Autophagy Impairment-Derived SQSTM1 Accumulation Promotes Ferroptosis in Corneal Epithelial Cells Through ACSL4 in Dry Eye

Abstract

Purpose

To reveal the function of autophagy impairment-derived sequestosome 1 (SQSTM1) in inducing ferroptosis in an experimental dry eye model and investigate the underlying mechanism.

Methods

To induce the dry eye animal model, 8-week-old C57BL/6 mice were subcutaneously injected with scopolamine and exposed to a desiccated environment. To build the in vitro dry eye model, human corneal epithelial cells (HCECs) were applied with desiccating stress. Cell viability was examined using a CCK-8 kit. Intracellular reactive oxygen species (ROS), oxidative lipid, and Fe²⁺ were detected using the H2DCFDA assay kit, C11 BODIPY probe, and FerroOrange probe. Gene expression was screened by RNA sequencing. Protein expression was evaluated by western blot and immunofluorescence staining. Corneal defect area was assessed by fluorescein sodium staining. Conjunctiva goblet cells were counted by periodic acid–Schiff staining. Tear secretion was measured using phenol red cotton thread.

Results

Desiccating stress induced ferroptosis and SQSTM1 accumulation in both HCECs and C57BL/6 mice. SQSTM1 knockdown alleviated ferroptosis in HCECs. In contrast, the overexpression of SQSTM1 promoted ferroptotic changes. Additionally, overexpression of SQSTM1 significantly increased acyl-CoA synthetase long chain family member 4 (ACSL4). Also, targeted inhibition of ACSL4 mitigated the dry eye symptoms and ferroptosis caused by both SQSTM1 overexpression and desiccating stress.

Conclusions

The accumulation of SQSTM1 triggers corneal epithelial cells ferroptosis through ACSL4 in dry eye disease.

Dry eye disease (DED) is a prevalent ocular surface disorder that affects millions globally and causes symptoms such as dryness, itching, pain, blurred vision, and vision loss.¹ DED arises from reduced tear production or excessive tear evaporation, leading to ocular surface dryness, inflammation, and damage to epithelial and goblet cells.² Accumulating evidence indicates that inflammation triggers various types of cell death in the corneal epithelium, which further amplifies inflammation through the release of pro-inflammatory cellular contents, creating a vicious cycle.^3,4 Recent studies highlight multiple forms of cell death in DED, including apoptosis, pyroptosis, necroptosis, and ferroptosis.⁴ Among these, ferroptosis has been identified as a key contributor to ocular surface inflammation.⁹ Despite its recognized role, the underlying mechanism of the initiation of ferroptosis in cornea epithelium remains unclear.

Ferroptosis is a nonapoptotic cell death driven by ferrous ion-dependent lipid peroxidation.⁵ The morphology change is characterized by shrinking mitochondria, swelling cells, and disrupted plasma membranes, which can be observed under various oxidative pressures.⁶ Its regulation involves iron homeostasis and lipid peroxidation. Transferrin receptor (TFRC) is a protein located on the cell membrane which mediates iron uptake by binding to the iron-carrying protein transferrin.^7,8 The upregulation of TFRC produce more ion influx, leading to Fe²⁺ accumulation in cellular plasma. The Fe²⁺ ion overload then oxidizes lipids in a Fenton-like manner and finally results in a large amount of reactive oxygen species (ROS) production.⁹ In addition, glutathione peroxidase 4 (GPX4), an antioxidant enzyme, plays a vital role in limiting lipid peroxidation.¹⁰ With the loss of GPX4, lipid peroxides cannot be metabolized. In particular, ACSL4 plays a role in promoting ferroptosis by incorporating PUFAs into phospholipids, which produce the substrate of lipid peroxidation. Thus, ACSL4 is recognized as a significant contributor to ferroptosis.³⁷ The initiation of ferroptosis has not been discussed before in the context of DED, to our knowledge. In this article, we mainly focus on the mechanism of ferroptosis.

Autophagy is a cellular self-monitoring system that maintains homeostasis by degrading cellular waste. However, autophagy dysfunction can lead to cell death. In our previous study, we found the induction of DNA damage-inducible transcript 4 (DDIT4) resulted in autophagy flux impairment.²⁶ We also revealed the importance of autophagy in cell death in DED,²⁷ and numerous studies have illustrated the relationship between autophagy and ferroptosis. Autophagy can regulate ferroptosis in both ion accumulation and lipid peroxidation. Ferritinophagy is the process of autophagic degradation of the iron-storage protein ferritin; it promotes cellular iron accumulation and therefore enhances the Fenton reaction.¹¹ Lipophagy, a selective autophagic process targeting lipid droplets, releases free fatty acids that serve as substrates for lipid peroxidation and ferroptosis^12,13 In our previous study, we observed that Atg5-dependent lipophagy induced the release of lipid droplets (LDs), which enhance ferroptosis in DED.¹⁴ However, the direct molecular link between autophagy and ferroptosis remain unclear in DED.

Sequestosome 1 (SQSTM1) is a cargo receptor in autophagy, connecting target proteins to microtubule-associated protein light chain 3 (LC3) autophagosomes.¹⁵ Nevertheless, emerging evidence suggests that SQSTM1 also takes part in several molecular pathways.^33,34 For example, SQSTM1 has been proven to modulate apoptosis by activating caspase-8.¹⁶ Also, a growing number of studies have demonstrated that SQSTM1 plays a vital role in inducing ferroptosis. Studies suggest that SQSTM1-mediated clockophagy can promote lipid peroxidation during ferroptosis.¹⁷ Other studies have investigated the role of p62/SQSTM1 in regulating ROS generation during ferroptosis^18,19; however, the exact function of SQSTM1 in DED remains to be elucidated but presents a potential therapeutic target to be studied.

In this study, we found that autophagic flux impairment leads to SQSTM1 accumulation in DED, promoting ferroptosis through acyl-CoA synthetase long chain family member 4 (ACSL4) activation. Additionally, we demonstrated that inhibiting ACSL4 mitigates ferroptosis, highlighting its potential as a therapeutic approach for DED.

Methods

Cell Culture and Desiccating Stress Model

The human SV40 immortalized corneal epithelial cell line (CRL-11135, human corneal epithelium [HCE-2]) was acquired from the American Type Culture Collection (Manassas, VA, USA). The HCE-2 cells were cultured on plates in Gibco Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Thermo Fisher Scientific, Waltham, MA, USA), 10% fetal bovine serum (FBS; Gibco), and 1% streptomycin /penicillin (Gibco). The cultural environment was controlled in an atmosphere containing 5% carbon dioxide (CO₂) at 37°C. The desiccating stress in vitro model was previously described.^20,30 The culture medium was completely removed from HCE-2 cells and dried in air for 10 minutes under 25°C and 40% relative humidity. Then, the culture medium was placed in plates for subsequent experiments (Supplementary Fig. 1).

Animal Model and Treatment

Eight-week-old C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Ethics Committee of Zhongshan Ophthalmic Center (Guangzhou, China; approval ID: Z2024011). Establishment of the DED mouse model was reported previously.^21,30 Briefly, the DED model was established by subcutaneously injecting the mice with 1.5 mg/0.3 mL of scopolamine hydrobromide (SCOP; MedChemExpress, Shanghai, China) three times daily for 7 consecutive days while maintaining a humidity level of 20% to 30%. Successful model induction was confirmed via sodium fluorescein staining and tear secretion tests.

Mice were randomly assigned to four groups. The control (CTRL) group received only a phosphate-buffered saline (PBS) injection. Another group received the ACSL4 inhibitor rosiglitazone (ROSI, 5 mg/kg; MedChemExpress) subperitoneally two times a day for 7 days. Both of these groups were maintained in a normal-humidity environment. The dry eye (DE) group was given subcutaneous injections of SCOP and received PBS subperitoneally. The DE and ACSL4 inhibition (DE+ROSI) group received both SCOP and ROSI. These last two groups were maintained in low-humidity environments. Slit-lamp examinations with cobalt blue light were used to examine ocular surface damage on days 0, 3, and 7. Tear secretion was measured on day 7. All mice were sacrificed on day 7 for further analysis. At least three biological replicates were conducted in each group.

Tear Secretion Test

Tear secretion was measured using phenol red thread (Jingming, Tianjin, China). The thread was gently placed in the conjunctival sac near the lateral canthus of each mouse for 15 seconds. The length of the red-stained part of the thread was measured; each eye was tested three times, and the average was recorded as the final result.

Sodium Fluorescein Staining of Cornea

Sodium fluorescein staining was chosen as the method to evaluate corneal surface damage. Each eye was wetted by 0.25% fluorescein sodium (Jingming) and washed with PBS. The corneal defect area was observed by microscope with cobalt blue light, and images were captured for future analysis. The defect area was quantified by ImageJ (National Institutes of Health, Bethesda, MD, USA).

Cell Counting Kit-8 Assay

Cell viability was measured by Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) following the instructions provided by the manufacturer. Briefly, 1 × 10⁴ human corneal epithelial cells (HCECs) per well were seeded in 96-well plates and cultured with 100 µL medium for 24 hours. Then, medium containing CCK-8 reagent (1:10) was added to the plates, which were cultured for 1 hour at 37°C, away from light. An enzyme-labeling instrument (Agilent Technologies, Santa Clara, CA, USA) was then used to measure the absorbance at 450 nm.

Measurement of Malondialdehyde Levels

An malondialdehyde (MDA) assay kit (S0131S; Beyotime, Jiangsu, China) was used to evaluate MDA production, following the manufacturer's instructions. 1 × 10⁶ HCECs were cultured in six-well plates for 24 hours. After the conditioned exposure, 200 µL radioimmunoprecipitation assay (RIPA) lysis buffer was added per well for 10 minutes on ice. Cell lysis was then collected and centrifuged at 15,000 rpm for 10 minutes. The supernatant was collected, mixed with the MDA working solution, and heated to 100°C for 15 minutes. After cooling down, the mixture was added to 96-well plates, and absorbance under 532 nm was measured using the Agilent enzyme-labeling instrument. Then, the final content of MDA was calculated and analyzed.

Measurement of ROS and Lipid Peroxidation

ROS were measured by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) assay kit (ab113851; Abcam, Cambridge, UK). Briefly, HCECs were cultured in 12-well plates for 24 hours. Cells were rinsed in PBS for three times and then incubated with medium containing 10-µM H2DCFDA per 1 mL per well at 37°C for 1 hour. After rinsing with PBS, cells were imaged under fluorescence microscopy. Lipid peroxidation levels were similarly examined. HCECs were seeded, placed in 12-well plates, and incubated with 5-µM C11-BODIPY 581/591 C11 (Lipid Peroxidation Sensor) medium solution (D3861; Thermo Fisher Scientific) at 37°C for 1 hour. After rinsing with PBS, images were captured using fluorescence microscopy, and fluorescence intensity was measured and calculated with ImageJ.

Measurement of Fe²⁺ Ions

Intracellular ferrous ions were measure by a Fe²⁺-specific probe (Dojindo Laboratories). In brief, HCECs were grown in 12-well plates and exposed to conditioned cultural medium. Subsequently, the cells were washed with PBS for 3 times, and the medium was changed to DMEM/F-12 containing 250-µM FerroOrange (Dojindo Laboratories) for 1 hour at 37°C. After rinsing, fluorescent pictures were captured under microscope and analyzed by ImageJ.

RNA Interference

Small interfering RNA (siRNA) was transfected into HCECs using Lipofectamine RNAiMAX Transfection Reagent (13778150; Thermo Fisher Scientific). Sequences of siRNA were as follows: SQSTM1 sense—5′-GGAUAACUGUUCAGGAGGATT-3′, anti-sense—UCCUCCUGAACAGUUAUCCTT-3′; ACSL4 sense—5′-GUAUGUAUCUCUUGGGAAATT-3′, anti-sense—5′-UUUCCCAAGAGAUACAUACTT-3′. A non-targeting siRNA sequence served as a negative control. Briefly, the siRNA was dissolved in diethylpyrocarbonate (DEPC) water at a concentration of 20 µM. The siRNA was mixed with Lipofectamine RNAiMAX in Gibco Opti-MEM I Reduced Serum Medium (31985062; Thermo Fisher Scientific) and incubated at room temperature for 5 minutes. Cells were digested and resuspended, then mixed with the siRNA solution. The cell suspension was divided equally among six-well plates. Each well contained 2 mL of cultural medium, 4.5 µL Lipofectamine RNAiMAX, and 1.5 µL siRNA (60 pmol). The transfection time was 48 hours, and knockdown efficiency was verified by western blot analysis.

Western Blot Analysis

Cell samples were collected from six-well plates (three repeats per group), and corneas from two eyeballs were collected as one sample (three repeats per group). The samples were lysed by 100 µL RIPA lysis buffer (P0013B; Beyotime) containing 1% proteinase inhibitor (P1006; Beyotime) for 10 minutes on ice. The samples were then centrifuged at 15,000 rpm for 5 minutes. The supernatant was collected, and protein concentrations were measured by bicinchoninic acid (BCA) assays (P0010; Beyotime). Protein was denatured in SDS-PAGE loading buffer (P0015L; Beyotime) at 100°C for 10 minutes. All of the samples were then loaded on 10% SDS-PAGE gels for 1-hour electrophoresis under 130 V and transferred to 0.22-µm polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Bedford, MA, USA) for 1 hour under 250 mA. The membrane was blocked in QuickBlock (P0252; Beyotime) for 15 minutes at room temperature. Then, the membrane was incubated at 4°C overnight with the following primary antibodies: anti-β-actin (1:3000, 66009-1-Ig; Proteintech, Rosemont, IL, USA), anti-SQSTM1 (1:2000, 18420-1-AP; Proteintech), anti-LC3B (1:1000, 12741; Cell Signaling Technology, Danvers, MA, USA), anti-BECN1 (1:1000, 495; Cell Signaling Technology), anti-TFRC (1:1500, 46222; Cell Signaling Technology), anti-GPX4 (1:2000, ab125066; Abcam), anti-4HNE (1:1000, ab46545; Abcam), and anti ACSL4 (1:2000, 22401-1-AP; Proteintech). After rinsing with Tris-buffered saline with Tween 20 (TBST) three times, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000, SA00001-2; Proteintech) or HRP-conjugated goat anti-mouse IgG (1:5000, SA00001-1; Proteintech) for 1 hour at room temperature. The bands were then illuminated using an enhanced chemiluminescence (ECL) solution (Beyotime), and expression levels were analyzed by ImageJ.

RNA Sequencing Analysis

A TruSeq Stranded mRNA Library Prep Kit (20020594; Illumina, San Diego, CA, USA) was used to construct the RNA sequencing (RNA-seq) libraries, which were sequenced on Illumina PE150 sequencers. Sequencing reads were mapped to hg19 using STAR. Transcripts per million (TPM) values were determined using RNA-Seq by Expectation-Maximization (RSEM). An absolute value of log₂ fold change > 1 and P < 0.05 was used to identify differentially expressed genes using DESeq2 analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed with the the Bioconductor package pathview.

Immunofluorescence Staining

HCECs were cultured in 12-well plates with cell slides and fixed with 4% paraformaldehyde for 15 minutes, and each eye ball was fixed and sectioned. Following fixation, samples were permeabilized with 0.1% Triton X-100 in PBS for 20 minutes. Then all samples were blocked using 3% bovine serum albumin (BSA, 9048-46-8; Solarbio, Beijing, China) solution at room temperature for 30 minutes. The slides were incubated with anti-SQSTM1 (1:1000, 18420-1-AP; Proteintech), anti-LC3B (1:500, 12741; Cell Signaling Technology), anti-TFRC (1:1000, 46222; Cell Signaling Technology), anti-GPX4 (1:1000, ab125066; Abcam), and anti-4HNE (1:500, ab46545; Abcam) at 4°C overnight. After the slides were washed three times in PBS with Tween 20, Alexa Fluor 488- and Alexa Fluor 594 conjugated secondary antibodies (1:500, 4412 and 8889; Cell Signaling Technology) were then applied for 1 hour at room temperature, and then stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes. Following three additional washes, all slides were photographed with a fluorescence microscope (Axio Imager Z2; Carl Zeiss Microscopy, Oberkochen, Germany).

TUNEL Staining

Cell death in corneal epithelium was examined using a TUNEL apoptosis kit (A111; Vazyme Biotechnology, Nanjing, China), following the manufacturer's instructions. Slides of eyeballs were deparaffinized and permeabilized by proteinase K for 20 minutes. The slides were equilibrated with an equilibration buffer for 20 minutes at room temperature and then washed in PBS. TUNEL reaction mixture was added, and the slides were incubated at 37°C for 1 hour in the dark. After reaction, the sections were observed and photographed under fluorescent microscopy, and cell deaths were counted, with three repeats in each group.

Periodic Acid–Schiff Staining

Eyeballs were fixed in 4% paraformaldehyde and sliced. The slices were then stained with a periodic acid–Schiff (PAS) staining kit (G1008-20ML; ServiceBio, Wuhan, China). Images of conjunctiva were captured using an Eclipse 50i microscope (Nikon, Tokyo, Japan). The number of goblet cells were counted with three repeats for each group.

Results

Desiccating Stress Induces Corneal Epithelial Cells Ferroptosis in DED

To investigate whether desiccating stress induces ferroptosis in corneal epithelial cells, we performed RNA-seq and observed significant changes in gene expression indicative of ferroptosis (Fig. 1A). To verify the role of ferroptosis in this process, we conducted rescue experiments using Fer-1, a ferroptosis inhibitor. Fer-1 significantly alleviated cell death and increased MDA levels caused by desiccating stress (Figs. 1C, 1D).

Figure 1.

Desiccating stress induces cornea epithelial cells ferroptosis in dry eye. (A) RNA-seq heat map of the ferroptotic genes changes in HCECs after desiccating stress (n = 3). (B) Western blot and quantitative analyses of TFRC and GPX4 in HCECs (n = 3). (C) Fer-1 rescued HCECs from cell death caused by desiccating stress, as measured by CCK-8 (n = 5). (D) Fer-1 reduced the elevated MDA levels caused by desiccating stress in HCECs (n = 3). (E) Representative fluorescein sodium staining and defect area analysis of desiccating stress–induced DE in mice (n = 6). (F) Tear secretion measure of DE in mice (n = 6). (G) Western blot and gray value analysis of DE in mice (n = 3). (H) TUNEL apoptosis staining in the cornea epithelium of control and DE groups (n = 3). Scale bar: 100 µm. Error bars: SD. P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

To explore the ferroptosis level in vivo, we established the animal model by combining subcutaneous injection of SCOP with exposure to a low-humidity environment. Fluorescence sodium staining showed cornea staining in the DE group compared to the CTRL group (Fig. 1E, Supplementary Fig. 2). Tear secretion was also reduced in the DE group (Fig. 1F). Western blot analysis showed similar changes in both the in vitro and in vivo models of TFRC and GPX4 and elevated 4-hydroxynonenal (4HNE) levels (Figs. 1B, 1G). The TUNEL assay identified more stained points in the DED model, further confirming cell death in the corneal epithelium (Fig. 1H).

In Vitro SQSTM1 Knockdown Alleviates Ferroptosis Caused by Desiccating Stress

Our early studies demonstrated that autophagy flux is impaired in DED. Immunofluorescence colocalization staining revealed an increase in SQSTM1-positive LC3B autophagosome puncta in desiccated HCECs (Fig. 2A). Consistently, RNA-seq and western blot revealed elevations of SQSTM1 and LC3B (Figs. 2B, 2C). Similar changes in SQSTM1 and LC3B levels were observed in an animal model of DED (Figs. 2D, 2E).

Figure 2.

The obstruction of autophagy flux causes the accumulation of SQSTM1. (A) HCECs were exposed to desiccating stress for 10 minutes and fixed after 2, 4, and 6 hours for immunofluorescence staining of SQSTM1 and LC3. SQSTM1 and LC3 colocalized puncta (yellow) were counted and analyzed (n = 10). Scale bar: 10 µm. (B) Heat map showing the transcriptional changes in genes related to autophagy in HCECs after desiccating stress (n = 3). (C) Western blot and gray values of Beclin 1 (BECN1), SQSTM1, and LC3 in HCECs (n = 3). (D) Western blot analysis of SQSTM1 and LC3 from the corneal samples acquired from control (CTRL) and DE mice (n = 3). (E) Representative fluorescence images showing the expression of SQSTM1 and LC3 in the corneal epithelium of CTRL and DE mice. Scale bar: 100 µm. Error bars: SD. P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Given the established link between SQSTM1 and ferroptosis, we investigated the role of SQSTM1 in DED. HCECs were transfected with siRNA targeting SQSTM1 to knock down its expression. We measured the levels of ROS using a dichlorodihydrofluorescein diacetate (DCFH-DA) probe, ferrous ions using FerroOrange, and lipid peroxidation using the BODIPY 581/591 C11 probe (Fig. 3A). Desiccating stress significantly increased levels of ROS, ferrous ions, and lipid peroxidation. Knockdown of SQSTM1 mitigated these ferroptotic indicators and restored cell viability (Fig. 3B). Additionally, the elevated MDA levels were reduced upon SQSTM1 knockdown (Fig. 3C). Western blot analysis showed decreased 4HNE and TFRC expression and increased GPX4 levels after SQSTM1 knockdown (Fig. 3D). Immunofluorescence staining further confirmed these findings (Fig. 3E). To sum up, our results demonstrated that SQSTM1 knockdown can rescue HCECs from ferroptosis, at both metabolic and protein levels.

Figure 3.

In vitro SQSTM1 knockdown alleviates ferroptosis caused by desiccating stress. (A) Representative fluorescence images showing the ROS, Fe²⁺, and oxidative lipid levels in control (CTRL) and desiccating stress (DS) groups treated with scrambled siRNA (NC) or SQSTM1-targeted siRNA (si-SQSTM1). Fluorescence intensity was measured by ImageJ (n = 3). (B) SQSTM1 knockdown restored the cell viability in desiccated HCECs (n = 6). (C) MDA levels decreased in the si-SQSTM1 group compared with the NC group (n = 3). (D) Western blot analysis of 4HNE, TFRC, and GPX4 in HCECs. Protein expression levels were determined by ImageJ (n = 3). (E) Representative fluorescence images showing the expression of 4HNE, TFRC, and GPX4 in HCECs. Scale bar: 100 µm. Error bars: SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

SQSTM1 Aggravates Ferroptosis in Corneal Epithelial Cells

To further determine the function of SQSTM1 in ferroptosis in DED, we overexpressed SQSTM1 by infecting HCECs with lentivirus. The R pathview package (R Foundation for Statistical Computing, Vienna, Austria) revealed the changes in the ferroptosis pathway, including an increase in ferroptosis-related proteins (Fig. 4A). Western blot analysis confirmed elevated levels of SQSTM1 and LC3 (Fig. 4B). The infection efficiency was verified by fluorescence microscopy, which showed widespread expression of green fluorescent protein (GFP) in the infected cells (Fig. 4C).

Figure 4.

SQSTM1 aggravates ferroptosis in corneal epithelial cells. (A) KEGG pathway view showing the major gene changes in the ferroptotic pathway after SQSTM1 overexpression. (B) Western blot verified the protein expression level of SQSTM1 and LC3 after HCECs infected with SQSTM1 overexpression lentivirus (n = 3). (C) Representative fluorescence images showing the successfully infected HCECs labeled with GFP. (D) CCK-8 showed that Fer-1 restored the cell viability loss caused by SQSTM1 overexpression (n = 5). (E) MDA test showed that Fer-1 alleviated the lipid peroxidation caused by SQSTM1 (n = 3). (F) RNA-sequencing heat map shows transcriptional changes in ferroptotic genes (n = 3). (G) The ferroptotic protein markers TFRC and GPX4 in HCECs were measured by western blot after SQSTM1 overexpression (n = 3). (H) Western blot indicating a significant increase of ACSL4 in SQSTM1 overexpressed HCECs compared to the blank group (n = 3). Scale bar: 200 µm. Error bars: SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

To validate ferroptosis in SQSTM1-overexpressing cells, we treated the cells with Fer-1. Fer-1 significantly improved cell viability and reduced MDA levels, confirming the presence of ferroptosis (Figs. 4D, 4E). RNA-seq showed multiple downregulation of protective genes (GPX4, SLC39A14, FTH1, etc.) and upregulation of harmful genes (TFRC, LPCAT3, GCLC, etc.) (Fig. 4F). Western blot analysis further confirmed increased TFRC and decreased GPX4 expression, consistent with ferroptosis induction (Fig. 4G).

To identify the key connection between SQSTM1 and ferroptosis, we analyzed RNA-seq data and observed significant upregulation of lipid peroxidation–related genes. Notably, ACSL4 was highly upregulated in SQSTM1-overexpressing cells compared to controls, as confirmed by western blot (Fig. 4H). This suggests that ACSL4 may act as a downstream effector of SQSTM1 in ferroptosis.

Ferroptosis in Corneal Epithelial Cells Can Be Ameliorated by Knocking Down ACSL4

Monitoring ACSL4 protein expression in desiccated HCECs via western blot revealed a significant increase, mirroring the results observed with SQSTM1 overexpression (Fig. 5A, Supplementary Fig. 2). Given the established role of ACSL4 in lipid peroxidation during ferroptosis, we hypothesized its importance in our study. To validate the role of ACSL4, we used siRNA to silence ACSL4 in HCECs, confirming the knockdown efficiency with western blot for three different siRNA sequences (Fig. 5B).

Figure 5.

Ferroptosis in corneal epithelial cells can be ameliorated by ACSL4 knockdown. (A) Western blot and gray value analysis depicting ACSL4 elevation after HCECs were exposed to desiccating stress (n = 3). (B) The knockdown efficiency of three sequences of siRNAs was tested by western blot (n = 3). (C) Representative fluorescence images show the ROS, Fe²⁺, and oxidative lipid levels in control (CTRL) and desiccating stress (DS) groups treated with scrambled siRNA (NC) or ACSL4-targeted siRNA (si-ACSL4). Fluorescence intensity was measured by ImageJ (n = 3). (D) ACSL4 knockdown restored the cell viability in desiccated HCECs (n = 5). (E) Lipid peroxidative levels measured by MDA assay were decreased in the si-ACSL4 group compared with the NC group (n = 3). (F) Western blot analysis of 4HNE, TFRC, and GPX4 in HCECs. Protein expression levels were determined by ImageJ (n = 3). Scale bar: 100 µm. Error bars: SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

We evaluated the effects of ACSL4 knockdown on ferroptosis by evaluating ROS levels, ferrous ion levels, and lipid peroxidation. Knockdown of ACSL4 significantly reduced ROS levels, ferrous ion levels, and lipid peroxidation, as confirmed by fluorescent microscopy and quantified by fluorescence intensity analysis (Fig. 5C). Simultaneously, cell death and MDA levels were reduced after silencing ACSL4 (Figs. 5D, 5E). Western blot analysis showed that 4HNE generation was decreased in the si-ACSL4 groups, also indicating mitigation of lipid peroxidation. Also, the ferroptotic markers TFRC and GPX4 both recovered. (Fig. 5F). Overall, ACSL4 knockdown had a similar inhibitory effect on ferroptosis as SQSTM1 knockdown, suggesting the pivotal role of ACSL4 in mediating ferroptosis under desiccating stress.

SQSTM1 Promotes Ferroptosis Via ACSL4 in DED

To further establish that ACSL4 operates downstream of SQSTM1 in the context of ferroptosis, we first observed that ACSL4 levels were significantly reduced following SQSTM1 knockdown in desiccated HCECs (Fig. 6A). Next, we transfected siRNA targeting ACSL4 into SQSTM1-overexpressed HCECs. Silencing ACSL4 can reverse the harmful effect on the cell viability of SQSTM1 overexpression (Fig. 6B). Lipid peroxidation was measured by determining the content of MDA. Compared to negative controls, si-ACSL4 reduced MDA levels elevated by SQSTM1 overexpression (Fig. 6C). Cytoplasmic Fe²⁺ levels were measured using the FerroOrange indicator. ACSL4 knockdown prevented Fe²⁺ accumulation induced by SQSTM1 overexpression (Fig. 6D). Additionally western blot analysis confirmed the reduction of ferroptosis, as evidenced by decreased 4HNE and TFRC levels and increased GPX4 expression (Fig. 6E). Immunofluorescence staining further supported these findings, providing visual evidence of reduced ferroptotic signaling (Fig. 6F). In summary, these findings provide compelling evidence that ACSL4 is a downstream target of SQSTM1 in the ferroptotic pathway induced by SQSTM1 elevation in desiccated HCECs.

Figure 6.

SQSTM1 promotes ferroptosis via ACSL4 in dry eye. (A) Western blot depicts the ACSL4 expression level after SQSTM1 knockdown (n = 3). (B) CCK-8 indicated that si-ACSL4 restored the cell viability in SQSTM1-overexpressed HCECs (n = 5). (C) ACSL4 knockdown reduced lipid peroxidative products induced by SQSTM1, as measured by MDA (n = 3). (D) FerroOrange showed that SQSTM1 overexpression led to intracellular Fe²⁺ accumulation, and, in si-ACSL4 groups, Fe²⁺ decreased compared to the NC group. Fluorescence intensity was determined by ImageJ (n = 3). (E) Protein expression levels of ACSL4, 4HNE, TFRC, and GPX4 were measured by western blot (n = 3). (F) Representative fluorescence images show the expression of TFRC, GPX4, and 4HNE. Scale bar: 100 µm. Error bars: SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Inhibition of ACSL4 In Vivo Mitigates Ferroptosis in DED

Based on our cellular findings, we revealed the significance of SQSTM1 in inducing ferroptosis in HCECs and identified ACSL4 as a downstream effector. To further demonstrate this result, we conducted experiments on the DED in vivo model. Mice in the DE group were injected with SCOP (1.5 mg/0.3 mL) subcutaneously three times a day for 7 consecutive days under low-humidity conditions (20%–30%). PBS was injected as a control (CTRL). ROSI, an ACSL4 inhibitor, was administered subperitoneally at 5 mg/kg twice a day for 7 days.

Cornea damage is evaluated by fluorescein sodium staining with photographs taken under cobalt blue light. The DE group exhibited significant corneal defects, which were alleviated by ROSI treatment (Fig. 7A). Tear secretion recovered after ROSI treatment but without statistical significance (Fig. 7B). Eyeballs and eyelids were sampled and sectioned for histological analysis. PAS staining revealed a loss of goblet cells in the conjunctiva of DE mice compared to controls, with slight recovery observed in ROSI-treated mice (Fig. 7C) but again without statistical significance. Hematoxylin and eosin staining showed a minor decrease in thickness in the corneal epithelium but no apparent change after ROSI treatment (Fig. 7C). A TUNEL apoptosis kit was used to detect dead cells in the cornea. The results showed a significant reduction in dead cells in the ROSI-treated DED model, indicating a recovery of corneal damage (Fig. 7D). Western blot and immunofluorescence analyses confirmed that ROSI treatment reversed the elevation of 4HNE and TFRC caused by desiccating stress and restored GPX4 levels (Figs. 7E, 7F). In summary, our in vivo study confirmed the role of ACSL4 in ferroptosis-induced DED and demonstrated the potential therapeutic benefits of targeting ACSL4 to protect the corneal epithelium from desiccation-induced damage.

Figure 7.

Inhibition of ACSL4 in vivo mitigates ferroptosis in dry eye. (A) Representative fluorescein sodium staining of samples from control mice and desiccating stress-induced DE mice with or without ROSI injection (n = 6). (B) Phenol thread tear production test in mice (n = 6). (C) Representative PAS staining and HE staining of conjunctival goblet cells and cornea. Goblet cells were counted and statistically analyzed (n = 3). (D) Representative TUNEL staining in the corneal epithelium of control mice and DE mice with or without ROSI injection (n = 3). (E) Western blot and quantitative analyses showed the protein expression levels of 4HNE, TFRC, and GPX4 (n = 3). (F) Representative immunofluorescence staining of TFRC, GPX4, and 4HNE. Scale bar: 100 µm. Error bars: SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Discussion

The prevalence of DED has risen sharply and is still increasing. As a major ocular surface disease, DED can cause different levels of frustration to patients, ranging from discomfort to damaged vision.²² Cell death has been demonstrated to play an essential role in the DED vicious cycle, leading to corneal epithelium damage.²³ In our previous research, we explored the role of ferroptosis in the pathogenesis of DED and highlighted the protective function of AKR1C1 against ferroptosis-induced cellular damage and inflammation.²⁴ In the current study, we further demonstrated that desiccating stress is a significant factor in inducing ferroptosis in HCECs. However, the precise triggers for ferroptosis remain elusive, prompting us to investigate the underlying mechanisms in DED.

Autophagy, a vital process for maintaining intracellular homeostasis, involves the recycling and degradation of damaged proteins and organelles.²⁵ Myriad studies have demonstrated the protective role of autophagy in human diseases; however the abnormality of autophagy may lead to disease genesis. In our earlier work, we discovered that the autophagy flux is impaired in the DED process, primarily due to the induction of DDIT4.²⁶ We then determined the potential role of S100A9-TLR4 axis in autophagy-derived DED.²⁷

Additionally, a series of studies have proven the relationship between autophagy and ocular surface disease.²⁸ In this research, we found that a change in autophagy flux leads to unusual elevations of SQSTM1. The accumulation of LC3B puncta indicates a blockage in autophagosome degradation, and changes observed in RNA sequencing further support the notion of impaired autophagy. These findings suggest that the mechanisms by which autophagy contributes to the pathogenesis of DED warrant further investigation.

The various types of cell death are classified based on the different morphological changes. The diverse initiation of cell death is widely studied, and autophagy, in some instances, has been identified as the cause of cell death.²⁹ In the realm of DED, pyroptosis has been shown to be activated by KCNK5-induced autophagy impairment.³⁰ Yet, ferroptosis, a type of ion-dependent cell death, has also demonstrated crosstalk with autophagy.³¹ In our study, we illustrated the molecular pathway for autophagy to induce corneal epithelium ferroptosis.

SQSTM1 has been extensively studied for its function in sensing oxidative stress and acting as a autophagy receptor for ubiquitinated cargos.³² Disruption of autophagy flux often leads to the accumulation of SQSTM1, which is commonly used as an indicator of autophagosome formation due to its role in autophagosome transport. LC3 is commonly used as an indicator of autophagosome because of its function for autophagosome transportation. In our previous study, we noticed that the autophagy flux was blocked.²⁶ Here, our immunofluorescence colocalization indicated that the accumulated SQSTM1 was combined with undegraded autophagosome, marked as LC3. This suggests that the elevation of SQSTM1 in DED is a result of a blockage of autophagy.

In recent years, SQSTM1 studies have not been limited to investigating its role as an autophagic substrate but have also investigated its role in many molecular pathways.³³ SQSTM1 has been reported in many studies for its crosstalk between autophagy and ferroptosis, and it has been reported that SQSTM1 is crucial in aiding ubiquitin-specific peptidase 8 (USP8)-regulated ferroptosis.³⁴ SQSTM1 has also been identified as a cargo receptor responsible for ARNTL degradation which results in clockophagy-induced ferroptosis.³⁵ In alignment with these findings, in our study we found that overexpressing SQSTM1 enhanced ferroptosis in HCECs, but SQSTM1 knockdown ameliorated ferroptosis caused by desiccating stress. This finding underscores the pivotal role of SQSTM1 in mediating the interplay between autophagy and ferroptosis in DED.

Lipid peroxidation is a hallmark of ferroptosis. Under various types of environmental stress, ROS generate free ferrous ions, resulting in the phospholipid peroxidation which marks ferroptosis.³⁶ Initiation of peroxidation of polyunsaturated fatty acid chains (PUFAs) requires two critical molecules, ACSL4 and lysophosphatidylcholine acyltransferase 3 (LPCAT3).^37,38 ACSL4 facilitates the ligation of long-chain PUFAs with coenzyme A, allowing LPCAT3 to esterify PUFAs into phospholipids (PLs), which are then peroxidized into phospholipid hydroperoxides (PL-OOH), compromising membrane integrity and leading to cell death.^6,13 Studies have found that deletion of ACSL4 can preserve the proliferation of GPX4-null cells.¹⁶ Meanwhile, we discovered a significant increase of ACSL4 in HCECs under desiccating stress analyzed by RNA-seq and western blot. We postulated that ASCL4 may have an important role in HCEC ferroptosis. Correspondingly, deletion of ACLS4 reduced oxidative stress, ion load, and lipid peroxidation levels, based on the results of our use of the ROS probe, FerroOrange, and BODIPY 581/591 C11. Also, we showed that the ACSL4 inhibitor ROSI alleviated ocular surface damage in DED animal models.

One study has reported that extracellular SQSTM1 exacerbated ferroptosis in acute pancreatitis via ACSL4.¹⁸ In our study, we observed a similar increase of ACSL4 in SQSTM1-overexpressed HCECs compared to desiccated HCECs. Simultaneously, we found that ACSL4 knockdown was able to rescue the ferroptotic changes caused by SQSTM1 overexpression.

In conclusion, our study showed that the abnormal autophagy process results in the accumulation of SQSTM1, which is a key promoter of ferroptosis in HCECs under desiccating stress conditions. Silencing SQSTM1 and ASCL4 indicated a similar inhibition in vitro, and inhibiting ACSL4 in vivo prevented ferroptotic damage in DED. Additionally, we discovered that SQSTM1 contributes to ferroptosis via the downstream factor ACSL4. SQSTM1 plays a role in autophagy-related ferroptosis in corneal epithelial cells that provides a wealth of opportunity to elucidate both autophagy and execution mechanisms of ferroptosis in DED.