The role of the KEAP1-NRF2 signaling pathway in form deprivation myopia guinea pigs
The etiology of myopia is complicated . Despite significant efforts by many researchers to elucidate the causes of myopia, a comprehensive understanding remains elusive. Previous studies found that oxidative stress (OS) responses and alterations in associated signaling pathways due to hypoxia may contribute to myopia, especially in high myopia . Additionally, recent research indicates that the scleral hypoxia caused by reduced choroidal blood flow perfusion, which influences scleral remodeling by up-regulating the expression of hypoxia-inducible factor-1α (HIF-1α), thereby promoting myopic progression . Due to hypoxia plays a crucial role in inducing OS, which suggests that OS might regulation the myopic progression. Furthermore, the excessive accumulation of HIF-1α leading to the inactivation of prolyl hydroxylase and subsequent stimulation of reactive oxygen species (ROS) released, result ROS and HIF-1α jointly induces OS . These results further suggest that OS was closely connection with the myopic progression, but the role of OS related signaling pathways in myopia remains poorly understood. Superoxide dismutase (SOD) activity was decreased in the retinas of FDM guinea pigs, and antioxidant levels were significantly reduced in the aqueous humor of myopic patients . These findings suggest that OS contributes to reduced antioxidant capacity might play a critical role in myopia development. The Kelch-like ECH-associated protein 1 (KEAP1) - nuclear factor erythroid 2-related factor 2 (NRF2) pathway is a critical regulator of OS, but few studies reports about its role in myopia progression. KEAP1 binds tightly to NRF2, maintaining stable expression in the cytoplasm . Upon the occurrence of OS, tyrosine kinase rapidly facilitates the separation of KEAP1 from NRF2 in the cytoplasm, resulting in KEAP1 degradation. Subsequently, NRF2 translocates into the nucleus and accumulates, where it regulates downstream antioxidant genes, thereby exhibiting an anti-OS function . When OS severity exceeds the antioxidant capacity, tissue damage ensues. Research has shown that reduced SOD activity in the retinas of form deprivation myopia (FDM) guinea pigs may lead to pathological changes such as retinal thinning and structural disorganization . As a downstream target gene activated by NRF2, SOD works to alleviate OS and reduce retinal damage . These findings suggest that activation NRF2 and regulation SOD could enhance retinal antioxidant capabilities and affect myopic progression. Therefore, activating the KEAP1-NRF2 signaling pathway is a vital strategy to regulate SOD and protect the retinas from OS-induced damageIt might become a new intervention method for the myopic progression. This study aims to conduct a preliminary investigation that provides novel insights into the pathogenesis of myopia. This study constructed the FDM guinea pig model to demonstrate the influence of KEAP1, NRF2, and SOD to myopic development. Specifically, alterations in KEAP1-NRF2 signaling in myopia provide indirect evidence that OS contributes to myopia’s development. Activating NRF2 to enhance SOD expression could potentially improve retinal antioxidant capacity and decelerate myopia progression.   Animal grouping This research received approval from the Animal Care and Ethics Committee at the North Sichuan Medical College ( NSMC2022036 ). It complied with the Association for Research in Vision and Ophthalmology statement for using animals in ophthalmic and vision research. Guinea pigs aged three weeks weighing 120 g ~ 150 g were selected for the study. After examining the diopter of all guinea pigs, those with congenital myopia were excluded. A total of 45 guinea pigs were retained and randomly assigned to different groups. The blank control group without any treatment was the negative control (NC, n = 15). The experimental group had a white translucent mask covering their right eyes to induce form deprivation myopia (FDM, n = 15), and their left eyes were without intervention as the self-control (SC, n = 15) . The intervention group was treated with tert-butylhydroquinone (TBHQ, HY-100489, USA) (TBHQ, n = 15), which was dissolved in a mixture of normal saline with 10% DMSO and delivered into the animal by intraperitoneal injection (10 mg/kg) for 48 h intervals. All guinea pigs were housed in a 12 h light/12 h dark room for four weeks, and the temperature was maintained at 23 ± 2℃, and the lighting was 500 lx . After four weeks, all animals were humanely euthanized using excessive isoflurane (RWD Life Science Co., R510-22-10, China) inhalation followed by cervical dislocation. Subsequently, the eyeballs were removed for further experiments. Measurement spherical equivalent & axial length The spherical equivalent (SE) and axial length (AL) were examined at five time points: before the covered mask and after being treated with different methods at one week, two weeks, three weeks, and four weeks. The Biological parameters were measured in a darkroom in the morning at 8:00 without the mask. The SE was used at least three times using a streak retinoscopy (66 Vision-Tech Co., China) by two experienced optometrists. The AL is defined as the distance from the anterior cornea center to the retina and was measured using an A-scan ultrasound image (Cinescan, France) by a proficient ophthalmic technician. The AL data were derived from the average of ten measurements for each eye. The mean values of SE and AL were calculated for analysis. Immunohistochemistry Eyeballs were taken from each group and fixed in a 4% paraformaldehyde solution. Then, it is dehydrated with gradient alcohol and embedded in paraffin. Then, the paraffin-embedded tissues were sectioned into 4 μm thick slices, which were subsequently deparaffinized in dimethyl benzene and rehydrated in gradient alcohol. Antigen retrieval was performed using EDTA (pH 8.0, Biosharp, China) for 15 min under heat induction. Sections were incubated in a 3% hydrogen peroxide-methanol solution at 37 °C for 15 min and rinsed thrice with PBS. Blocking was carried out with 3% goat serum for 30 min, followed by overnight incubation at 4 °C with primary antibodies (anti-KEAP1, 1:100, Proteintech, China; anti-NRF2, 1:100, Huabio, China; anti-SOD1, 1:50, Omnimabs, Canada). After incubation, sections were washed three times with PBS and incubated with secondary antibodies (Boster, China) at room temperature for 1 h. Protein immune activity was detected using DAB chromogen (ZSGB-BIO, China). Sections were stained with hematoxylin, differentiated with hydrochloric alcohol, and dehydrated in gradient alcohol. Finally, the sections were mounted using neutral resin and examined for protein distribution under a microscope (Leica, France). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Retinal samples were pre-prepared for RNA extraction using Trizol. The extraction followed the detailed steps outlined in the provided specifications kit (Takara, Japan). The extracted RNA was reverse transcribed into cDNA and then mixed with SYBR Green II (Takara, Japan) in the wells for amplification using the instrument (Light Cycler480, USA) for Real-time PCR. The amplification protocol was as follows: initial denaturation at 95 °C for 30s, followed by 40 cycles of denaturation at 95 °C for 5s, annealing at 60 °C for 30s, and extension at 97 °C for 1s. The relative mRNA levels of KEAP1, NRF2, and SOD were normalized to the internal control gene GAPDH and calculated using the 2 −ΔΔCT method. The sequences used in this study are shown in Table . Western blot Retinas were isolated from eyeballs and lysed in RIPA buffer (Beyotime Biotechnology, China) containing 1% PMSF (Beyotime Biotechnology, China). Protein extracts were obtained from the retinal supernatant, and their concentrations were determined using a BCA kit (Beyotime Biotechnology, China). The proteins were then mixed with 5× protein loading buffer (Solarbio, China), heated at 90℃ for 10 min, and subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred the proteins to nitrocellulose membranes, which were then cut based on the locations of the target proteins. The NRF2 is located at 100kd, the KEAP1 is located at 70kd, the SOD1 is located at 26kd, and the GAPDH is located at 36kd. Next, the membranes were blocked with 5% nonfat milk at room temperature for 1 h and hybridization overnight at 4℃ with primary antibodies (anti-KEAP1, 1: 2000, Proteintech, China; anti-NRF2, 1: 1000, Huabio, China; anti-SOD1,1: 1000, WanleiBio, China; and anti-GAPDH, 1: 5000, Huabio, China). After incubation with mouse anti-rabbit secondary antibody (1: 10000; Boster, China) and incubated for one hour on a shaker, the membranes were developed using ECL developer solution (Biosharp, China) and imaged via a chemiluminescence system (Vilber Lourmat, France). Densitometry was quantified using Image J software. Statistical analyses All data are presented as mean ± standard deviation (SD) and were analyzed using SPSS software, version 28.0. An unpaired Student’s t-test was used to assess the significance between the two groups, while one-way ANOVA was used to analyze multiple groups. Statistical significance was established at p < 0.05. Graphical representations were created with Prism software, version 9.0 (GraphPad, San Diego, CA, USA).   This research received approval from the Animal Care and Ethics Committee at the North Sichuan Medical College ( NSMC2022036 ). It complied with the Association for Research in Vision and Ophthalmology statement for using animals in ophthalmic and vision research. Guinea pigs aged three weeks weighing 120 g ~ 150 g were selected for the study. After examining the diopter of all guinea pigs, those with congenital myopia were excluded. A total of 45 guinea pigs were retained and randomly assigned to different groups. The blank control group without any treatment was the negative control (NC, n = 15). The experimental group had a white translucent mask covering their right eyes to induce form deprivation myopia (FDM, n = 15), and their left eyes were without intervention as the self-control (SC, n = 15) . The intervention group was treated with tert-butylhydroquinone (TBHQ, HY-100489, USA) (TBHQ, n = 15), which was dissolved in a mixture of normal saline with 10% DMSO and delivered into the animal by intraperitoneal injection (10 mg/kg) for 48 h intervals. All guinea pigs were housed in a 12 h light/12 h dark room for four weeks, and the temperature was maintained at 23 ± 2℃, and the lighting was 500 lx . After four weeks, all animals were humanely euthanized using excessive isoflurane (RWD Life Science Co., R510-22-10, China) inhalation followed by cervical dislocation. Subsequently, the eyeballs were removed for further experiments.   The spherical equivalent (SE) and axial length (AL) were examined at five time points: before the covered mask and after being treated with different methods at one week, two weeks, three weeks, and four weeks. The Biological parameters were measured in a darkroom in the morning at 8:00 without the mask. The SE was used at least three times using a streak retinoscopy (66 Vision-Tech Co., China) by two experienced optometrists. The AL is defined as the distance from the anterior cornea center to the retina and was measured using an A-scan ultrasound image (Cinescan, France) by a proficient ophthalmic technician. The AL data were derived from the average of ten measurements for each eye. The mean values of SE and AL were calculated for analysis.   Eyeballs were taken from each group and fixed in a 4% paraformaldehyde solution. Then, it is dehydrated with gradient alcohol and embedded in paraffin. Then, the paraffin-embedded tissues were sectioned into 4 μm thick slices, which were subsequently deparaffinized in dimethyl benzene and rehydrated in gradient alcohol. Antigen retrieval was performed using EDTA (pH 8.0, Biosharp, China) for 15 min under heat induction. Sections were incubated in a 3% hydrogen peroxide-methanol solution at 37 °C for 15 min and rinsed thrice with PBS. Blocking was carried out with 3% goat serum for 30 min, followed by overnight incubation at 4 °C with primary antibodies (anti-KEAP1, 1:100, Proteintech, China; anti-NRF2, 1:100, Huabio, China; anti-SOD1, 1:50, Omnimabs, Canada). After incubation, sections were washed three times with PBS and incubated with secondary antibodies (Boster, China) at room temperature for 1 h. Protein immune activity was detected using DAB chromogen (ZSGB-BIO, China). Sections were stained with hematoxylin, differentiated with hydrochloric alcohol, and dehydrated in gradient alcohol. Finally, the sections were mounted using neutral resin and examined for protein distribution under a microscope (Leica, France).   Retinal samples were pre-prepared for RNA extraction using Trizol. The extraction followed the detailed steps outlined in the provided specifications kit (Takara, Japan). The extracted RNA was reverse transcribed into cDNA and then mixed with SYBR Green II (Takara, Japan) in the wells for amplification using the instrument (Light Cycler480, USA) for Real-time PCR. The amplification protocol was as follows: initial denaturation at 95 °C for 30s, followed by 40 cycles of denaturation at 95 °C for 5s, annealing at 60 °C for 30s, and extension at 97 °C for 1s. The relative mRNA levels of KEAP1, NRF2, and SOD were normalized to the internal control gene GAPDH and calculated using the 2 −ΔΔCT method. The sequences used in this study are shown in Table .   Retinas were isolated from eyeballs and lysed in RIPA buffer (Beyotime Biotechnology, China) containing 1% PMSF (Beyotime Biotechnology, China). Protein extracts were obtained from the retinal supernatant, and their concentrations were determined using a BCA kit (Beyotime Biotechnology, China). The proteins were then mixed with 5× protein loading buffer (Solarbio, China), heated at 90℃ for 10 min, and subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred the proteins to nitrocellulose membranes, which were then cut based on the locations of the target proteins. The NRF2 is located at 100kd, the KEAP1 is located at 70kd, the SOD1 is located at 26kd, and the GAPDH is located at 36kd. Next, the membranes were blocked with 5% nonfat milk at room temperature for 1 h and hybridization overnight at 4℃ with primary antibodies (anti-KEAP1, 1: 2000, Proteintech, China; anti-NRF2, 1: 1000, Huabio, China; anti-SOD1,1: 1000, WanleiBio, China; and anti-GAPDH, 1: 5000, Huabio, China). After incubation with mouse anti-rabbit secondary antibody (1: 10000; Boster, China) and incubated for one hour on a shaker, the membranes were developed using ECL developer solution (Biosharp, China) and imaged via a chemiluminescence system (Vilber Lourmat, France). Densitometry was quantified using Image J software.   All data are presented as mean ± standard deviation (SD) and were analyzed using SPSS software, version 28.0. An unpaired Student’s t-test was used to assess the significance between the two groups, while one-way ANOVA was used to analyze multiple groups. Statistical significance was established at p < 0.05. Graphical representations were created with Prism software, version 9.0 (GraphPad, San Diego, CA, USA).   Changes in SE and AL of guinea pigs in each group The baseline SE among all groups was moderate hyperopia with no statistically significant differences. By the second week, the SE differences among the groups had become statistically significant. After four weeks of treatment, the SE in both the NC and SC groups continued to show hyperopia, whereas it shifted to myopia in the FDM and TBHQ groups. However, the myopia degree in the TBHQ group was lower than in the FDM group. Initially, there were no significant differences in AL among the groups. By the second week, significant differences in AL emerged. Following treatment, the AL in both the FDM and TBHQ groups was longer compared to the NC and SC groups, with the TBHQ group exhibiting a shorter AL than the FDM group (Table ; Fig. ). Protein localization and expression in each group of guinea pigs After four weeks of FD, this study investigated the distribution of KEAP1, NRF2, and SOD1 to determine which part of the retina were affected by those genes. These proteins are predominantly localized in the retinal ganglion cells (RGC) (Fig. ). The mRNA expression in the retinas of guinea pigs The results indicated that KEAP1 mRNA expression was elevated in both the NC and SC groups but significantly decreased in the FDM group. Conversely, NRF2 expression was significantly higher in the FDM group compared to the lower levels observed in the NC and SC groups. Similarly, SOD expression decreased in the FDM group (Fig. A). This study investigated the impact of activators on mRNA by comparing the NC, FDM, and TBHQ groups. Progressive decrease in KEAP1 mRNA expression was noted across the NC, FDM, and TBHQ groups. In contrast, NRF2 expression gradually increased among these groups. SOD expression in the TBHQ group was lower than in the NC group but higher than in the FDM group (Fig. B). The protein expression in the retinas of guinea pigs After comparing the mRNA expression levels of KEAP1, NRF2, and SOD1, the protein levels of these genes were subsequently analyzed. The results revealed that the expression of KEAP1 and SOD1 was significantly elevated in both the NC and SC groups compared to the FDM group. Conversely, NRF2 expression was significantly higher in the FDM group than in the NC and SC groups (Fig. A-B). This study assessed the differential effects among the NC, FDM, and TBHQ groups to elucidate the activator’s impact on protein expression. KEAP1 expression was higher in the NC group than in the FDM and TBHQ groups. In contrast, NRF2 expression was lower in the NC group but was upregulated in the FDM group and significantly enhanced following TBHQ treatment. SOD1 showed higher expression in the NC group, which decreased in the FDM group and increased TBHQ treatment (Fig. C-D).   The baseline SE among all groups was moderate hyperopia with no statistically significant differences. By the second week, the SE differences among the groups had become statistically significant. After four weeks of treatment, the SE in both the NC and SC groups continued to show hyperopia, whereas it shifted to myopia in the FDM and TBHQ groups. However, the myopia degree in the TBHQ group was lower than in the FDM group. Initially, there were no significant differences in AL among the groups. By the second week, significant differences in AL emerged. Following treatment, the AL in both the FDM and TBHQ groups was longer compared to the NC and SC groups, with the TBHQ group exhibiting a shorter AL than the FDM group (Table ; Fig. ).   After four weeks of FD, this study investigated the distribution of KEAP1, NRF2, and SOD1 to determine which part of the retina were affected by those genes. These proteins are predominantly localized in the retinal ganglion cells (RGC) (Fig. ).   The results indicated that KEAP1 mRNA expression was elevated in both the NC and SC groups but significantly decreased in the FDM group. Conversely, NRF2 expression was significantly higher in the FDM group compared to the lower levels observed in the NC and SC groups. Similarly, SOD expression decreased in the FDM group (Fig. A). This study investigated the impact of activators on mRNA by comparing the NC, FDM, and TBHQ groups. Progressive decrease in KEAP1 mRNA expression was noted across the NC, FDM, and TBHQ groups. In contrast, NRF2 expression gradually increased among these groups. SOD expression in the TBHQ group was lower than in the NC group but higher than in the FDM group (Fig. B).   After comparing the mRNA expression levels of KEAP1, NRF2, and SOD1, the protein levels of these genes were subsequently analyzed. The results revealed that the expression of KEAP1 and SOD1 was significantly elevated in both the NC and SC groups compared to the FDM group. Conversely, NRF2 expression was significantly higher in the FDM group than in the NC and SC groups (Fig. A-B). This study assessed the differential effects among the NC, FDM, and TBHQ groups to elucidate the activator’s impact on protein expression. KEAP1 expression was higher in the NC group than in the FDM and TBHQ groups. In contrast, NRF2 expression was lower in the NC group but was upregulated in the FDM group and significantly enhanced following TBHQ treatment. SOD1 showed higher expression in the NC group, which decreased in the FDM group and increased TBHQ treatment (Fig. C-D).   Recent studies have demonstrated that hypoxia-induced upregulation of HIF-1α enhances scleral agonist protein expression and promotes differentiation of scleral fibroblasts, ultimately contributing to myopia formation. Additionally, hypoxia increases matrix metalloproteinase levels by stimulating the secretion of scleral inflammatory factors, leading to significant degradation of collagen fibers, decreased scleral rigidity, and accelerated myopic progression . These findings underscore hypoxia as a pivotal factor in myopic development. Hypoxia results from an imbalance in oxygen metabolism; disruption in the balance between oxygen supply and consumption could induce OS by increasing the release of ROS and free radicals and by regulating HIF-1α transcription through the inhibition of prolyl hydroxylase, thus promoting excessive accumulation of HIF-1α and subsequent damage . Given that both HIF-1α and ROS are influenced by OS, these findings suggest that OS contributes to myopia. A recent study has found significant increases in ROS in the retinas of FDM animals , further confirming that OS was a important reason of myopia. Therefore, understanding these pathways in myopia could reveal additional targets for future research aimed at preventing or delaying myopia, thereby enhancing the effectiveness of myopia prevention and control strategies. As a crucial signaling pathway regulating OS, the KEAP1-NRF2 pathway could enhances the antioxidant capacity of the retina. It mitigates damage caused by OS, primarily through increasing SOD activity and the expression of other antioxidative genes . SOD was a crucial antioxidant, eliminates excess ROS and free radicals, thereby reducing apoptosis . Additionally, decreased SOD activity has been implicated in the development of myopia . Previous studies have identified the primary electrophilic structures of SOD1 as copper and zinc ions, while SOD2 involves manganese ion. They coexist in the retinas and regulate the OS. But deficiency in SOD1 was a major factor that induces the retinal antioxidant capacity decreased and leads to structural damage . Thus, this study examined the distribution of KEAP1, NRF2, and SOD1, the IHC revealing they are predominantly located in the retinal ganglion cell (RGC) layer of guinea pig retinas. RGCs as vital photoreceptors of the eyes, regulation through regulate ON, OFF signaling pathway influence myopic progression . Other studies found that the ipRGC participated in myopia development by regulating melanopsin . Indicating that different types of RGCs could affect the myopic progression. Moreover, RGCs have high oxygen consumption, are rich in polyunsaturated fatty acids and have a high mitochondrial density, making them extremely sensitive to changes in oxygen metabolism. Dysregulation in oxygen supply predisposes to lipid peroxidation, causing OS response and resulting retinal damage . These findings suggest that antioxidant genes in RGCs play a crucial role in myopia regulation. This study analyzed the changes of KEAP1 and NRF2 in the retinas of myopic animals, observing decreased KEAP1 expression and increased NRF2 expression in the FDM group. Due to the small sample size, this study doesn’t direct detection of ROS content changes, so only provides only indirect evidence of OS changes. Because under normal conditions, KEAP1 expression is higher and NRF2 expression is lower, it is even degraded in the cytoplasm. OS accelerated KEAP1-NRF2 dissociation and induced KEAP1 degradation in the cytoplasm, followed by NRF2 expression was increased and translocation to the nucleus, where it activates antioxidant genes to combat OS . Therefore, this study conducted a preliminary exploration of its role in myopia, and the results indirectly showed that OS might be one of the mechanisms regulating myopia development and that KEAP1-NRF2 participated in the progression of myopia. While previous studies confirmed that increased ROS levels due to factors such as hypoxia could accelerate KEAP1-NRF2 dissociation and enhance NRF2’s regulation of downstream elements like SOD, HO-1, TGF, and HIF-1α, which is involved in myopia progression , there were few studies revealed that KEAP1-NRF2 signaling pathways in myopia. Therefore, further studies elucidating the role of KEAP1-NRF2 signaling in myopia and its interactions with other known pathways are essential for a comprehensive understanding. Another study has found that the retinal SOD activity was decreased, combined with damaged retinal structure in myopic animals . As downstream of keap1-nrf2, OS induced by hypoxia leads to SOD1 deficiency and attenuates the retinal antioxidant capacity; this results in irreversible oxidative modifications to retinal proteins and lipids, subsequently impairing visual function by disrupting retinal structures, a change particularly notable in high myopia . The result showed that the SOD1 decreased in the retinal of the FDM group, further suggesting that the decreased retinal antioxidant capacity is related to myopia development. Still, their dynamic changes during form-deprivation myopia needed depth observation. TBHQ is an exogenous antioxidant compound that specifically activates NRF2 and promotes the expression of its downstream antioxidant gene SOD, thereby enhancing tissue antioxidant capacity . Treatment with TBHQ resulted in more significant changes in SE and AL compared to the NC group, but less so than in the FDM group. Molecular experiments demonstrated that TBHQ upregulated retinal NRF2 in FDM guinea pigs and mitigated myopia progression by increasing SOD1 expression. These results confirm that activating NRF2 and enhancing SOD1 expression can bolster retinal antioxidant capability and decelerate experimental myopic progression. Nonetheless, other second-phase antioxidant genes, such as HO-1 and NQO-1, are also regulated by the KEAP1-NRF2 signaling pathway, indicating that the antioxidant properties of SOD1 not unique . Moreover, earlier studies indicate that inflammatory mediators like NF-κB, TNF-α, and IL-6, which are regulated via the KEAP1-NRF2 pathway, contribute to myopia progression . This evidence suggests that the KEAP1-NRF2 mechanism is complex, and additional mechanisms still require exploration. Future research should isolate tissues from myopia models for proteomic analysis and further investigating potential interactions among several key pathways through protein interaction analysis and the Kyoto Encyclopedia of Genes and Genomes. In conclusion, this study is the first to identify changes in KEAP1-NRF2 in the FDM. Activation of KEAP1-NRF2 could promote downstream SOD1 expression and enhance retinal antioxidant capacity, offering a novel method to inhibit the progression of myopia.   This study examined the distribution and expression of KEAP1, NRF2, and SOD1 in the retina. The results showed that they were distribute in RGC. The levels of KEAP1 and SOD1 were reduced in the FDM group compared to the NC and SC groups, while NRF2 expression was increased. Treatment with TBHQ upregulated NRF2, which subsequently elevated SOD1 expression and mitigated myopia progression. Overall, this study demonstrated the involvement of the KEAP1-NRF2 pathway in form-deprivation myopia progression, suggesting that the underlying mechanisms warrant further investigation.   Supplementary Material 1.   