NRF2 signalling pathway: New insights and progress in the field of wound healing

Abstract As one of the most common pathological processes in the clinic, wound healing has always been an important topic in medical research. Improving the wound healing environment, shortening the healing time and promoting fast and effective wound healing are hot and challenging issues in clinical practice. The nuclear factor‐erythroid–related factor 2 (NFE2L2 or NRF2) signalling pathway reduces oxidative damage and participates in the regulation of anti‐oxidative gene expression in the process of oxidative stress and thus improves the cell protection. Activation of the NRF2 signalling pathway increases the resistance of the cell to chemical carcinogens and inflammation. The signal transduction pathway regulates anti‐inflammatory and antioxidant effects by regulating calcium ions, mitochondrial oxidative stress, autophagy, ferroptosis, pyroptosis and apoptosis. In this article, the role of the NRF2 signalling pathway in wound healing and its research progress in recent years are reviewed. In short, the NRF2 signalling pathway has crucial clinical significance in wound healing and is worthy of further study.

F, as well as bZip proteins, to form heterodimers and activate gene transcription. The kelch domain of kelch-like ECH-related protein 1 (KEAP1) specifically interacts and mediates the ubiquitination, and the degradation of NRF2 occurs at the Neh2 domain, which mainly contains ETGE and DLG motifs and is the core structure of the NRF2 area. The remaining domains, when combined with various components in the transcription mechanism, act as transcription activation domains, mediate the degradation of NRF2 in oxidatively stressed cells or bind to other receptors to regulate the activity of NRF2. [1][2][3][4][5] The NRF2 regulatory pathway plays a vital role in protecting cells from oxidative damage. After exposure to electrophilic and oxidative stress, the reactive cysteine residues of Keap1 are modified, resulting in reduced E3 ligase activity, stable accumulation of NRF2 in cells and robust induction of a series of cytoprotective genes. 6 During wound healing, free radicals, such as reactive oxygen species (ROS), are considered the main driving force of oxidative damage.
The healing process of diabetic wounds is affected by the internal pathophysiology and external factors (repeated infection and trauma), such as reduced blood supply, matrix migration and wound contraction. Pretreatment with NRF2 activator reduced the oxidative stress level of diabetic wounds and promoted wound healing. 7,8 Given the high cost of wound treatment, it is essential to study this process regarding physical health and economic costs. Therefore, it is necessary and urgent to study the specific role of the NRF2 factor in wound healing.

| The process of wound healing
Wound healing and treatment are enduring topics in medical research, and although there have been numerous advances in understanding the steps involved in wound healing, the exact mechanisms underlying wound healing remain unclear. As one of the most complicated and miraculous physiological processes, wound repair occurs regularly in all living organisms 9,10 In current studies, the process of wound repair has been divided into four stages: haemostasis, inflammation, proliferation and remodelling ( Figure 1).
Directly after tissue injury, the wound healing process begins.
The wound starts to stop bleeding, and the damaged tissue cells release vasoactive substances to constrict the local blood vessels.
At the same time, platelets aggregate and activate the coagulation system, and fibrinogen forms an insoluble fibrin network, produces blood clots, seals damaged blood vessels and protects the wound to prevent further bacterial contamination and loss of body fluids.
Inflammation, a complex and massive body defence response with the purpose to remove or inactivate harmful substances, removes necrotic tissue and creates the right conditions for the subsequent proliferation process. 11 The proliferative stage of wound healing is also called the granulation stage. It starts in the first week after the trauma and lasts about two to three weeks. New epidermal cells proliferate and divide to replace injured dead cells. The whole proliferative stage mainly includes three parts: cell proliferation and migration (re-epithelialization), cell recruitment, and granulation tissue production. The epidermal cells at the edge of the wound migrate to the central area of the wound after temporary dedifferentiation. During the migration process, these cells rest in contact with the adjacent cells rather than immediately getting in contact with the wound edges. Therefore, epidermal cells continuously migrate and stagger in the wound and finally connect to form a complete epidermal layer. In detail, under the action of various growth factors, dermal fibroblasts can further differentiate into myofibroblasts and secrete large amounts of collagen and extracellular matrix proteins to the central area of the wound. It is well known that new granulation tissue is mainly composed of new capillary networks, fibroblasts, myofibroblasts and extracellular matrix protein (ECM), as well as inflammatory cells. A large number of fibroblasts and inflammatory cells often gather around capillaries. Moreover, inflammatory cells are usually dominated by macrophages and include many neutrophils and lymphocytes.. 9,12 F I G U R E 1 The process of wound healing. The process of wound healing includes four stages: haemostasis, inflammation, proliferation and remodelling. The cells involved in wound healing include macrophages, neutrophils, keratinocytes, monocytes, endothelial cells and fibroblasts As the amounts of blood vessels and water in the granulation tissue decrease, the tissue in the wound hardens gradually to finally form scars. After wound repair, the hardened tissue softens and flattens, and the tissue intensity strengthens. Epithelial cells cover the wound due to proliferation and migration from the edge of the wound to its centre to form new epithelial cells. Decomposition and remodelling of the wound starts with the decomposition process, which comprises the three stages of wound healing and terminates with the end stage.
The structures assembled in previous stages are removed or modified.
As the edge of the migration layer aggregates, epidermal cell migration stops, and proliferation decreases. Extracellular matrix proteins are reshaped, and granulation tissue is eliminated. By dissolving the inside of the wound, a scar is formed by the aligned ECM filaments. Although failure or termination of any of the above processes can have catastrophic consequences due to bleeding or infection, a series of severe problems may also occur if any process takes longer than expected.
For example, excessive or long-term inflammation may slow down the process or even lead to aggravation of the original wound because the neutrophil collateral damage produced large amounts of ROS. 12 In the process of normal wound healing, the level of reactive oxygen species is in a relatively balanced state. The redox signal mediated by the reactive oxygen species participates in different processes such cell recruitment and cytokine and growth factor production and plays a vital role in wound healing. 13 When the body is in a state of disease, such as diabetes, this balance is broken, and the mitochondria produce considerable amounts of reactive oxygen species. By up-regulating the polyol, hexosamine, and protein kinase C pathways and increasing the formation of glycation end products, endothelial cells are damaged, leading to local ischaemia and hypoxia. 14 Simultaneously, oxidative stress injury results in reduced proliferation and migration of keratinocytes and fibroblasts, decreased synthesis of collagen and fibronectin, and imbalance of matrix metalloproteinases and their inhibitors, which further increases the wound healing difficulty. 15 In addition, the sustained high ROS level causes chronic and long-time inflammation of the wound, which further causes cell damage and delays wound healing. 13 Therefore, oxidative stress injury is one of the critical factors of non-healing wounds. Therefore, wound repair is an extremely complex biological process in which transcription factors play a vital role. 16,17 NRF2 is one of the most representative transcription factors in the process of oxidative stress. Furthermore, the NRF2 signalling pathway takes part in the wound healing process. This article describes the role of the NRF2 signalling pathway in wound healing.

| E XPRE SS I ON AND AC TIVIT Y OF NRFIN WOUNDS
Oxygen (O 2 ) is one of the substrates required for mitochondria to drive adenosine triphosphate (ATP) synthesis. In the process of wound repair, oxygen continuously supplies energy for tissue renewal and metabolism. At the same time, ROS, that is free radical derivatives of oxygen, are crucial. Members of the ROS family are molecules that usually contain O 2 and are reduced to highly active free radical forms due to the electrons at the surface of these molecules. Typical members of the ROS family are the superoxide anion (⋅O 2 − ), peroxide ion (⋅O 2 −2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH) and hydroxyl ion (OH − ). Under normal circumstances, the endogenous ROS source may be the endoplasmic reticulum or the oxidative phosphorylation of mitochondria during ATP production.
The role of ROS in cell homeostasis shows that low ROS levels may inhibit cell growth and cause cell cycle arrest, while excessive ROS will induce the activation of pro-apoptotic proteins and further induce cell death. 18,19 Therefore, many studies focused on the role of NRF2 in wound healing. Keratinocytes of the hyperproliferative epithelium in skin wounds showed a strong expression of NRF2, but NRF2 gene expression was also observed in granulation tissue cells. 20  However, the mode of action of NRF2 is still uncertain, but NRF2 mainly targets the inflammation and proliferation phases of wound repair (Table 1).
It has recently been demonstrated that exosomes, as natural nanoparticles secreted from cells, play a crucial role in cell-to-cell communication. 38 35 research direction and whether it plays a role in the healing process of human wounds remains to be verified.

| EFFEC T OF NRF2 DEFI CIEN C Y ON WOUND HE ALING
The effect of NRF2 activation on wound healing has been described in detail above, and the lack of NRF2 in wound healing has also been studied in depth. Due to the lack of the KEAP1 binding domain to achieve the specific expression of NRF2, which does not affect the interaction between KEAP1 and other proteins, the caNRF2 mutant still has constitutive activity in the nucleus. 59,60 As one of the primary cells involved in the wound healing process, activated Nrf2 fibroblasts play an indispensable role in the whole wound healing process. Relevant studies have shown that the activation of NRF2 in fibroblasts accelerates the senescence of fibroblasts in vivo or in vitro. The main reason is that, mediated by NRF2, the senescence-promoting factor plasminogen activator inhibitor 1 (PAI-1, serpine1) in the matrix accelerates the senescence of cells, although it reduces the ROS level and DNA damage.
However, in mice expressing caNRF2 fibroblasts, the wound healing time is shortened, and senescent cells can directly promote the proliferation of keratinocytes. 52 The activation of NRF2 in inflammatory cells seems to have a relatively small effect on wound healing. Mice expressing caNRF2 in bone marrow cells showed no significant difference in the wound closure rate and even failed to show a significant increase in the expression of NRF2 target genes. 53 A possible reason is that, even under steady-state conditions, the NRF2 expression level in these immune cells (such as neutrophils) is still high, so the specific expression of caNRF2 is not evident or does not occur.
Combined with the above studies, in the case of adverse effects on wound healing (such as oxidative stress and increased ROS levels), activating the NRF2/HO-1 pathway is a potential treatment strategy to promote wound repair.

| Regulation of calcium
In homeostasis, due to the Na + -K + -ATP and Ca 2+ pumps on the bimo-

| Relationship between mitochondrial oxidative stress and NRF2 pathway
Among the various sources of reactive oxygen species (ROS) in cells, electrons released by the mitochondrial electron transport chain present the most important ROS source. In mitochondria, molecular oxygen is reduced to the superoxide anion (O2 •-), which is then converted into H 2 O 2 to produce more active hydroxyl groups (•OH  and ferroptosis, as well as the synthesis of the inhibitor GPx4. 65,66 Furthermore, these target genes are also involved in iron metabolism proteins, such as transferrin receptor (TfR1), iron transporter (Fpn) and ferritin-1 (Fer-1), as well as the regulation of haeme oxygenase 1 (HO-1).

| NRF2 signalling pathway in ferroptosis
Michele et al showed that Gclc-deficient mice exhibited epidermal hyperkeratosis, reduced adhesiveness of the corneocytes and glutathione-deficient keratinocytes. Additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione-dependent. 64 Thus, Michele et al hypothesized that the cell-protective effect of NRF2 is strongly induced by enzymes involved in glutathione synthesis and recovery. In the absence of Gclc, this protective effect is no longer present, so the regulation of Nrf2 in Gclc-deficient cells does not result in a functional rescue. Therefore, in addition to the regulatory relationship between Gclc and Nrf2, the crosstalk between NRF2 and ferroptosis still needs further research and discovery in the field of wound healing.

| NRF2 signalling pathway in pyroptosis
Pyroptosis, which is characterized by the release of inflammasomes, the formation of ASC (apoptosis-associated spot-like protein) and the activation of pro-inflammatory caspase, is a widely recognized form of programmed cell death. 67

| NRF2 signalling pathway in autophagy
Autophagy is a biological process by which eukaryotic cells maintain cell homeostasis and perform self-renewal. This process has become another important research field after apoptosis. Autophagy can be divided into three types: macroautophagy, microautophagy and chaperon-mediated autophagy, but currently, it usually refers to the research of macroautophagy. As a dynamic biological process, autophagy includes several stages: initiation, expansion and maturation. Autophagy-related (ATG) genes are regulators in this process that take part in every stage of the process and strictly regulate each stage. 71 The induction of autophagy promotes migration under cer- feedback loop enhances the NRF2 activity. 76 As a possible reason, they hypothesized that the degradation of KEAP1 is reduced, and oxidized phosphatidylcholines directly affect NRF2. 6 In this case, studies on genetically modified mice have shown that NRF2 is a double-edged sword, and persistently high levels of NRF2 may not have a protective effect. NRF2 may also interfere with the homeostasis of the cell epidermis. 77 In conclusion, autophagy is a relevant mechanism for limiting the NRF2 activity in the dermis.

| NRF2 signalling axis in programmed cell necrosis
Programmed cell necrosis is a new type of cell death, which usually has similar morphological characteristics to cell necrosis but is regu- The very close relationship between the NRF2 pathway and procedural necrosis presents a potential research direction in the field of wound repair.

| GPX
GPX is a strong reductant. In its oxidized state, it simultaneously releases electrons by GSSH and mainly catalyses the formation of peroxide or organic hydrogen peroxide. 84,85 The activity of GPX is regulated by the NRF2-ARE axis.

| SODs and CAT
Studies have shown that both SODs and CAT are downstream target genes of NRF2. SOD is the oxygen free radical with the highest oxidation potential among ROS molecules. Therefore, a two-step process is required for the elimination of SOD, and SODs and CAT are the main regulators of these two reactions. First, SODs are decomposed into hydrogen peroxide and water, and then CAT further decomposes the formed hydrogen peroxide into water and oxygen, completely scavenging the oxygen free radicals. 18

NQO-1
In cells, quinone compounds are usually converted into relatively unstable semi-quinone compounds by cytochrome P450 and NADPH, which can react with the sample to generate reactive oxygen damage to cells. The main function of NQO-1 is to prevent cellular oxidative damage induced by quinone. NQO-1 competes with NADPH and cytochrome P450 to maintain intracellular redox homeostasis by converting quinones into relatively stable hydroquinones through a two-electron reduction process wherein NADH or NADPH acts as a reduction cofactor. 90 However, studies have found that the regulatory role of NRF2 leads to methylation of the promotor region of NQO-1 to activate its transcription. Therefore, NQO-1 is also regulated by NRF2 transcription. 88

| SUMMARY AND FUTURE PER S PEC TIVE S
As the most prominent molecule of the oxidative stress pathway, NRF2 and its anti-oxidative stress effect have been the focus of an immense number of studies. In addition to its role in wound healing, NRF2 also plays a critical role in preventing cancer and treating chronic diseases. 91,92 However, it is worth noting that, in addition to its cytoprotective effect, NRF2 promotes the senescence of fibroblasts in the skin, the proliferation of keratinocyte stem cells, the proliferation of sebaceous glands and the abnormal deposition of ECM in STZ-induced diabetic mice. 52,93 Keratinocytes, as the primary cell type involved in the wound healing process, can promote their own proliferation and migration and inhibit cell apoptosis when NRF2 is activated. 8 As a multi-organ protection pathway, the NRF2 signalling pathway has anti-oxidative stress and anti-apoptosis effects, promotes the re-epithelialization and mediates matrix remodelling. The NRF2 signalling pathway has become a potential research direction for studying the process of wound healing. Complex regulatory mechanisms occur in oxidative stress-related wound healing, such as the reduction of mitochondrial damage, regulation of calcium ions in the cell, programmed cell death, autophagy and ferroptosis ( Figure 3). Activation and suppression of the NRF2 signalling pathway have a vital impact on wound healing. The indepth study of the NRF2 signal transduction pathway can provide reliable theoretical support for clinical research on wound healing. Therefore, it needs to be clarified if NRF2 plays a crucial role in the human wound.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (grant IDs 81772094 and 81974289).

F I G U R E 3
The main regulation modes of NRF2 signalling pathway. The regulation of NRF2 signalling pathway mainly includes the following: regulation of calcium ion (the overexpression of HO-1 is caused by the increase of calcium ion concentration, and the overexpression of HO-1 can inhibit the transduction of PI3K/AKT signalling pathway), mitochondria oxidative stress (NRF2 is the main transcription factor expressed by SIRT3, so the expression of SIRT3 can be enhanced by regulating NRF2 to optimize the therapeutic effect of mesenchymal stem cells on skin wound healing), ferroptosis (NRF2 downstream target genes HO-1, GCLC, NQO-1 and SLC7A11, by participating in lipid peroxidation), pyroptosis (activate the Nrf2 pathway, promote cell apoptosis and inhibit the activation of inflammasome NLRP3), autophagy (arsenite affects the expression of Nrf2 and mTOR through the PI3K/AKT pathway, as well as its effect on P62 and ATG genes), programmed cell necrosis (the regulatory relationship between Nrf2 and receptor-interacting proteins RIP1 and RIP3) and oxeiptosis (activates oxygen free radicals through the KEAP1-PGAM5-AIFM1 pathway, independent of caspase)