tumors. However, peripheral neuropathy (PNP) is one of the most common side effects of bortezomib and leads to dose modification and discontinuation of therapy. One hallmark of the mechanism of action of bortezomib in tumor cells is NF-κB, while bortezomib treatment may induce neuropathy by inhibiting NF-κB in non-neuronal types or targeting other signaling pathways. It has been found that animals with impaired NF-κB activation developed significantly less neuropathy, suggesting that the NF-κB activation played an active role in bortezomib-induced neuropathy. There was also evidence to identify the NF-κB signaling pathway mediating neurotoxicity when investigating the molecular factors associated with PNP in patients with MM induced by bortezomib. Thus, inhibition of NF-κB may be a promising strategy to protect against bortezomib-triggered neurotoxicity.

48PTX is an anti-tumor drug with high efficacy, especially for breast cancer, ovarian cancer, bladder cancer, lung cancer, and other types of solid tumors. Despite its dramatic effect, PTX can cause severe peripheral neuropathy (usually manifested as painful neuropathy), affecting up to 50% of cancer patients. Huangqi Guizhi Wuwu Decoction (HGWD) was found to have a therapeutic effect on oxaliplatin-induced peripheral neuropathy. It was revealed that HGWD significantly inhibited PTX -induced activation of TLR4/NF-κB and decreased the expression of IκKα, and phosphorylation of NF-κB to inhibit PTX-evoked inflammatory and oxidative responses in the peripheral nervous system. It also promoted activation of the PI3K/Akt-Nrf2 signaling pathway, alleviating PTX-induced oxidative stress to some extent. Moreover, other protective drugs have also been shown to regulate the downstream Nrf2/HO-1 pathway. Another chemotherapeutic drug, oxaliplatin, also often causes peripheral neuropathy afflicting patients. However, some studies have found that L-carnosine exerted a neuroprotective effect against oxaliplatin-induced peripheral neuropathy in colorectal cancer patients via the Nrf2 and NF-κB pathways.

49According to the results of another study, we reasonably hypothesize that the protective mechanism against chemotherapy-induced neurotoxicity may be that protective drugs and their metabolites increase the DNA-binding activity of Nrf2, thereby enhancing the ability to counteract oxidative stress. On the other hand, the potential inhibitory effect of Nrf2 on NF-κB inhibits the inflammatory response and enhances the alleviation of neurotoxicity. However, it is noteworthy that elevation of Nrf2 signaling by protective agents can also activate the phase I, II and III response in tumors. Increased expression of xenobiotic metabolism and efflux pumps due to compounds (protective agents mentioned above) that elevate Nrf2 in cancer maybe diminish anti-cancer agent efficacy. These concerns may be diminished due to co-administration agents possessing, such as pharmacokinetic properties which cause differential temporal effects, and treatment regimens that cause increased metabolism/efflux transporter expression only after anti-cancer agents have already bound to their target. Other effects such as organ-specific compound uptake and other variables could also be explained. Notably, differences between genetic and pharmacologic activation of Nrf2 signaling also exist.

50Suppression of Nrf2 can cause the development of neurotoxicity in animal models. Previous studies have reported that Nrf2 deficient mice developed vacuolar (spongiform) leukoencephalopathy along with extensive astrogliosis, revealing a possible physiological role for Nrf2 in maintaining myelin in the central nervous system. A dysfunctional Nrf2 system may increase the risk of chronic diseases in humans, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). A study in neurodegenerative disease showed that the amount of Nrf2 was decreased in hippocampal astrocytes which were one of the brain regions where neurodegeneration began in AD patients. Also, in PD patients, the nuclear localization of Nrf2 is strongly induced, but this response may not be sufficient to protect neurons from degeneration. In addition, genetic variants in the Nrf2 gene have been associated with the progression of AD and PD. Several studies have shown that NF-κB activation in astrocytes could aggravate neuroinflammation and produce neurotoxic effects. In addition, the expression of various NF-κB target genes and NF-κB DNA-binding activity increased in the brains of AD patients. Several genetic, cell biology, biochemical and animal studies supported that inhibiting NF-κB could play a critical role in preventing the development of AD pathology.

51Consistent with the above content, a study showed that DMF is involved in protecting β-amyloid-induced cytotoxicity and alleviating neurotoxicity by reducing the pro-inflammatory pathway: NF-κB, as well as regulating the Nrf2 pathway to increase the production of antioxidant enzymes. A study found that DMF exerted neuroprotective effects through the crosstalk between Nrf2 and NF-κB pathways in vitro and in vivo, besides regulating antioxidant enzymes such as Nrf2, Mn-SOD, and HO-1, thereby reducing the levels of pro-inflammatory cytokines such as IL-1 and iNOS. The study also has elucidated the crosstalk between Nrf2 and NF-κB pathways in acrylamide-induced neurotoxicity and found that the protective effect of oxidant N-Acetyl-L-Cysteine (NAC) against acrylamide was to resist cell injury by activating the Nrf2 pathway and inhibiting NF-κB pathway. In addition, MAPKs, as the central mediators that transmit extracellular signals from cell membrane to nucleus, can regulate the activation of Nrf2 and NF-κB pathways to reduce cell injuries. Vincristine was proved to increase NF-κB levels in sciatic nerves and consequently cause oxidative stress and neuroinflammation through the suppression of the Nrf2 pathway. Meanwhile, the quercetin treatment increased Nrf2 levels and downregulated NF-κB levels to alleviate oxidative stress and neuroinflammation. Another research highlights that acrylamide, a common food contaminant formed during food heat processing, induces oxidative stress in astrocytes and microglia by regulating the Nrf2/ARE and NF-κB pathways, leading to neurotoxicity. These results reveal the importance of the crosstalk of Nrf2 and NF-κB signal pathways in neurotoxicity.

52Furthermore, it has been proved that mice bearing Nrf2 deficiency were suffered from neuroinflammation and lost dopaminergic neurons. By comparison, the neuroprotection appeared in a mutant alpha-synuclein (αSyn; a small protein with 140 amino acids abundant in presynaptic nerve terminals) transgenic mouse model with an overexpression level of Nrf2 gene. At the same time, the increased activation of NF-κB is related to the strong nuclear p65 immunoreactivity in PD patients, which suggests that NF-κB participates in neurodegenerative disorder. Also, evidence has shown that we could take NF-κB as an ideal therapeutic target due to its critical role in forming inflammatory mediators in models of PD-induced neurotoxicity. Thus, it is obvious that Nrf2 and NF-κB interplay in neurodegenerative disorders, in which the increase of NF-κB aggravates neuroinflammation while the increase of Nrf2 affords neuroprotection.

53Here, we summarize the protective agents that may alleviate injury in different organ toxicities as well as their effects in anti-oxidative and anti-inflammatory systems (Table 1).

54In clinical applications, the treatment of many drugs, including APAP and antineoplastic agents, is limited by their potential organ toxicity. However, the mechanism of drug-induced tissue injury and the corresponding protective strategies have not been fully discussed. Meanwhile, there is increasing interest in investigating the relevance of Nrf2 and NF-κB signaling pathways in the development/progression of drug-induced injury. Therefore, this review summarizes and discusses the role of common agents in the induction of multiple organ toxicities. We found that the unbalanced relationship between Nrf2 and NF-κB may induce or aggravate toxic responses, while substances that regulate the relationship between Nrf2 and NF-κB may play a protective role-restoring Nrf2 activity, thereby enhancing antioxidant capacity and reducing NF-κB-mediated inflammatory responses. The crosstalk of both may serve as a central hub for drug-induced toxic responses. At the same time, drugs or therapeutic approaches that target the crosstalk between Nrf2 and NF-κB appear to be promising pointcuts for future clinical responses to drug-induced toxicity (Figure 4). In addition, further study and therapeutic exploration of potential molecular mechanisms will help explain the specific regulatory mechanisms of Nrf2 and NF-κB signaling pathways in various drug-induced organ toxicities, and ultimately standardize rational and safe medicine usage and reduce the occurrence of toxic reactions. In summary, we urgently need signaling network studies of Nrf2 and NF-κB in pathological situations, as well as new treatments and new drugs targeting the crosstalk between the two.