Nrf-2 as a novel target in radiation induced lung injury

Abstract

Radiation-induced lung injury (RILI) is a common and fatal complication of chest radiotherapy. The underlying mechanisms include radiation-induced oxidative stress caused by damage to the deoxyribonucleic acid (DNA) and production of reactive oxygen species (ROS), resulting in apoptosis of lung and endothelial cells and recruitment of inflammatory cells and myofibroblasts expressing NADPH oxidase to the site of injury, which in turn contribute to oxidative stress and cytokine production. Nuclear factor erythroid 2-related factor 2 (Nrf-2) is a vital transcription factor that regulates oxidative stress and inhibits inflammation. Studies have shown that Nrf-2 protects against radiation-induced lung inflammation and fibrosis. This review discusses the protective role of Nrf-2 in RILI and its possible mechanisms.

1. Introduction

Radiotherapy is one of the main treatments for malignant tumors, especially lung cancer. It can be potentially helpful in different types and stages of lung cancer, both in controlling cancer progression and palliative care [1,2]. With advances in treatment techniques and improvements in radiotherapy (RT), the adverse effects of RT have gradually decreased, and treatment outcomes have improved. However, radiation-induced lung injury (RILI) is inevitable in sensitive, normal lung tissue [3]. RILI can be divided into early radiation pneumonitis (RP) and late radiation pulmonary fibrosis (RPF). Early RILI is usually short-term and occurs approximately six months after the end of radiation. The pathological manifests as alveolar fluid exudation, alveolar wall congestion, edema, inflammatory cell exudation, megakaryocytic interstitial infiltration, and alveolar membrane damage, and the imaging manifests as ground-glass opacity. Advanced RPF usually appears 6–12 months after radiation exposure and is characterized by an irreversible alveolar wall or interstitial fibrosis [4]. Current treatment techniques are minimal because of the widespread involvement of the lungs in pulmonary fibrosis (PF), and the treatment mainly consists of glucocorticoids in combination with antibiotics, cough and sputum suppression, and other symptomatic treatments; there is no precise, effective treatment, which seriously affects the survival and quality of life of patients [3,5]. Therefore, reducing and preventing RILI represents a critical and unmet medical need that will provide significant clinical benefits to numerous patients.

Nrf-2, a critical regulator of antioxidant, drug, carbohydrate, lipid, heme, and iron metabolism, is a transcription factor susceptible to oxidative stress. It binds to the nucleus's antioxidant response element (ARE) and promotes the transcription of various antioxidant genes. The antioxidant pathway of Nrf-2 is essential in multiple lung diseases, including acute lung injury/acute respiratory distress syndrome, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma, and allergy. It is widely considered a new therapeutic target for inflammatory lung diseases [6]. It has been shown that Nrf-2 is involved in radiation-induced oxidative stress and has protective effects against radiation-induced acute lung injury and inflammation [[7], [8], [9]]. The regulation of Nrf-2 could serve as a novel and more efficacious approach to treating radiation-induced lung damage (RILI). This article discusses the possible mechanism of RILI and the protective role of Nrf-2 in lung damage. It also explores the potential protective mechanism of activating Nrf-2 against RILI and provides an update on the progress of antioxidant therapy research in this field.

2. The mechanisms of radiation-induced lung injury

It is generally accepted that there are two main mechanisms of ionizing radiation damage: direct damage to the deoxyribonucleic acid (DNA) and indirect damage through the production of reactive oxygen species (ROS) [10,11]and release of corresponding cytokines and molecules through intracellular signal transduction to promote inflammation and the immune response [12].

In a very short period after radiation exposure, water molecules ionize to produce ROS, such as nitrogen species (NGS), hydroxyl radicals, and superoxide, which interact with proteins, nuclei, organelles, and the extracellular matrix, leading to DNA damage [[13], [14], [15]]. Damage to the alveolar epithelial cells and vascular endothelial cells following radiation exposure can lead to impaired barrier function. Most of these damaged cells can repair themselves, and the rest may undergo apoptosis or mutations [16]. These damaged cells are sensed by inflammatory cells, causing the proliferation of leukocytes and lymphocytes along with the release of inflammatory cytokines, such as tumor necrosis factor (TNF-α), interleukin family members, and transforming growth factor (TGF-β1) [17]. The persistence of the inflammatory state eventually leads to early reversible toxicity (RP), which can progress to irreversible late toxicity (RPF).

After apoptosis, cells release damage-associated molecular patterns (DAMPs), activating the innate immune system (neutrophils, macrophages, white blood cells, and lymphocytes); immune effector cells are recruited and infiltrate the damaged lung tissues. The inflammatory factors produced by these cells lead to the activation and proliferation of fibroblasts [18,19]. Simultaneously, the utilization of oxygen by the immune cells leads to tissue hypoxia. Hypoxia increases ROS production, regulates TGF-β, and promotes collagen formation, thereby reducing alveolar elasticity [20]. In addition, ROS can cause cell loss, alveolar wall edema, increased vascular permeability, and protein exudation, further reducing lung elasticity, destroying vascular integrity, and increasing the apoptosis of alveolar type I epithelial cells, thereby promoting alveolar type II epithelial cell proliferation and aggravating lung inflammation [10,21]. Under long-term cytokine action, fibroblast recruitment and myofibroblast proliferation lead to the remodeling of the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT), resulting in fibrogenesis and scar formation, eventually replacing the normal lung tissue with the development of advanced PF [22,23]. The specific signaling pathways are shown in Fig. 1.

Fig. 1.

Signaling pathways in radiation-induced lung injury. Radiation induces lung injury in these ways, showed in the figure. Under ionizing radiation, ROS are rapidly produced, and ROS can activate TGF-β. Activated TGF-β can bind to TGF-βRII, phosphorylating Smad2 and Smad3, which can form a complex with Smad4. Additionally, Radiation-induced ROS stimulates downstream signaling via the ERK/GSK-3β/snail axis. Increased GSK-3β then activates TGF-β and leads to an increase in β-catenin levels, which maintains stemness of type II AEC and promotes its differentiation into fibroblasts. IGFBP7 is enhanced by TGF-β and is involved in the EMT of AECs through the ERK signaling pathway. The complex can regulate gene expression to promote fibrosis. Activated HMGB1 leads to NF-kB into the nucleus and interacts with DNA, promoting TNF-α, IL-6, and IL-1 expression, which can cause lung inflammation.

Abbreviations: ROS: Reactive oxygen species; HMGB1: High-mobility group box 1; TNF: Tumor necrosis factor; IL: Interleukin; TGF: Transforming growth factor; AEC: Alveolar epithelial cells; VEGF: Vascular endothelial growth factor; ERK: Extracellular regulated protein kinases; IGFBP7: Insulin-like growth factor binding protein 7; GSK-3β: Glycogen synthase kinase-3 beta; EMT: Epithelial-mesenchymal transformation; ET-1: Endothelin-1.

With the emergence of stereotactic radiotherapy (SBRT) or stereotactic ablative radiotherapy (SABR), the indications for thoracic RT are expanding; however, RILI remains the most common complication after RT in patients with thoracic tumors. RILI is classified into early- and delayed-radiation PF. Although RP occurs in the early stages of RT and may be cured, there is a limited treatment currently for widespread RP, and late PF is considered irreversible. There is still no effective treatment for the prevention and treatment of RILI and radiation-induced PF [24,25]. However, although researchers have a new understanding of the mechanism, clinical evaluation, and treatment of RILI, more is needed to solve the need for effective clinical treatment of RILI. Hence, while radiotherapy may effectively treat cancer patients, the occurrence of severe RILI can significantly impact their quality of life and perhaps lead to fatal outcomes. Suppose somebody can explore more targeted therapy can be explored based on the known mechanism of RILI. In that case, minimizing radiation damage to normal lung tissue may enable a broader application of chest radiotherapy.

3. Introduction to Nrf-2 and its role in disease models

Nrf-2 is an intracellular transcription factor that is degraded in the cytoplasm under normal conditions by interacting with Kelch ECH-binding protein 1 (Keap1) inhibitors and then acts as an activator of ubiquitination factor [26]. The Nrf-2/Keap1 axis plays a significant role in the cellular regulation of redox homeostasis, mitochondrial physiology, autophagy, protein homeostasis, the immune system, and metabolism [27].

Studies in mouse models have shown that Nrf-2-mediated gene expression is an essential regulator of cellular response to radiation. Recent research indicates that cellular Nrf-2 absence enhances the sensitivity to radiation, resulting in reduced radioresistance. This effect is achieved by activating Nrf-2 overexpression, which helps mitigate radiation-induced damage. In conclusion, several preclinical experiments have verified that increased Nrf-2 expression can minimize radiation damage to normal tissues. However, the current study has not yet answered whether Nrf-2 expression affects the radiation sensitivity of normal human tissues, as it does in mice. Nevertheless, we expect additional relevant clinical trials to be carried out in the next few years, which may provide new treatment options for RILI. We briefly summarized some preclinical studies on the relationship between Nrf-2 expression and tissue radiation injury response, as shown in Table 1.

Table 1.

The role of Nrf-2 in the response to normal tissue radiation injury.

	Intervention	Effect	Mechanism	Outcome	Reference
Hematopoietic system	Theaflavin	Nrf-2 activation	Alleviated radiation-induced DNA damage	Ameliorate radiation-induced hematopoietic system injury	[28]
	Vam3	Nrf-2 activation	Decreased the cellular ROS level	Ameliorate radiation-induced hematopoietic system injury	[29]
	TMC	Nrf-2 activation	Notch pathway activation	Ameliorate radiation-induced hematopoietic system injury	[30]
Tongue	CDDO-Im	Nrf-2 activation	Alleviated radiation-induced DNA damage	Ameliorate radiation-induced oral mucositis	[31]
Gastrointestinal tract	3,3′-Diindolylmethane	Nrf-2 activation	Decreased the cellular ROS level	Ameliorate radiation-induced intestinal injury	[32]
	CDDO	Nrf-2 activation	Alleviated radiation-induced DNA damage	Ameliorate radiation-induced intestinal injury	[33]
	Quercetin	Nrf-2 activation	Decreased the cellular ROS level	Ameliorate radiation-induced intestinal injury	[34]
Skin	Curcumin	Nrf-2 activation	Down-regulation of both inflammatory and fibrogenic cytokines	Ameliorate radiation-induced cutaneous cytotoxicity	[35]
Lung	Thalidomide	Nrf-2 activation	Inhibition of TGF-β1/Smad3 pathway	Ameliorate radiation-induced lung fibrosis	[36]
	EASM	Nrf-2 activation	Inhibition of TGF-β1/Smad3 pathway	Ameliorate radiation-induced lung fibrosis	[37]

Abbreviations:Nrf-2:nuclear factor erythroid 2-related factor 2; ROS:Reactive oxygen species; TBI:total body irradiation; TMC:2-trifluoromethyl-2′-methoxychalone; TGF: Transforming growth factor; HSC:hematopoietic stem cell; CDDO:1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl); EASM:Salvia miltiorrhiza.

Numerous studies have demonstrated that Nrf-2 protects against oxidative lung disorders such as COPD, asthma, IPF, ARDS, respiratory syncytial virus disease, etc. Based on the above studies, Nrf-2 also has a specific protective effect against RILI, which undoubtedly provides new prevention and treatment ideas for RILI.

COPD is a progressive respiratory disease characterized by permanent alveolar wall destruction with loss of lung elasticity and ultimately irreversible airflow limitation, which is associated with a high mortality rate [38]. Smoking and oxidative stress are considered to be significant risk factors for COPD [39,40]. Impaired Nrf-2 has been shown to contribute potentially to the development of COPD [41]; this could be due to the Nrf-2 pathway's role in increasing antioxidant defense and decreasing lung inflammation and alveolar apoptosis. These mechanisms help protect alveolar cells from the harmful effects of tobacco smoke [[42], [43], [44]]. At the same time, recent studies have also shown that activation of the Nrf-2 pathway can balance redox reactions in COPD and restore macrophage function, thus playing a protective role [45].

Asthma is a genetic disease characterized by chronic inflammation and extensive and variable reversible airway obstruction [46]. It has been reported that in mouse models, disruption of the Nrf-2 pathway results in enhanced severity of the asthmatic response, possibly due to reduced basal Nrf-2 expression, leading to reduced antioxidant activity in the lungs, as well as significant attenuation of the transcription of multiple antioxidant genes [47,48]. Several recent studies have demonstrated the protective effects of sulforaphane against asthma. Sulforaphane-activated Nrf-2 signaling plays improves the bronchial protective response to methacholine (MCh) [48,49].

IPF is a chronic progressive lung disease associated with fibroplasia and excessive ECM deposition, which can eventually lead to irreversible interstitial lung fibrosis and respiratory failure. Early studies using mouse models revealed that patients with Nrf-2 deficiency are more susceptible to IPF-like PF and bleomycin-induced effects, resulting in more pronounced lung inflammation and fibrosis [50]. Nrf-2 signaling was also found to enhance antioxidant activity and inhibit bleomycin-induced inflammation in experimental PF [51,52].

ARDS is a severe clinical condition characterized by dyspnea, refractory hypoxemia, and noncardiogenic pulmonary edema. Nrf-2-deficient mice are more likely to develop ARDS with enhanced lung permeability, epithelial damage, and inflammation in response to stimulation than wild-type mice [53]. Recently, several studies have reported the protective effects of Nrf-2 activators against ARDS [[54], [55], [56]].

The respiratory syncytial virus (RSV) is currently considered the leading cause of acute respiratory infections in infants and children. RSV infection can significantly reduce the Nrf-2 levels and antioxidant enzymes in the airways and lungs of mice and nasopharyngeal secretions in children, which may be due to RSV-induced Nrf-2 degradation [57,58]. Previous experiments have demonstrated that Nrf-2-deficient mice develop more severe bronchopulmonary inflammation, epithelial damage, and reduced viral clearance in the presence of RSV infection [59].

4. The potential role and mechanisms of Nrf-2 in radiation induced lung injury

The Nrf-2 signaling pathway is key in regulating cell and tissue homeostasis and protecting cells from oxidative and pro-electrical stresses [60,61]. Electrophilic reagents or ROS can lead to conformational changes in Keap1, resulting in the dissociation of Nrf-2 from Keap1 and its subsequent translocation to the nucleus to activate the antioxidant genes [62,63]. Nrf-2 deficiency impairs ΔNp63 stem/progenitor cell mobilization after irradiation and promotes EMT of alveolar type 2 cells to myofibroblasts [36,64,65]. In addition, recent studies have shown that Nrf-2 plays a crucial role in oxidative homeostasis and inhibition of ferroptosis [[66], [67], [68]], strongly suggesting that Nrf-2 is closely associated with the onset and development of RILI.

4.1. Nrf-2 is related to radiation-induced inflammatory response

Several studies have shown that the Keap1-Nrf-2 pathway plays an essential role in the cytoprotective response to oxidative and pro-electrical stress, and the critical signaling factor in this pathway is the transcription factor Nrf-2 [[69], [70], [71]]. As previously described, Nrf-2 remains inactive by forming a complex with Keap1 in the mammalian cytoplasm. Keap1 is a cysteine-rich protein. When exposed to ionizing radiation, the reactive cysteine residues in Keap1 are covalently modified, damaging the structural integrity of the Keap1-Cul3 E3 ligase complex [72,73]. Subsequently, Nrf-2 dissociates from Keap1, translocates to the nucleus, heterodimerizes with the small Maf protein, and activates the target gene through the antioxidant/electrophilic response element (ARE/EpRE). It participates in glutathione synthesis, eliminates ROS, and inhibits oxidative stress to promote cell protection [[74], [75], [76]]. Thus, Nrf-2 can mitigate radiation-induced acute lung injury.

The lethal damage caused by RT is primarily the result of direct DNA damage by ionizing radiation [11]. It has also been suggested that Nrf-2 can repair radiation-induced DNA damage through the basal excision repair pathway and repair of broken DNA duplexes [77,78]. The basal excision repair pathway has been described in a study by Singh et al. Nrf-2 binds to the OGG1 promoter, and Nrf-2 deficiency inhibits OGG1 expression [79]. It has been demonstrated that the human OGG1 promoter has an ARE of 29 bp from the transcription start site. That alternate splicing of OGG1 leads to the expression of mitochondrial and nuclear proteins [80,81]. Nrf-2 repairs broken DNA duplexes primarily by regulating 53BP1 [33]; this is in line with previous studies showing that 53BP1 is involved in the repair of DNA double-strand breaks and that 53BP1 deficiency increases radiosensitivity [[82], [83], [84], [85]].

Furthermore, it has been shown that Nrf-2 deficiency decreases the levels of radiation-induced serum anti-inflammatory cytokines, IL-10, and antioxidant proteins, exacerbating the radiation-induced imbalance of serum inflammatory cytokines, thus, the inflammatory response [86,87]. All the above findings suggest that Nrf-2 has a considerable protective effect against RILI and inflammation.

4.2. Nrf-2 is involved in the regulation of pulmonary fibrosis

In the late stage of RILI, PF is a consequence of delayed radiation effects and is often considered an irreversible hazard [4,88]. Recent clinical studies have shown that Nrf-2 is associated with radiation-induced PF, and Nrf-2 activators have demonstrated anti-fibrotic effects [89]; this is mainly because Nrf-2 deficiency inhibits the mobilization of ΔNp63 stem/progenitor cells while amplifying the tendency of alveolar type 2 cells to convert into myofibroblasts under radiation induction [64]. In the injured lung, Δp63+/Krt5+ stem cells are significantly mobilized and proliferate. Diphtheria toxin targets the stem cells to damage the regeneration of injured lungs and pulmonary oxygenation while promoting fibrosis. Studies have shown that targeting Nrf-2 can promote epithelial cell repair and activate the BRCA1/Nrf-2/miR-140 signaling pathway to reduce the self-renewal of lung fibroblasts while increasing their migration and contraction, thereby reducing the risk of pulmonary fibrosis [9,90,91]. Xi et al. claimed that under hypoxic conditions in the lung, Notch signaling and Krt5POS basal-like cell expansion are driven by hypoxia-inducible factor (HIF1α) to re-encode Δp63+/Krt5+ cells to form basal-like metaplasia [92]. Nrf-2 activation improves the antioxidant capacity of fibroblasts and myofibroblast dedifferentiation in IPF [93].

The anti-fibrotic function of Nrf-2 is also reflected in the inhibition of EMT and TGF-β1/Smad signaling [[94], [95], [96]]. Nrf-2 deficiency promotes TGF-β/Smad signaling, a key factor in promoting EMT. At the molecular level, it has been shown that Nrf-2 can form a nuclear complex with nuclear pSmad3 at the CAGA site of the proximal promoter of the TGF-β target gene, thereby inhibiting gene expression [97,98]. TGF-β/Smad signaling induces ATF3. The ATF2/Nrf-2 complex binds to the ARE site in the Nrf-2 target gene promoter and inhibits the recruitment of CBP to the ARE and expression of Nrf-37 target genes [99,100]. It is generally believed that EMT is another major source of myofibroblasts and that Nrf-2 alleviates PF by blocking EMT progression [101]. Moreover, Nrf-2 attenuates TGF-β1-induced EMT by down-regulating high mobility group box 1 (HMGB1) [102]. Nrf-1, Nrf-148, and Nrf-155 also help to alleviate EMT progression [96,103,104]. The current Nrf-2-related signaling pathways and lung inflammation and fibrosis-related signaling pathways can be seen in Fig. 2.

Fig. 2.

Nrf-2 and signaling pathways. In classical TLRs/NF-κB signal transduction, TLR activates the TAK1-TAB1 kinase complex, leading to the synergistic release of NF-κB dimers and IκB phosphorylation by the IKK complex. However, the activation of Nrf-2 can inhibit the phosphorylation of IκB in the typical NF-κB pathway, thereby reducing the nuclear accumulation of NF-κB dimers and inhibiting the expression of downstream immune response genes, such as IL-1, IL-6, TNF-α, iNOS, and COX-2. Activation of the TGF-β1/Smad pathway leads to a disturbance in the steady-state microenvironment, essential for promoting FMD and EMT processes. Activation of Nrf-2 specifically blocks these two processes and protects against pulmonary fibrosis.

Abbreviations: TLR: Toll-like receptor; IκB: inhibitory kappa B; NQO1: NADH quinone oxidoreductase 1; HO-1: Heme oxygenase-1; IL: Interleukin; TNF: Tumor necrosis factor; iNOS: inducible nitric oxide synthase; COX-2: Cyclooxygenase-2; Ub: Ubiquitination; TGF: Transforming growth factor; EMT: Epithelial-mesenchymal transformation; FMD: fibroblast-myofibroblast differentiation.

Mont et al. demonstrated that iso-L-prostaglandin (IsoLG) adducts of proteins are formed in radiation-induced and oxidant-mediated lung injury and that oxidative stress caused by the loss of Nrf-2 or NADPH oxidase activity can promote IsoLG adduct formation [105]. Chronic oxidative stress leads to the accumulation of IsoLG adducts, which can lead to protein toxicity or apoptotic events [64]. While this theory presents an alternative hypothesis for regulating pulmonary fibrosis by Nrf-2, it is essential to note that both studies are small-sample preclinical studies, which inherently possess certain limitations regarding objectivity. This argument would be more convincing if more large-scale clinical studies based on this theory could be carried out.

4.3. Nrf-2 regulates radiation induced cell ferroptosis

Ferroptosis is an oxidative stress-dependent cell death process characterized by iron accumulation and lipid peroxidation [106]. Research has shown that ferroptosis is important in radiation-induced cell death [107]. ROS overload is the basis of ferroptosis, which kills cells by amplifying oxidative stress or inhibiting the antioxidant system [108]. Studies have shown that after treatment of acute RILI with ferroptosis inhibitors, the level of ROS in the lungs and serum inflammatory cytokines (TNF-α, IL-6, IL-10, and TGF-β1) decreased significantly, leading to reduced radiation damage [109,110]. Ferroptosis can be induced by inhibiting GSH synthesis and disrupting the redox balance, thereby increasing the radiosensitivity of tumor cells [111]. A report by Ling et al. suggested that ferroptosis plays a vital role in regulating EMT in PF [112]. A recent report also demonstrated that miR-let-7, an exosome derived from menstrual blood stem cells, can inhibit ferroptosis and improve pulmonary fibrosis through the Sp3/HDAC2/Nrf2 signaling pathway [113].

According to recent research, Nrf-2 is also closely involved in ferroptosis [114,115]. Firstly, Nrf-2 is essential for iron metabolism [116]. Nrf-2 positively regulates the transcription of heme oxygenase 1 (HMOX1), increases the storage of iron, and reduces intracellular free iron by rapidly upregulating the transcription of ferritin light chain (FLH) and ferritin heavy chain (FTH) [117]. Nrf-2 can control FTL/FTH, and the iron transporter (SLC40A1) is responsible for iron transport out of cells [[118], [119], [120]]. Secondly, Nrf-2 can participate in the catabolism/detoxification of reactive intermediates, such as AKR1B1, inhibiting the de novo synthesis of glutathione [121] and ALDH1A1, enhancing DNA repair [122]. In addition, Nrf-2 directly promotes the expression of GPX4, regulates the GCH1/BH4 pathway to mediate the cell redox reaction, and inhibits ferroptosis [123,124]. Studies have revealed that Nrf-2 can also mediate glutathione synthesis by promoting the expression of SLC7A11, glutamate cysteine ligase (GCLC/GLCM), and glutathione synthetase (GSS), which play crucial roles in preventing ferroptosis [[125], [126], [127]]. Therefore, Nrf-2 can activate downstream genes to regulate ferroptosis, and its target genes can be divided into three categories, as shown in Fig. 3.

Fig. 3.

Nrf-2 is involved in ferroptosis. Under stress-free conditions, Keap1 homodimers promote Nrf-2 ubiquitination. Under oxidative or electrophilic stress, the reactive cysteine residues in Keap1 are covalently modified, allowing Nrf-2 to dissociate from Keap1 and translocate to the nucleus, where it regulates ferroptosis through activation of the target genes by the antioxidant ARE.

Abbreviations: Nrf-2: nuclear factor erythroid 2-related factor 2; Keap1: Kelch ECH binding protein 1; sMAF: small Maf; ARE: antioxidant response element; Ub: Ubiquitination.

In addition, HIF-1α acts as a regulator of ferroptosis. The up-regulation of HIF-1α can buffer radiation-induced ROS and reduce ferroptosis, thereby enhancing the radioresistance of cells [128]. Nrf-2 is also involved in HIF-1α-mediated ferroptosis inhibition. Nrf-2 silencing blocks the accumulation of HIF-1α in hypoxic cancer cells, weakens its regulatory effect on cell metabolism, and leads to an imbalance of ROS homeostasis [129]. These results suggest that Nrf-2 can inhibit ferroptosis by regulating proteins associated with iron metabolism and ROS-scavenging pathways, thereby reducing radiation-induced oxidative stress and enhancing radiosensitivity.

5. Therapeutic potential of targeting of Nrf-2 in RILI

In conclusion, Nrf-2 is an essential predictor of RILI and may play an important role in preventing RILI. Nrf-2 and its target genes play a vital role in the development of RILI, and studies have suggested that inhibition of Nrf-2 activity may be a viable strategy for improving the radiation response and reducing radiation resistance in cancer [130]. Therefore, antioxidant therapy that activates Nrf-2 may be an effective intervention for preventing and treating RILI. Table 2 summarizes the common activators and inhibitors of Nrf-2 and their mechanisms of action.

Table 2.

Activators and inhibitors of Nrf-2.

	Compound	Mechanism of action	Reference
Nrf-2 activators	Resveratrol	Modification of Keap1-Cys-151	[94,131]
	Sulforaphane	Modification of Keap1-Cys-151	[98]
	Oltipraz	Modification of Keap1-Cys-151	[132]
	Dimethyl fumarate	Modification of Keap1-Cys-151	[133]
	CDDO; CDDO-Im	Modification of Keap1-Cys-151	[134,135]
	Curcumin	Modification of Keap1-Cys-151	[136]
	Diallyl trisulfide	Modification of Keap1-Cys-288	[137]
	Apigenin	Epigenetic modifications of Nrf-2	[138]
	Epigallocatechin-3-gallate	Oxidizing the cysteine thiols of Keap1	[139]
Nrf-2 inhibitors	Retinoic Acid	Prevention of nuclear translocation of Nrf-2	[140,141]
	Trigonelline	Prevention of nuclear translocation of Nrf-2	[142]
	Chrysin	Prevention of nuclear translocation of Nrf-2	[143]
	Luteolin	Nrf-2 mRNA degradation, Reduction of Nrf2 binding to AREs	[144]
	Brusatol	Stimulation of Nrf-2 poly-ubiquitination	[145]

Abbreviations: Nrf-2: nuclear factor erythroid 2-related factor 2; Keap1: Kelch ECH binding protein 1; ARE: antioxidant response element; Cys: cysteine.

Antioxidants known to help prevent and treat RILI include thiol compounds, plant antioxidants, antioxidant enzymes, etc [146].The commonly used sulfhydryl compounds mainly include GSH and its precursor N-acetylcysteine (NAC). The primary mechanism is to use the active sulfhydryl group of its side chain to combine with free radicals and neutralize free radicals to protect the sulfhydryl group on its protein from oxidation. As mentioned above, Nrf-2 can regulate the expression of the downstream genes to mediate the synthesis of GSH from glutathione to regulate cellular ferroptosis; GSH can also be reduced to H2O by binding to H2O2 by the enzyme glutathione peroxidase to minimize oxidation. NAC, a precursor of GSH, can also act as a direct ROS scavenger and regulate the redox state of the cells [147]. Another study demonstrated that NAC may affect mucin expression and act as a mucolytic agent during oxidative stress and inflammation [148]. The application of NAC reportedly reduced sputum production in patients with RP, thereby reducing the use of expectorants [149].

At present, plant antioxidants have gradually gained attention in the prevention and treatment of RILI due to their advantages of comprehensive source, low side effects, and high patient acceptance, and have potential effects on radiosensitization of cancer cells and radiation protection of non-cancer cells [150]. Polyphenols in the diet, such as curcumin, resveratrol, and myrtol, can regulate the activation of Nrf-2 and biosynthesis of glutathione to remove ROS, thereby regulating inflammatory factors in macrophages and lung epithelial cells to play a therapeutic role [7,151,152]. Hesperidin and naringenin, antioxidants found in citrus peels and seeds, have been shown to significantly reduce the inflammatory response of the lung tissue after RT in rats and are potential therapeutic drugs for RILI [153,154].

The antioxidant enzyme system maintains the redox balance and protects against ROS, including heme oxygenase (HO-1), catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). Increased levels of ROS activate Nrf-2 signaling, induce the expression of antioxidant enzymes, and protect cells from oxidative stress. At present, there are many studies on SOD in the field of RILI prevention and treatment, especially on SOD small-molecule analogs (AEOL 10150). Several studies have revealed that SOD can reduce lung injury caused by radiation and significantly improve survival rates of lung cancer patients [155,156]. Antonic et al. found that subcutaneous injection of 15 mg/kg bovine SOD 15 h after radiotherapy significantly improved the increased respiratory rate, pathological changes in the lung tissue, oxidative stress, macrophage activation, and TGF-β expression in rats [157]. Although this study verified that superoxide dismutase (SOD) can reduce reactive oxygen species (ROS) levels, the production of ROS may vary with the volume, dose, segmentation, course of treatment, and type of concurrent radiotherapy. If this study had included a comparison of the results at different administration times after radiotherapy, it would be of greater relevance.

Based on these results, we can infer that Nrf-2 promotes the expression of SOD in RILI, which is clinically significant since it can reduce the damage and inflammatory response of the normal lung tissue to ROS. These results indicate that Nrf-2 enhances the expression of SOD in RILI; this has clinical significance as it could reduce the adverse effects and inflammatory reactions caused by ROS in healthy lung tissue. Although Nrf-2 has significant therapeutic potential, somebody should adjust the timing and course of treatment with antioxidants to achieve absolute radiation protection. Therefore, the issues of individualization and standardization need to be further explored.

6. Perspective and conclusion

Injury to normal lung tissue is almost inevitable when cancer patients receive chest radiotherapy. The clinical needs for effective prevention or treatment of RILI have not been met. Therefore, it is necessary to consider previously unrecognized mechanisms of RILI to determine new effective therapies. The expression of Nrf-2 affects the recovery of radiation injury in a tissue-dependent manner. It has a specific protective effect on the skin, hematopoietic system, oral cavity, gastrointestinal tract, and lung. Especially in the lungs, Nrf-2 attenuates inflammatory response, oxidative stress, fibrosis, and cell death, ultimately having a protective effect against the development of lung diseases. It is considered an important target for clinical prevention of RILI.

At present, increasing evidence shows that targeting Nrf-2 may be very effective in the treatment or prevention of RILI. However, further standardized and large-scale clinical research evidence is needed to clarify the role of Nrf-2 in RILI after radiotherapy. In addition, although it has been determined that Nrf-2 is involved in the up-regulation pathway of inhibiting ROS production and down-regulating the expression of numerous genes, there is still a lack of understanding of the many downstream pathways mediated by Nrf-2; this hinders the transformation of preclinical evidence into clinical practice, providing suggestions for our further research ideas. Suppose somebody can control the expression or activity of Nrf-2 to increase radiotherapy's damage to tumor tissues and reduce the radiation damage to normal lung tissues. In that case, it will be of great clinical significance for treating and prognosis patients with thoracic tumors.

Funding

This work was supported by Hubei Province Natural Science Foundation of China (2022BCE038 to Jun Cai).

Ethics declarations

All participants provided informed consent to participate in the study.Review and/or approval by an ethics committee was not needed for this study because it does not directly involve humans or animals. It is a comprehensive analysis and summary of the existing literature.

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

CRediT authorship contribution statement

Yuan-Yuan Chen: Visualization, Writing – original draft. Meng Wang: Investigation, Writing – original draft. Chen-Yang Zuo: Investigation. Meng-Xia Mao: Visualization. Xiao-Chun Peng: Supervision, Conceptualization, Writing – review & editing. Jun Cai: Project administration, Writing – review & editing, Funding acquisition.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.