Human corneal epithelial cells (HCECs), a human SV40 immortalized corneal epithelial cell line (CRL-11135, HCE-2; ATCC, Manassas, VA, United States), between passages 15 and 20, were cultured as previously described. The immortalized HCECs were then treated with different osmolarities, ranging from 312 to 550 mOsM, which was achieved by adding 0, 70, 90, or 120 mM sodium chloride (NaCl) (Sigma-Aldrich, St. Louis, MO, United States). Lipid ROS scavenger Fer-1 (MCE, Shanghai) and iron chelator DFO (MCE, Shanghai) were used as cell death inhibitors 4 h prior to NaCl exposure. Dimethyl sulfoxide (DMSO; Wako) was used as the vehicle and the control for AST and Fer-1 treatments. DFO and Fer-1, regarded as known ferroptosis inhibitors, were used to further confirm the presence of ferroptosis and as a reference to further illustrate the anti-ferroptosis effect of astaxanthin.

In this study, 6–8-week-old female BALB/c mice (Pengyue Laboratory, Jinan, China) were used for this study. All animals were maintained according to the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research. The mice were randomly divided into four groups as follows (n = 5): 1) control, 2) DE, 3) DMSO + DE, and 4) AST + DE. DE indicates the dry eye group. Astaxanthin was dissolved in DMSO to obtain a mixture of 10 μM. The mice in groups 2, 3, and 4 were subcutaneously injected with 0.5 mL of scopolamine hydrobromide (2.5 mg/mL; MedChemExpress) three times a day for 14 days to induce ocular surface injury. Among them, groups 3 and 4 received 1 μL AST and DMSO, respectively, four times a day in both eyes. The eyes were immediately removed from each animal, some of which were processed with paraformaldehyde and stored at 4°C for tissue embedding and sectioning. Cornea tissues were stored at −80°C, and homogenates were processed with formaldehyde and glutaraldehyde for protein and histological analysis. All mice were euthanized through quick cervical dislocation. The experiments were performed at least three times to verify the reproducibility of the result.

Cells were seeded and grown in 96-well plates for various treatments. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; DOJINDO, Kumamoto, Japan) assay. Then, 10 μL of the CCK-8 solution was added to the 100-μL fresh medium. Absorbance was measured at 450 nm after 2 h of incubation in the cell incubator.

Corneas and cells were lysed with RIPA lysis buffer (Solarbio, Beijing, China). Protein concentrations were measured by BCA assay and mixed with SDS sample buffer (1:1) and boiled for 10 min at 95°C. The protein samples were separated by 10%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skimmed milk at room temperature for 2 h and then incubated with a primary antibody diluted by antibody dilution buffer (Beyotime, Shanghai, China) at 4°C overnight. Primary antibodies included the antibody to GPX4 (Abmart, Shanghai, China), ferritin (Abmart, Shanghai, China), SLC7A11 (Abmart, Shanghai, China), LC3 (CST, United States), P62 (CST, United States), and GAPDH (CST, United States). The following day, the membranes were washed with 1 × Tris-buffered saline and Tween 20 (TBST) three times and incubated with secondary antibodies at room temperature for 1 h. Protein expression levels were tested with chemiluminescence assay (Thermo Fisher Scientific, Waltham, MA, United States). The ratio of the gray value of the target band to GAPDH was representative of the relative protein expression.

Total RNA was extracted from HCECs using the TRIzol reagent (Vazyme Biotech Co., Ltd., Nanjing, China), according to the manufacturer’s instructions. The RNA was reverse-transcribed to cDNA using a HiScript^® III Reverse Transcription SuperMix for qPCR (Vazyme, Nanjing, China), and then, cDNA was used for PCR using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The relative expression of RNA was normalized to the endogenous control GAPDH using the 2^−ΔΔCT method (Pan et al., 2023). The primers used in this study are as follows:

Mouse eye sections were fixed with 4% paraformaldehyde in PBS for 15 min, then permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 10% goat serum for 1 h. The samples were stained with a rabbit monoclonal anti-GPX4 antibody (1:250; Abmart) in antibody dilution buffer (Beyotime, Shanghai, China) at 4°C overnight. Secondary staining was performed with Alexa Fluor^TM 488 goat anti-rabbit IgG (1:200; Invitrogen, United States) and Alexa Fluor^TM 555 goat anti-rabbit IgG (1:200; Invitrogen, United States) for 1 h at room temperature, and the nuclei were counterstained with DAPI for 7 min.

Intracellular Fe²⁺ was assessed by treatment with 5 μM FeRhoNox-1 (Goryo Chemical, Inc., Sapporo, Japan) in Hank’s balanced salt solution (HBSS; Thermo Fisher Scientific). After washing, the cells were observed under a fluorescence microscope (BZ-9000; Keyence). At least four fields per chamber were imaged, and the fluorescence intensities were analyzed using ImageJ (https://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, United States).

HCECs seeded into six-well plates were used. Cells digested by trypsin were collected and washed twice with PBS, and a reagent was added (GSSG/GSH quantification kit). First, the cells were resuspended and then lysed by repeated freezing and thawing 2–3 times. Then, the cells were centrifuged, and the supernatant was collected at 4°C for measurement. The total protein concentration was detected using the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Shanghai, China), and the GSH concentration was analyzed using the GSSG/GSH quantification kit (GSH assay; Solarbio). The corrected GSH content of the samples is the ratio of the supernatant concentration to protein concentration obtained using a 96-well plate according to the manufacturer’s protocol, and a microplate reader was used to measure absorbance at 412 nm.

HCECs were seeded into 96-well plates and pre-treated with 10 μM Fer-1, 100 μM DFO, or 10 μM AST for 4 h and then with 120 mM NaCl for 24 h. Intracellular ROS was determined using a Reactive Oxygen Species Assay Kit (Solarbio, CA1410) through the DCFH-DA ROS probe, according to the manufacturer’s instructions. After washing, adherent cells were observed under a fluorescence microscope. The collected suspended cells were detected using the FL1 channel of flow cytometry and then analyzed using FlowJo v10.6.2.

The MDA amount often reflects the degree of lipid peroxidation in the body, which indirectly indicates the degree of cell damage. The brain tissues or cells were mixed using normal saline in a ratio of 1:9 and placed on ice. After homogenization, the brain tissues were centrifuged at 12,000 rpm at 4°C for a quarter. The MDA content was detected following the manufacturer’s protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) (Du et al., 2022; Yu et al., 2022). The absorbance at 532 nm was assessed using a microplate reader (Thermo Fisher Scientific, United States).

HCECs were cultured and stimulated according to experimental needs. At a confluent density of 90%, HCECs were harvested and fixed with 2.5% glutaraldehyde (Servicebio, China) for 30 min at room temperature and stored at 4°C. The cell mass after centrifugation was then rinsed three times with 0. 1M PB (pH 7.4), and the fixed HCECs were post-fixed using 2% osmium tetroxide for 2 h at room temperature. After washing with PB three times, the cells were extracted and suspended in 1% agarose solution to be solidified for pre-embedding. After washing another time, the cells were dehydrated in a graded series of alcohol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) before being embedded in EMBed 812 (SPI, United States), and ultrathin sections (60–80 nm) were prepared using a Leica ultramicrotome (Leica Microsystems, United States) and fished out onto the 150-mesh cuprum grids with a formvar film. The prepared sections were then subjected to double staining with uranyl acetate and lead citrate. The cuprum grids were observed under a transmission electron microscope (TEM; HT7800, Hitachi, Japan), and the images were obtained.

Data are presented as the mean ± standard error of the mean (SEM). All statistical analyses were performed using the GraphPad Prism 7.0 program (GraphPad Software Inc., San Diego, CA). Values with p < 0.05 were considered statistically significant. The quantitative experiments were all repeated three times.

Imaging of Fe²⁺ using FeRhoNox-1 showed that hyperosmolarity significantly enhanced intracellular Fe²⁺ levels in HCECs at 24-h exposure. The increase in Fe²⁺ levels in HCECs caused by hyperosmolarity is concentration-dependent (Figures 1A, B). qRT-PCR and Western blotting were used to analyze the expression of ferroptosis-related genes and proteins, respectively. Immunoblotting analysis indicated that 120 mM NaCl elicited a significant decrease in protein levels of ferritin (Figures 1C, D). Transferrin (TF) traps ferric iron (Fe³⁺) and binds to protein TF receptor 1 (TFR1) on the cell membrane to endocytose Fe³⁺. Fe³⁺ is then reduced to Fe²⁺ by a six-transmembrane epithelial antigen of the prostate 3 (STEAP3), and divalent metal transporter 1 (DMT1) delivers Fe²⁺ from endosomes to the cytoplasm. Partial ferric iron is stored in the ferritin, which consists of ferritin light chain (FTL) and ferritin heavy chain 1 (FTH1). Ferritinophagy modulated by a cargo protein called nuclear receptor coactivator 4 (NOCA4) causes ferritin degradation and thereby releases Fe²⁺. Ferroportin (FPN) acts synergistically with ferroxidase ceruloplasmin (CP) or hephaestin (HEPH) to export excessive Fe²⁺ out of the cells. The mRNA levels of FPN, TFRC, FTL, STEAP3, and CP were significantly upregulated by 120 mM NaCl for 24 h. NCOA4 mRNA levels were upregulated by 90 or 120 mM NaCl. Hyperosmolarity downregulated the mRNA levels of FTH, HEPH, and DMT1 (Figure 1E).

The data from the CCK-8 assay showed that 120 mM NaCl for 24 h caused lower cell viability (Figure 2A). Intracellular GSH levels showed a decreasing trend when exposed to NaCl at a concentration of 70–120 mM and indicated statistical significance at 120 mM (Figure 2B). Hyperosmolarity significantly increased lipid peroxide MDA levels in HCECs in a concentration-dependent manner (Figure 2C). Immunoblot analysis showed that the relative protein level of SLC7A11 was not changed. In contrast, the protein expression of GPX4 in HCECs was significantly downregulated by treating with 120 mM NaCl for 24 h (Figures 2D, E). Intracellular ROS generation visualized using the fluorescent probe H2DCFDA by fluorescence microscopy showed that total ROS levels were increased at 90 mM NaCl and substantially elevated at 120 mM NaCl (Figure 2F). Fluorescence quantization and flow cytometry yielded the same results (Figures 2G, H).

The results of the CCK-8 assay revealed that at a concentration of 1 μM–100 μM, the three chemicals begin to cause a decrease in cell viability to preliminarily determine the intervention concentration (Figure 3A). The treatment with AST at concentrations of 50 μM and 100 μM significantly decreased the cell viability in a concentration-dependent manner. The corneal epithelial cell viability remarkably reduced by Fer-1 at concentrations of 25 μM, 50 μM, and 100 μM. The intervention with DFO at 100 μM maintains constant cell viability (Figure 3A). The cells were pretreated with the three reagents for 6 h before incubation with NaCl. The viability of HCECs, following 24-h exposure to 120 mM NaCl, was significantly increased from 50.51% to 83.26% by 5 μM and from 50.51% to 96.73% by 10 μM Fer-1 (Figure 3B). So, the intervention concentration of Fer-1 to inhibit ferroptosis was 10 μM. Fe²⁺ production caused by 120 mM NaCl was significantly attenuated by 10 μM Fer-1 in HCECs (Figures 3C, D). Moreover, it also inhibited intracellular ROS fluorescence intensity detected by a microscope and flow cytometry (Figures 3E–G). Similarly, the MDA levels indicated that the accumulation of lipid peroxidation in HCECs caused by treatment with either 90 mM or 120 mM NaCl for 24 h was significantly cleared by 10 μM Fer-1 (Figure 3F). These findings imply that Fer-1 attenuates ferroptosis induced by hyperosmolarity in HCECs.

The viability of HCECs after 24 h of incubation with 120 mM NaCl was still increased from 48.93% to 70.84% by 100 μM DFO (Figure 4A). The MDA levels indicated that the accumulation of lipid peroxidation in HCECs caused by treatment with either 90 mM or 120 mM NaCl for 24 h was significantly reduced by 100 μM DFO (Figure 4B). Fluorescence microscopy with FeRhoNox-1 staining revealed that 100 μM DFO significantly decreased the levels of Fe²⁺ induced by 120 mM NaCl (Figures 4C, D). As expected, 100 μM DFO significantly inhibited the production of ROS in HCECs after 24 h of exposure to 120 mM NaCl (Figure 4E). Moreover, we also used flow cytometry to quantify ROS in cells exposed to 120 mM NaCl and found that it was significantly reduced by 100 μM DFO (Figures 4F, G).

The viability of HCECs with 120 mM NaCl for 24 h was improved from 48.93% to 87.24% by 10 μM AST and from 48.93% to 70.54% by 25 μM AST (Figure 5A). AST also remarkably increased the GSH level in hyperosmolarity-induced HCECs (Figure 5B). Western blot analysis also demonstrated that 10 μM AST increased protein levels of ferritin and GPX4 in HCECs and has no statistic influence on SLC7A11 (Figures 5C, D). The MDA levels in HCECs caused by treatment with 90 mM for 24 h were slightly but not significantly reduced by 10 μM AST (Figure 5E). However, 10 μM AST can remarkably relieve MDA caused by 120 mM NaCl (Figure 5E). Moreover, 10 μM AST significantly inhibited the production of ROS in HCECs after exposure to 120 mM NaCl (Figure 6A). Fluorescence quantization and flow cytometry results showed the same (Figures 6B, C). Confocal microscopy with FeRhoNox-1 staining showed that 10 μM AST significantly decreased the levels of Fe²⁺ induced by 120 mM NaCl in HCECs (Figures 6D, E). These results imply that AST may inhibit ferroptosis by upregulating GPX4 to protect hypertonic corneal epithelial cells.

We further investigated the alterations in mitochondria. Transmission electron microscopy demonstrated that mitochondria presented considerable shrinkage with a ruptured outer mitochondrial membrane and reduced or disappeared mitochondrial cristae, similar to morphological characteristics of ferroptosis, in hyperosmolarity-induced HCECs. However, Fer-1 and AST can restrain the detrimental damage to mitochondrial morphology (Figure 7).

Our previous literature reported that autophagy was not activated in HCECs treated with 120 mM NaCl (Pan et al., 2023). In the present study, Western blot results showed that the protein of LC3B increased and P62 reduced in hyperosmolarity-exposed HCECs pretreated with DFO, Fer-1, and AST compared with hyperosmolarity-exposed HCECs pretreated with DMSO (Figures 8A–E). The LC3 molecule is attached to autophagosomes, so its increase may be due to an increase in autophagosomes or a lack of degradation of autolysosomes. In order to know which one it is, HCECs were further infected with mRFP-GFP-LC3 to dynamically detect the state of autophagic flux. Under normal conditions, the autophagosome is labeled with yellow signals (mRFP and GFP), and after autophagosome–lysosome fusion, GFP is rapidly quenched by the low pH of lysosomes, and the autophagosome only appears as a red dot. In hyperosmolarity-induced HCECs pretreated with DMSO, we observed significant yellow fluorescence and no red dot, indicating that autophagic flux was inhibited. However, AST-protected cells showed yellow punctum reduction and red fluorescence appearance in the cytoplasm, revealing that AST recovered autophagic flux (Figure 8F). Similarly, TEM showed that the number of autophagosomes increased in HCECs in the AST + DE group compared with the DMSO + DE group (Figure 8G). Collectively, these results suggested that the inhibition of ferroptosis promotes autophagy in hyperosmolarity-exposed HCECs.

Previous studies have reported that ferroptosis occurred in the corneal epithelial cells in dry eye disease mice induced by scopolamine hydrobromide (Zuo et al., 2022). We established a dry eye disease mouse model to explore the effect of AST on ferroptosis in vivo. The corneal fluorescein staining score in the DE group was significantly higher than that in the control group, and AST rescued ocular surface defects (Figures 9A, B). The GSH expression of corneal tissue in the DE group was significantly lower than that in the control group. As expected, GSH levels in the AST-treated group improved (Figure 9C). Furthermore, it is found that the protein expression of SLC7A11 and GPX4 was downregulated in the DE and DMSO pretreated mice, and AST effectively increased both molecules, as demonstrated by Western blot and immunofluorescence staining (Figures 9D, E; Figure 10). MDA, considered a byproduct of lipid peroxidation (Yan et al., 2021), was used to indicate the content of lipid peroxidation in vivo. The MDA level was remarkably higher in the DE mice than that in the control mice, and AST decreased the MDA expression significantly compared with the DMSO + DE group (Figure 9F).