Corneal diseases are one of the major causes of blindness worldwide. Conservative medical agents, which may prevent sight-threatening corneal disease progression, are urgently desired. Numerous evidences have revealed the involvement of oxidative stress in various corneal diseases, such as corneal wound healing and Fuchs endothelial corneal dystrophy (FECD). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like erythroid-cell-derived protein with CNC homology- (ECH-) associated protein 1 (Keap1)/antioxidant response element (ARE) signaling is well known as one of the main antioxidative defense systems. To the best of our knowledge, this is the first review to elucidate the different expression profiles of Nrf2 signaling as well as the underlying mechanisms in corneal diseases, implicating that Nrf2 may serve as a potentially promising therapeutic target for corneal diseases.
The cornea is the transparent front part of the eye and contributes estimated two-thirds of the optical power. Human cornea is mainly composed of corneal epithelium (the outer layer), stroma (the middle layer), and endothelium (the inner layer). Normal morphologies and functions of these cells maintain the transparency of the cornea. The ocular surface mucosa (mainly the cornea) is the first layer of the eye that is exposed to environmental stress. The cornea is susceptible to be damaged by varieties of external stresses due to its constantly direct exposure to harmful factors, such as physical or chemical injuries, UV radiation, and air pollutants (gases, vapors, or cigarette smoke). Oxidative stress is characterized by the generation of reactive oxygen species (ROS), which contains superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), and is considered to be involved in these external stresses [
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2 or NFE2L2), encoded by the gene of NFE2L2, is a vital nuclear transcription factor for the systemic antioxidant defense system. In basal conditions, Nrf2 binds to Kelch-like erythroid-cell-derived protein with CNC homology- (ECH-) associated protein 1 (Keap1) as a complex and is restricted to the cytoplasm where it undergoes ubiquitination and proteasomal degradation. Under stressed condition, Nrf2 separates from Keap1 (a primary Nrf2 inhibitor) and is translocated into the nucleus, where it binds to the phase 2 of antioxidant response element (ARE) in the DNA promoter region and initiates the transcription of ARE controlled antioxidative enzymes, such as superoxide dismutase (SOD), catalase, glutathione S-transferase (GSTP) [
Normally, there is a balance between the systemic generation of oxidants and biological antioxidants’ capacity to remove the oxidants or to repair the oxidative stress-induced damage. Overproduction of ROS or dysfunction of antioxidative enzymes can result in oxidative stress and lead to cellular damages (e.g., lipid peroxidation of cell membranes and oxidative damage to DNA and proteins). Oxidative stress is known as a vital pathogenesis underlying aging and many human ocular diseases, such as corneal diseases (injuries, keratoconus, Fuchs’ endothelial dystrophy (FECD), etc.), dry eyes, cataracts, glaucoma, age-related macular degeneration, and other oxidative-related diseases in the eyes [
To the best of our knowledge, this is the first review to elucidate the specific role and the underlying mechanism of Nrf2-mediated antioxidative defense in corneal diseases, including wound healing, Fuchs’ endothelial dystrophy, and corneal regenerative projects.
Located in the foremost layer of the cornea, corneal epithelial cells are easily injured by external factors such as physical injuries, chemical injuries, and oxidative stress (UV radiation). Delayed corneal wound healing is often found in the cornea of diabetic patients. It was revealed that the topical application of a high dose of carnosol (1 mM), a well-established Nrf2 activator, accelerated the corneal wound healing in diabetic rats with the corneal epithelium mechanically removed [
Corneal keratocytes are activated and transformed into myofibroblasts and fibroblasts upon injury, which is an important biological event during corneal wound repair with scar formation [
4-Hydroxynonenal (4-HNE), a major endogenous product of lipid peroxidation and a key marker of oxidative stress, is considered to play oxidant roles in corneal diseases, such as Fuchs endothelial corneal dystrophy (FECD) and keratoconus [
Schematic diagram of Nrf2 signaling and regulation in the corneal epithelial cells. 4-HNE or H2O2 induces excessive ROS generation (by upregulation of 3-NT, NOX4 protein expression), leading to oxidative stress in the cultured HCE cells and resulting in cell apoptosis and death of corneal epithelial cells. 4-HNE activates Nrf2/ARE-controlled antioxidant enzyme (NQO1 and GSTP) transcription, which facilitates ROS degradation. On the other hand, H2O2 decreases Nrf2/ARE-controlled SOD and catalase transcription, leading to ROS degradation suppression. NAC serves as an antioxidant by antagonizing 4-HNE overexpression and reversing the cell viability of HCE cells. SA3K blocks H2O2-induced ROS, 3-NT, and NOX4 overexpression and upregulates ROS degradation by activating Keap1-Nrf2-ARE pathway.
H2O2 is a main ROS product and known as an oxidative stressor in experimental researches. Various factors, such as Keap1-Nrf2 pathway and NOX4 (an isoform of NADPH oxidase), are considered to be involved in the complex process of oxidative stress. SERPINA3K (SA3K) belongs to the serine proteinase inhibitor family and possesses antioxidant effect. It prevented against H2O2-triggered apoptosis and ROS overproduction as well as repressed GSTP and NQO1 in cultured HCE cells [
Under normal circumstance, the injured corneal epithelium is regenerated by the corneal limbal stem cells. The physiological regenerative function of stem cells is lost in corneal limbal stem cell deficiency (LSCD), leading to corneal opacity and vision impairment. Transplantation of stem cells is applied to treat LSCD, using tissue-engineered epithelial cell sheets, such as human oral mucosal epithelial cell (hOEC) sheets [
Ebselen, an organic selenium-containing redox compound and a well-known Nrf2 activator, has exhibited great potentials as a promising medium for the preservation of tissue-engineered cell sheets and the stem/progenitor cells under hypothermia during preservation. The expression of two tight junction-relevant proteins (ZO-16 and MUC165), which maintain the barrier function of the corneal epithelium, were enhanced, and the reduction of p63 (an epithelial stem cell marker) was prevented by Ebselen in the hOEC sheet under hypothermic stress [
It was also reported that murine corneal epithelial progenitor cell line (TKE2) is more resistant to H2O2-induced oxidative stress with enhanced atrophy than cultured mature murine corneal epithelial cells (MCE). It was regulated by decreasing ROS production, reducing oxidative enzymes, such as NADPH oxidase 4 (NOX4), and increasing dual specificity phosphatase 6 (DUSP6). TKE2 also activated Nrf2 signaling and upregulated the expression of antioxidative enzymes (SOD and GSTP), indicating that Nrf2 is involved in maintaining the different hemostasis of corneal stem cells and exhibits strong antioxidant capacity against oxidative stress by regulating ROS generation and elimination [
FECD is a blinding disease and a primary reason of endogenous corneal endothelial degeneration. It is a gradually progressing disease with the accumulation of extracellular excrescences (guttae) [
Ultraviolet A (UV-A) with the wavelengths of 320 to 400 nm is the main source of solar radiation, which plays an important role in ROS production, and therefore may be related to the etiology of FECD. The cornea is radiated daily by solar ultraviolet (UV) rays, which can lead to DNA damage and induce oxidative stress [
Schematic diagram of Nrf2 signaling and regulation in the corneal endothelial cells. TBHP and H2O2, both high and low fluences of UVA, can induce excessive ROS generation in corneal endothelial cells, leading to apoptosis and cell death. SFN can inhibit tBHP-induced ROS generation, apoptosis, and cell death. Meantime, it can increase DJ-1 protein expression and Nrf2 translocation and induce Nrf2/ARE-dependent antioxidant enzyme (HO-1 and NQO1) transcriptions.
DJ-1, encoded by the PARK7 gene, is a multifunctional protein and is universally expressed in most human cells and tissues. Under the influence of oxidative stress, DJ-1 plays an important role in antioxidant defense by regulating several antioxidant gene expressions [
SFN is an Nrf2 level enhancer, which is found in green cruciferous vegetables like broccoli [
SLC4A11 is an anion transporter and a dimer located in the plasma membrane. It encodes a 100 kDa protein containing 14 domains [
Accumulating evidences denote that oxidative stress is one of the major mechanisms involved in the corneal diseases, which increases the injury in the corneal epithelial and endothelial cells via oxidation of proteins, DNA damage, apoptosis, cell death, and so forth. The Nrf2/Keap1/ARE signaling pathway is related to cell defense mechanisms against oxidative stresses. Therefore, the initiation of the Nrf2-ARE signaling pathway has been estimated as an important target for the design and synthesis of new agents for corneal diseases. Selected studies on the relationship between Nrf2 inducer/suppressor and corneal diseases reviewed in this article have shown notable effects on protecting or deteriorating corneal epithelial or endothelial cells against oxidative stress, decreasing the aberrant proteins and preventing or causing the corneal diseases (Table
Selected studies on the relationship between Nrf2 inducer/suppressor and corneal diseases.
Inducer | Suppressor | Type | Models | Results | Reference |
---|---|---|---|---|---|
4-HNE | NAC | In vitro | Cultured HCE cells | NAC antagonized the 4-HNE-induced oxidant effects in the cultured HCE cells by the reversed cell viability of HCE cells and reduced the 3-NT, NOX4, and Nrf2 protein expression induced by 4-HNE. | [ |
H2O2 | SA3K | In vitro | Cultured HCE cells | SA3K reversed H2O2-induced cell apoptosis. SA3K upregulated H2O2-induced downregulation of SOD2 and catalase gene expression. SA3K blocked H2O2-induced ROS and NOX4 overexpression and activated Keap1-Nrf2 pathway by suppressing Keap1-Nrf2-ARE pathway. | [ |
In vivo | Rat corneal epithelium | SA3K ameliorated H2O2-induced corneal epithelium death and decreased the H2O2-induced ROS, 3-NT, NOX4, and Nrf2 overexpression. | |||
H2O2 | In vitro | Cultured TKE2 and MCE | TKE2 cells have different homeostasis and strong antioxidant properties compared to MCE by decreasing ROS production and NOX4 and increasing DUSP6, Nrf2, SOD, and GSTP. | [ | |
Ebselen | In vitro | hOEC sheet, human corneal limbal tissue | Ebselen maintained the high ATP levels, normal morphology, viability, and function of the hOEC sheets by reducing ROS generation, inducing the Nrf2 activation, decreasing the lactate dehydrogenase (LDH) releasement, and increasing the glutathione (GSH)/oxidized glutathione (GSSG) ratio. Ebselen maintained the normal morphology of limbal epithelial layer and showed meaningfully higher colony-forming efficiency. | [ | |
Carnosol | In vivo | Diabetic rat corneal epithelium injury model | Carnosol accelerated the corneal epithelial wound healing. | [ | |
EP | In vitro | Cultured keratocytes and myofibroblasts | EP enhanced the Nrf2-mediated antioxidant response and induced phenotypic changes of quiescent corneal stromal keratocytes into contractile myofibroblasts. | [ | |
TSA | TGF- |
In vitro | Cultured corneal fibroblasts (HTK) | TSA inhibited of TGF- |
[ |
Lower fluences of UV-A | In vitro | CECs | Lower fluences of UV-A activated the antioxidant defense regulated by Nrf2 and higher fluences initiated p53 and caspase-3. UV-A may be related to the etiology of FECD. | [ | |
In vitro | FECD endothelium | Nrf2 is downregulated in FECD endothelium; higher levels of oxidative DNA damage and apoptosis of CE were also detected in FECD endothelium in contrast with normal controls. | [ | ||
tBHP | In vitro | HCECi and FECDi; FECD corneal buttons | Declined levels of DJ-1 in FECD at baseline and under the condition of oxidative stress were in relation with weakened Nrf2 nuclear translocation and improved cell susceptibility to apoptosis. | [ | |
UV-A | In vitro | CECs | Downregulation of DJ-1 resulted in decreased Nrf2 gene expression as well as its target genes HO-1 and NQO1, which inhibits translocation of Nrf2, leading to attenuate the expression of antioxidant gene and increase oxidative damage. The decrease of DJ-1 level results in enhanced CECs susceptibility to UV-A light via inducing p53-dependent apoptosis. | [ | |
SFN | In vitro | HCECi and FECDi | SFN increased the activation of Nrf2 in FECD specimens under the condition of oxidative stress. Pretreatment with SFN improved cell viability via diminishing the production of intracellular ROS in FECD. | [ | |
SLC4A11 mutations | In vitro | HEK 293 cells | Mutations in the |
[ |
4-HNE: 4-hydroxynonenal; 3-NT: 3-nitrotyrosine; NOX4: NADPH oxidase 4; HCE: human corneal epithelial cells; GSTP: glutathione S-transferase P; NQO1: NAD(P)H dehydrogenase (quinone 1); NAC: N-acetylcysteine; H2O2: hydrogen peroxide; SA3K: SERPINA3K; TKE2: murine corneal epithelial progenitor cell line; MCE: mature murine corneal epithelial cells; NOX4: NADPH oxidase 4; DUSP6: dual specificity phosphatase 6; SOD: superoxide dismutase; GSTP: glutathione S-transferase P; EP: ethyl pyruvate; TSA: trichostatin A; TGF-
Xiu-Fen Liu and Dan-Dan Zhou are co-first authors.
The authors declare that they have no conflicts of interest.
This study was funded by The First Hospital of Jilin University Grant (JDYY72016055) and Development and Reform Commission of Jilin Province (2015Y031-1). The authors thank Dr. Om Prakash Pant (ompant201@gmail.com) for the English language polishing.