December 2024
Volume 65, Issue 14
Open Access
Cornea  |   December 2024
Endoplasmic Reticulum Stress Induces ROS Production and Activates NLRP3 Inflammasome Via the PERK-CHOP Signaling Pathway in Dry Eye Disease
Author Affiliations & Notes
  • Zhiwei Zha
    The Affiliated Ningbo Eye Hospital of Wenzhou Medical University, Ningbo, Zhejiang, China
    State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Decheng Xiao
    State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Zihao Liu
    State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Fangli Peng
    State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    The First Affiliated Hospital of Soochow University, Suzhou, China
  • Xunjie Shang
    State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Zhenzhen Sun
    State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Yang Liu
    Department of Ophthalmology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China
  • Wei Chen
    State Key Laboratory of Ophthalmology, Optometry and Vision Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Ningbo Eye Institute, Ningbo Eye Hospital, Wenzhou Medical University, Ningbo, China
  • Correspondence: Yang Liu, Department of Ophthalmology, Zhongnan Hospital of Wuhan University, 169 Donghu Rd., Wuhan 430071, China; [email protected]
  • Wei Chen, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, 270 Xueyuan Rd., Wenzhou 325027, China; [email protected]
  • Footnotes
     ZZ and DX contributed equally to this work.
Investigative Ophthalmology & Visual Science December 2024, Vol.65, 34. doi:https://doi.org/10.1167/iovs.65.14.34
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      Zhiwei Zha, Decheng Xiao, Zihao Liu, Fangli Peng, Xunjie Shang, Zhenzhen Sun, Yang Liu, Wei Chen; Endoplasmic Reticulum Stress Induces ROS Production and Activates NLRP3 Inflammasome Via the PERK-CHOP Signaling Pathway in Dry Eye Disease. Invest. Ophthalmol. Vis. Sci. 2024;65(14):34. https://doi.org/10.1167/iovs.65.14.34.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The purpose of this study was to investigate the potential roles of endoplasmic reticulum (ER) stress in the development of dry eye disease (DED).

Methods: Single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database, derived from corneal tissues of a dry eye mouse model, was processed using the Seurat R program. The results were validated using a scopolamine-induced dry eye mouse model and a hyperosmotic-induced cell model involving primary human corneal epithelial cells (HCECs) and immortalized human corneal epithelial (HCE-2) cells. The HCE-2 cells were treated with 4-phenylbutyric acid (4-PBA) or tunicamycin (TM) to modulate ER stress. TXNIP and PERK knockdown were performed by siRNA transfection. Immunofluorescence, Western blotting, and real-time PCR were used to assess oxidative stress, ER stress, unfolded protein response (UPR) marker proteins, and TXNIP/NLRP3 axis activation.

Results: The analysis of scRNAseq data shows an increase in the ER stress marker GRP78, and the activation of the PERK-CHOP of UPR in DED mouse. These findings were confirmed both in vivo and in vitro. Additionally, HCE-2 cells treated with 4-PBA or TM showed significant effects on the production of reactive oxygen species (ROS) and the activation of the TXNIP/NLRP3-IL1β signaling pathway. Furthermore, siRNA knockdown of PERK or TXNIP, which alleviated the TXNIP/NLRP3-IL1β signaling axis, showed protective effects on HCECs.

Conclusions: This study explores the role of ER stress-induced oxidative stress and NLRP3-IL-1β mediated inflammation in DED, and highlights the therapeutic potential of PERK-CHOP axis and TXNIP in the treatment of DED.

Inflammation plays a critical role in the initiation and development of dry eye disease (DED).1,2 A vicious cycle of this response exacerbates clinical severity through persistent disruption of ocular surface integrity.3,4 Additionally, oxidative stress often accompanies and exacerbates this response, which is due to an imbalance between oxidants and antioxidants, which exacerbates inflammation.5,6 
Inflammation and oxidative stress are closely linked and interact in certain diseases. During this interaction, Thioredoxin-interacting protein (TXNIP) plays a significant role in upregulating of inflammatory factors through acting as a crucial sensor of oxidative stress in many diseases.7,8 Increases in oxidative stress and reactive oxygen species (ROS) upregulate TXNIP expression which in turn impairs the antioxidant activity of thioredoxin7,9 Our previous studies showed that the activation of the ROS-NLRP3-IL-1β signaling pathway is essential for induced innate immunity in DED.10 Furthermore, lowering ROS production suppresses the NOD-like receptor family, pyrin domain containing 3 (NLRP3), which can reduce DED symptoms.1113 These results highlight the significant role of oxidative stress in DED. However, it remains unclear whether TXNIP plays a role in this process. 
An association exists between increased TXNIP expression and endoplasmic reticulum (ER) stress in many diseases.1416 ER stress is a conserved adaptive intracellular response that is induced by various environmental insults, such as exposure to chemicals, ultraviolet light, and hyperosmotic stress.1719 These risk factors are also often implicated in DED.20 Prolonged ER stress results in continuous activation of the ER-induced unfolded protein response (UPR) and transitions into an unresolved terminal-UPR, leading to heightened inflammatory signaling and cell death. Under ER stress conditions, the UPR is activated by the ER resident chaperone molecule glucose regulating protein 78 (GRP78; i.e. ER stress marker) through three key proteins, including PKR-like ER kinase (PERK), inositol-requiring enzyme-1α, and activating transcription factor-6 to promote cell survival.21 In the PERK pathway, prolonged ER stress activates the CCAAT/enhancer binding protein homolog (CHOP), a key marker of terminal-UPR.16 This activation leads to increased inflammatory signaling and cell death. Stimulation of the PERK/CHOP pathway can induce ROS generation and lead to the overexpression of TXNIP.22,23 Although ER stress has been implicated in a variety of disease conditions, the role of ER stress and altered UPR signaling in DED remains unknown. The increased expression of CHOP is a crucial marker of the terminal-UPR. 
In this study, we hypothesize that hyperosmotic-induced ER stress alters the PERK-CHOP pathway and UPR signaling, contributing to DED pathogenesis. To test our hypothesis, we first utilized single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database (GSE182582). This dataset was obtained from a mouse model of dry eye induced by unilateral lacrimal gland excision. We then analyzed this dataset to determine whether there is an association between increases in UPR and the upregulation of DED signs. Subsequently, we used a scopolamine-induced dry eye mouse model as well as primary human corneal epithelial cells (HCECs) and immortalized human corneal epithelial cells (HCE-2) to validate the sequencing results. We were able to confirm that ER stress activated the terminal-UPR through PERK/CHOP pathway. Furthermore, a relationship was identified between ER stress and elevated ROS production in DED, and it was confirmed that ER stress inhibition is an effective way to lower rises in ROS levels caused by hyperosmolarity. 
Materials and Methods
Single-Cell Sequencing Analysis
Single-cell transcriptomic data (GSE182582) analysis from the GEO database was performed to clarify cell type heterogeneity and identify corneal epithelial cell type expression of specific molecular markers in DED. Samples were obtained from the corneal epithelium of the DED mouse model (n = 6) and a control group (n = 7), which is a complete tear deficiency model established by extirpation of the lacrimal gland. After excluding those of poor quality, 33,809 cells were procured. The scRNA-seq count data from the GEO database was imported into Seurat (version 4.3.0) and filtered for cells containing above 500 genes. SCTransform and Harmony normalized and removed batch effects. Cells were grouped and displayed on UMAP dimensions using the top 17 PCA main components at 0.6 resolution. Then, the cell clusters were categorized into eight cell types using the original data’s canonical annotation technique.24 Using the Fast Gene Set Enrichment Analysis (fgsea) R-package with Hallmark gene sets from the molecular signatures database (MSigDB), Gene set enrichment analysis (GSEA) analysis calculated the normalized enrichment score (NES). The terminal-UPR signature gene set, comprising 192 genes, was obtained from the literature,25 with gene expression determined by the AverageExpression function. The ComplexHeatmap package was used to produce the heatmap. Gene set signature scores were calculated using Seurat’s AddModuleScore function. The results are presented in boxplots and density plot diagrams. 
In Vivo Animal Model Studies
A scopolamine-induced dry eye mouse model was used to explore the potential correlation between declines in tear secretion inducing ocular surface dryness and initiating ER stress. We assessed changes in the corneal fluorescein staining (CFS) score in vivo and measured ROS production in corneal samples. 
Generation of Animal Dry Eye Model
The Shanghai-based Jiesjie Laboratory Animal Company provided 30 female C57BL/6 mice aged 6 to 8 weeks. The mice were treated according to the Statement for Animal Use in Ophthalmic and Vision Research. They were randomly assigned to either the control group or the DED group and housed in a controlled environment with 50% to 70% relative humidity at a temperature of 22 ± 1°C. Scopolamine (SCOP; 0.5 mg/0.2 mL; Sigma-Aldrich, Germany) was subcutaneously injected 3 times a day for 5 days in the DED group, and an equal volume of solvent was injected subcutaneously in the control mice. All mice were subsequently maintained in a controlled environment with fan-generated airflow and relative humidity maintained below 30%.26 
Corneal Fluorescein Staining
CFS was used in vivo to evaluate the dry eye extent. For staining, 1 µL (0.1% fluorescein) was instilled into the inferior conjunctival sac using a micropipette, as described previously.27 A stereofluorescence microscope was used to examine the cornea 3 minutes after fluorescein instillation, and the images were graded according to a standard system.10 
Tear Production
The tear production was analyzed using phenol red cotton threads (Tianjin Jingming).28 The phenol red thread was positioned in the lateral canthus of the eye for 60 seconds, and then, the thread wetting measurements were recorded. 
Dihydroethidium Staining
To determine whether ocular surface desiccating stress can lead to ER stress, we measured ROS accumulation with dihydroethidium (DHE; MedChemExpress, Shanghai, China) in mouse corneal slices.29 Sections were incubated in 10 µM DHE in PBS for 30 minutes at 37°C. All samples were observed and photographed under a confocal microscope (ZEISS LSM 880). The area with positive staining was determined using ImageJ software (ImageJ 1.8; NIH, USA). 
Western Blotting and Immunofluorescence of Mouse Tissues
CHOP activation is achieved through phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α), and the activation of activating transcription factor-4 (ATF4) in the PERK/CHOP pathway. To determine the involvement of PERK/CHOP pathway, Western blot and immunofluorescence techniques were used to examine the expression levels of GRP78, an ER stress marker, and PERK, as well as phosphorylated eIF2α (p-eIF2α), ATF4, and CHOP in corneal tissue. The expression levels of IRE1α and ATF6, two key proteins mediating the stress-activated ER response induced by UPR signaling, were also evaluated. 
Western Blot Analysis
The cornea tissues were lysed using the lysis buffer (P0013B; Biyuntian Biological Co., Ltd). A BCA Protein Assay Kit quantified the protein content (P0009; Biyuntian Biological Co., Ltd). To transfer proteins, a nitrocellulose membrane was used from GE Healthcare Life Sciences (Chicago, IL, USA). The membranes were blocked with a fast-Blocking Western solution (12010020; BIO-RAD) for 10 minutes to avoid nonspecific binding. The primary antibodies were incubated at 4°C overnight. Afterward, they were incubated with secondary antibodies for 2 hours at room temperature. Finally, the proteins were observed using chemiluminescent materials from Beyotime in China, and digital photographs were taken with the GE AI680. Antibodies used included: anti-NLRP3 (AG-20B-0014-C100; Adipogen); anti-PERK (24390-1-AP; Protein tech); anti-ATF6 (24169-1-AP; Protein tech); anti-Tubulin (sc-166729; Santa Cruz); anti-ATF4 (ab184909; Abcam); anti-IL1β(ab254360; Abcam); anti-IRE1 (329), anti-TXNIP (14715), anti-p-eIF2α (3398T), anti-GRP78 (3177), and anti-CHOP (2895; Cell Signaling Technology). 
Immunofluorescent Staining
Immunofluorescent staining was performed on frozen corneal sections. Samples were fixed by 4% paraformaldehyde for 20 minutes. Subsequently, permeabilization was achieved by treating the tissues with a 0.5% Triton X-100 solution for 15 minutes at ambient temperature. Samples were blocked with 20% goat serum to prevent nonspecific binding. The samples were then treated overnight at 4°C with GRP78, CHOP, NLRP3, or TXNIP primary antibodies. Secondary antibodies were then added and incubated for 1 hour at ambient temperature under light restriction. ZEISS LSM 880 confocal microscope was used to photograph stained samples. 
Cell Culture In Vitro Studies
PERK-CHOP pathway activation and TXNIP expression levels were evaluated to determine the roles of ER stress in inducing UPR signaling in hyperosmotic stressed human corneal epithelial cell line (HCE-2). Because of the limited availability of primary HCECs, they were only used to confirm the expression of ER stress markers and UPR markers following exposure to the 500 mOsm hyperosmotic medium. 
Cell Culture and Model of Hyperosmotic Stress
Both the immortalized HCE-2 cells and the primary HCECs were cultured according to a previously established procedure.30,31 The HCE-2 cells were acquired from the American Type Culture Collection (Manassas, VA, USA), and the primary HCECs were obtained by culturing limbal tissue discarded after surgery from the eye bank of Wenzhou Medical University. These 2 different cell types were exposed to either the isotonic 312 or the hyperosmotic 500 mOsm medium for 24 hours. 
Staining and Imaging of ER
The ER tracker (Biyuntian Biological Co., Ltd., Shanghai, China) was used to visualize the ER morphological structure.32 HCE-2 cells were exposed to the 500 mOsm hyperosmotic medium for 6, 12, and 24 hours, respectively. After adding ER-tracker green (1:2000), the cells were incubated for 30 minutes before being fixed by 4% paraformaldehyde. Then, the sections were mounted with an antifade agent and photographed under a confocal microscope (ZEISS LSM 880). 
ROS Measurement
General Oxidative Stress Indicator 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Molecular Probes, Invitrogen) was used to measure ROS activity.33 HCE-2 cells were placed on black plates with clear bottoms at a density of 1.5 × 104 per well. The cells were treated with either the 500 mOsm hyperosmotic culture medium and/or 4-PBA (2 µM; MedChemExpress, Shanghai, China) for 16 hours, or with either an ER stress antagonist tunicamycin (TM; 2 µg/mL; MedChemExpress, Shanghai, China), or an ER stress inducer, 4-PBA, for 24 hours. Subsequently, the cells were incubated with 5 µM of CM-H2DCFDA for 30 minutes at 37°C in the dark. Fluorescence was then observed using a ZEISS fluorescence microscope (ZEISS, Jena, Germany). 
Western Blotting and Immunofluorescence in Cultured Cells
Western blotting and immunofluorescence were used to detect ER stress and UPR markers in both HCE-2 cells and the primary HCECs. These cells were treated with either 4-PBA or TM for 24 hours in an isotonic or a 500 mOsm hyperosmolar medium. The detailed procedures were similar to those used in the Western blotting and immunofluorescence analysis of mouse tissues. 
RNA Interference
To investigate the impact of PERK/CHOP pathway activation and increased TXNIP expression in HCE-2 cells following exposure to a 500 mOsm medium, relevant siRNAs were used to individually inhibit PERK and TXNIP expression whereas plasmids containing TXNIP constructs were utilized to enhance TXNIP expression. 
PERK and TXNIP Inhibition
The HCE-2 cells were transfected with siRNA targeting PERK and TXNIP at 50% confluence (Supplementary Table S3). Stealth small interfering RNA was used as a treatment for negative control. Transfections were achieved using LipoMAX (SUDGEN, 32011) according to the manufacturer's protocol.34 The TXNIP and PERK siRNAs were purchased from Suzhou Jima Gene Co. Ltd (Suzhou, China). 
TXNIP Overexpression
Suzhou Jima Gene Co. Ltd. (Suzhou, China) supplied the TXNIP overexpression plasmid. The pEX-3 (pGCMV/MCS/Neo) was the vector used for plasmid synthesis. As directed by the manufacturer, Lipojet (Signagen) was used for transient transfection.35 
Real-Time PCR
RNA extraction from tissues was performed using the RNeasy Plus Mini Kit (QIAGEN Sciences) following the manufacturer’s instructions.36 A Takara cDNA synthesis kit was used to generate cDNA.37 Afterward, a Thermo Fisher Scientific 7500 Real-Time PCR System was used to measure gene expression of TXNIP and PERK. We used Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Supplementary Tables S1 and S2 list the primer sequences. 
Statistical Analysis
All statistical analyses were performed using GraphPad software (La Jolla, CA, USA), and findings are presented as mean ± standard deviation (SD). To compare the differences between the two groups, the Student’s t-test was utilized. For comparisons of three or more groups, 1-way ANOVA was used. Statistical significance was defined as a P value of < 0.05. 
Results
ScRNA-Seq Analysis Uncovers the Involvement of ER Stress in a Mouse Model of DED
We implemented dimensionality reduction clustering and Uniform Manifold Approximation and Projection (UMAP) embedding (Fig. 1A). Eight cell types were resolved by following the data annotation strategy described in the original dataset publication. The annotation strategy involved clustering cells based on their gene expression profiles, and validating these clusters using established marker genes.24 They include six subtypes of epithelial cells: (1) limbal stem cells (LSCs); (2) two varieties of transit-amplifying cells (TACs) with TAC1 in S phase of the cell cycle; (3) TAC2 in the G2/M phase, (4) basal cells; (5) wing and squamous cells (Squam); and (6) conjunctival cells (Conj) and immune cells. GSEA analysis showed that DED enhanced apoptosis pathways and inflammatory response pathways, and the UPR pathway were both significantly upregulated (Fig. 1B). Different cell types were tested for terminal-UPR feature gene set expression incorporating regulatory components. DED terminal-UPR feature gene expression was significantly upregulated in the early-stages of epithelial cell expansion, such as basal, LSC, and TAC1/2 (Figs. 1C, 1D), especially among the LSCs and TAC1/2 (see Fig. 1D). 
Figure 1.
 
Augmentation and characterization of the UPR in a DED mice model. (A) UMAP plot of mice corneal epithelial cells showing major cell types in control (Ctrl) and DED groups. LSC, limbal stem cell; TAC, transit amplifying cell; Squam, squamous; Conj, conjunctiva. (B) Differences in pathway activities determined by Gene set enrichment analysis (GSEA) between DED and Ctrl groups based on the HALLMARK database. The pathway scores are normalized, with a threshold established at padj < 0.5 and absolute NES > 1.2. (C) Heatmap of the expression level of terminal-UPR signature genes across each subtype of stem/progenitor cells. Corresponding box plots for gene scoring analysis based on terminal-UPR signature genes for different cell types are plotted above the heatmap. The P values were determined by the Wilcoxon test. ****P ≤ 0.0001. (D) UMAP plot of terminal-UPR signature scores calculated by the AddModuleScore function.
Figure 1.
 
Augmentation and characterization of the UPR in a DED mice model. (A) UMAP plot of mice corneal epithelial cells showing major cell types in control (Ctrl) and DED groups. LSC, limbal stem cell; TAC, transit amplifying cell; Squam, squamous; Conj, conjunctiva. (B) Differences in pathway activities determined by Gene set enrichment analysis (GSEA) between DED and Ctrl groups based on the HALLMARK database. The pathway scores are normalized, with a threshold established at padj < 0.5 and absolute NES > 1.2. (C) Heatmap of the expression level of terminal-UPR signature genes across each subtype of stem/progenitor cells. Corresponding box plots for gene scoring analysis based on terminal-UPR signature genes for different cell types are plotted above the heatmap. The P values were determined by the Wilcoxon test. ****P ≤ 0.0001. (D) UMAP plot of terminal-UPR signature scores calculated by the AddModuleScore function.
ER Stress and PERK-CHOP Branch Activation Underlies Dry Eye Mouse Model Development
The CFS score was significantly higher in DED mice (Fig. 2A). The ROS level was elevated in the corneal epithelium of DED (see Fig. 2A). The production of tears decreased in DED mice, indicating this model simulates tear deficiency (Fig. 2B). Immunoblotting and immunofluorescent staining revealed a significant rise in the GRP78 expression level, which is an ER stress marker in the epithelial layer of DED mice (Figs. 2C, 2D). The expression levels of PERK and ATF6 in the UPR branches were significantly upregulated, whereas IRE1α expression was unchanged (see Fig. 2C). Within the PERK branch, the expression levels of p-eIF2α, ATF4, and CHOP were significantly elevated (Figs. 2E, 2F). 
Figure 2.
 
The protein level of ER stress and terminal-UPR increased in the cornea of DED mouse. (A) A mouse DED model was successfully established, with representative pictures of CFS of control group mice and Scop group mice and statistical results of CFS. (B) Tear secretion of the control group mice and Scop group mice was measured by phenol red cotton. The color of the cotton thread changes from yellow to red on absorbing tears. (C) Continuous subcutaneous injection of Scop for 5 days increased the expression of GRP78, PERK, and ATF6 among ER stress markers in mouse corneal tissue. (D) Representative immunofluorescence images of GRP78 in corneal tissue. Scale bar = 50 µm. (E) The expression of p-eIF2α, ATF4, and CHOP among the terminal-UPR markers in the cornea of mice in the Scop group was increased. (F) Representative immunofluorescence images of CHOP. Scale bar = 50 µm. Results are presented as mean ± SD (n = 4–6). *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 2.
 
The protein level of ER stress and terminal-UPR increased in the cornea of DED mouse. (A) A mouse DED model was successfully established, with representative pictures of CFS of control group mice and Scop group mice and statistical results of CFS. (B) Tear secretion of the control group mice and Scop group mice was measured by phenol red cotton. The color of the cotton thread changes from yellow to red on absorbing tears. (C) Continuous subcutaneous injection of Scop for 5 days increased the expression of GRP78, PERK, and ATF6 among ER stress markers in mouse corneal tissue. (D) Representative immunofluorescence images of GRP78 in corneal tissue. Scale bar = 50 µm. (E) The expression of p-eIF2α, ATF4, and CHOP among the terminal-UPR markers in the cornea of mice in the Scop group was increased. (F) Representative immunofluorescence images of CHOP. Scale bar = 50 µm. Results are presented as mean ± SD (n = 4–6). *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Hyperosmotic Induced ER Stress and PERK-CHOP Branch Activation
Morphological Changes
Exposure to the 500 mOsm medium induced ER morphological changes in the HCE-2 cells. The ER tracker identified notable changes after 24 hours of treatment. Specifically, the continuity of the ER was disrupted around the nucleus because of fading of the circular signal (Supplementary Fig. S1). These results suggest that the 500 mOsm stress triggers pathological changes in the ER of HCECs. 
Changes in the Protein Expression Levels of Three UPR Branches
The hyperosmolarity treatment upregulated the GRP78 expression after 24 hours in the HCE-2 cells (Figs. 3A–3C). The expression levels of PERK, p-eIF2α, ATF4, and CHOP in the PERK-CHOP signaling pathway also increased, but IRE1α and ATF6 were not changed (see Figs. 3A, 3B). These effects also occurred in the primary HCECs (Figs. 3D, 3E). 
Figure 3.
 
Hyperosmotic 500 mOsm stress induces ER stress and upregulates terminal-UPR in HCE-2 cells and primary HCECs. (A) Western blot analysis of ER stress and terminal-UPR in HCE-2 cells with or without NaCl (312 or 500 mOsm) treatment. (B) Statistical analysis of the data shown in A. (C) Representative images showing immunofluorescent staining of GRP78 (green) in HCE-2 cells with or without NaCl (312 or 500 mOsm) treatment. Scale bar = 50 µm. (D) Western blot analysis of ER stress and terminal-UPR in primary HCECs with or without NaCl (312 or 500 mOsm) treatment. (E) Statistical analysis of the data shown in D. Results are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, **P < 0.001.
Figure 3.
 
Hyperosmotic 500 mOsm stress induces ER stress and upregulates terminal-UPR in HCE-2 cells and primary HCECs. (A) Western blot analysis of ER stress and terminal-UPR in HCE-2 cells with or without NaCl (312 or 500 mOsm) treatment. (B) Statistical analysis of the data shown in A. (C) Representative images showing immunofluorescent staining of GRP78 (green) in HCE-2 cells with or without NaCl (312 or 500 mOsm) treatment. Scale bar = 50 µm. (D) Western blot analysis of ER stress and terminal-UPR in primary HCECs with or without NaCl (312 or 500 mOsm) treatment. (E) Statistical analysis of the data shown in D. Results are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, **P < 0.001.
ER Stress Activates the ROS-NLRP3-IL-1β Signaling Pathway in HCECs
The 500 mOsm stress upregulated the UPR and ROS generation (Fig. 4A) and increased the activity of the ROS-NLRP3-IL-1β signaling pathway axis. When HCE-2 were treated with the ER stress inhibitor 4-PBA (Supplementary Fig. S2), the rises in ROS level were significantly inhibited (see Fig. 4A). In contrast, TM treatment of HCE-2 cells strongly activated the ER stress, and upregulated the ATF4 and CHOP protein expression levels (Supplementary Fig. S3) which was accompanied by a considerable rise in the ROS expression levels compared to those in the control group (see Fig. 4A). The results of Western blot analysis indicated that the expression levels of ROS-NLRP3-pro-IL-1β, and cleaved-IL-1β rose in TM-treated HCE-2 (Figs. 4E, 4F). In addition, exposure of the HCE-2 cells to the 500 mOsm hyperosmotic medium increased the protein expression levels of NLRP3, pro-IL-1β, and cleaved-IL-1β stress (Fig. 4C, 4D) whereas 4-PBA reduced the expression levels of these proteins (see Figs. 4C, 4D). 
Figure 4.
 
Hyperosmotic-induced ER stress activates the ROS-NLRP3-IL - 1β signaling pathway in HCE-2. (A) Representative images of the staining of ROS in HCECs: HCE-2 cells treated with 4-PBA under hyperosmolarity conditions and HCE-2 treated with TM under normal culture conditions. (B) Statistical analysis of the mean fluorescence intensity data presented in A. (C) Treatment with 4-PBA reversed the increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β induced by hyperosmolarity in HCE-2. (D) Statistical analysis of the data shown in C. (E) Treatment of HCE-2 cells with TM resulted in increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β. (F) Statistical analysis of the data shown in E. Results are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 4.
 
Hyperosmotic-induced ER stress activates the ROS-NLRP3-IL - 1β signaling pathway in HCE-2. (A) Representative images of the staining of ROS in HCECs: HCE-2 cells treated with 4-PBA under hyperosmolarity conditions and HCE-2 treated with TM under normal culture conditions. (B) Statistical analysis of the mean fluorescence intensity data presented in A. (C) Treatment with 4-PBA reversed the increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β induced by hyperosmolarity in HCE-2. (D) Statistical analysis of the data shown in C. (E) Treatment of HCE-2 cells with TM resulted in increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β. (F) Statistical analysis of the data shown in E. Results are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
TXNIP Mediates Hyperosmolarity-Induced Activation of the ROS-NLRP3-IL-1β Pathway
In order to determine whether hyperosmolarity-induced ER stress induces TXNIP to promote NLRP3-related inflammation in DED, Western blot analysis was used to assess if DED development in the mouse model and exposure to hyperosmolarity in HCEC increased TXNIP expression. Immunofluorescent staining showed that exposure to the 500 mOsm medium elevated both TXNIP and NLRP3 expression (Figs. 5A–5C) and they co-localized with one another in the HCE-2 (Fig. 5D). Subsequently, we also determined the effects of exposure to the 500 mOsm stress on the expression levels of NLRP3 and IL-1β after knocking down TXNIP expression. TXNIP siRNA transfection decreased the expression levels of TXNIP, NLRP3, and IL-1β, as well as the expression of cleaved-IL-1β induced by NLRP3 inflammasome activation (Supplementary Fig. S4Figs. 5E, 5F). Conversely, overexpression of TXNIP in HCE-2 promoted the expression of NLRP3, IL-1β, and cleaved-IL-1β (Figs. 5G, 5H). 
Figure 5.
 
TXNIP knockdown suppresses hyperosmolarity-induced activation of NLRP3 pathway in HCE-2 cells. (A–C) Western blot analysis of TXNIP in DED mice cornea (n = 4) and immortalized and primary HCECs with or without NaCl (312 or 500 mOsm) treatment (n = 3). (D) Co-immunofluorescent staining of NLRP3 (red) and TXNIP (green) in HCE-2 cells treated with hyperosmolarity is shown in representative images. Scale bar = 50 µm (n = 3). (E) TXNIP knockdown in HCE-2 resulted in decreased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β upon hyperosmolarity treatment (n = 3). (F) Statistical analysis of the data presented in B. (G) Overexpression of TXNIP in HCE-2 cells led to increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β (n = 3). (H) Statistical analysis of the data shown in G. Results are presented as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 5.
 
TXNIP knockdown suppresses hyperosmolarity-induced activation of NLRP3 pathway in HCE-2 cells. (A–C) Western blot analysis of TXNIP in DED mice cornea (n = 4) and immortalized and primary HCECs with or without NaCl (312 or 500 mOsm) treatment (n = 3). (D) Co-immunofluorescent staining of NLRP3 (red) and TXNIP (green) in HCE-2 cells treated with hyperosmolarity is shown in representative images. Scale bar = 50 µm (n = 3). (E) TXNIP knockdown in HCE-2 resulted in decreased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β upon hyperosmolarity treatment (n = 3). (F) Statistical analysis of the data presented in B. (G) Overexpression of TXNIP in HCE-2 cells led to increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β (n = 3). (H) Statistical analysis of the data shown in G. Results are presented as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
PERK-CHOP Signaling Pathway Activation Enhances TXNIP Expression
To investigate whether the PERK-CHOP signaling pathway mediates TXNIP expression, we first confirmed that 4-PBA suppression of hyperosmotic-induced UPR increases was associated with a corresponding reduction in TXNIP expression. As shown in Figures 6A and 6C, this expectation was validated. We then confirmed that TM-induced upregulation of ER stress was accompanied by a similar increase in TXNIP expression in HCE-2 cells (Figs. 6B and 6D). Finally, we observed that the expression levels of p-eIF2α, ATF4, CHOP, and TXNIP all decreased following PERK knockdown (Supplementary Fig. S5Figs. 6E, 6F). 
Figure 6.
 
PERK-CHOP signaling pathway in the terminal-UPR upregulates TXNIP expression. (A) Treatment with 4-PBA reversed the increased expression of TXNIP induced by hyperosmolarity in HCE-2 cells. (C) Statistical analysis of the data shown in A. (B) Treatment of HCE-2 cells with TM resulted in increased expression of TXNIP. (D) Statistical analysis of the data shown in B. (E) Suppression of PERK in HCE-2 cells resulted in inhibition of hyperosmolarity-induced increase in TXNIP expression. (F) Statistical analysis of the data are presented as mean ± SD (n = 3). *P < 0.05, ***P < 0.001, ****P < 0.0001.
Figure 6.
 
PERK-CHOP signaling pathway in the terminal-UPR upregulates TXNIP expression. (A) Treatment with 4-PBA reversed the increased expression of TXNIP induced by hyperosmolarity in HCE-2 cells. (C) Statistical analysis of the data shown in A. (B) Treatment of HCE-2 cells with TM resulted in increased expression of TXNIP. (D) Statistical analysis of the data shown in B. (E) Suppression of PERK in HCE-2 cells resulted in inhibition of hyperosmolarity-induced increase in TXNIP expression. (F) Statistical analysis of the data are presented as mean ± SD (n = 3). *P < 0.05, ***P < 0.001, ****P < 0.0001.
Figure 7.
 
Hypertonic-induced ER stress increases ROS production, augments the PERK-CHOP pathway of terminal-UPR and ultimately triggers activation of TXNIP/NLRP3 in corneal epithelial cells.
Figure 7.
 
Hypertonic-induced ER stress increases ROS production, augments the PERK-CHOP pathway of terminal-UPR and ultimately triggers activation of TXNIP/NLRP3 in corneal epithelial cells.
Discussion
Single-cell RNA-sequencing data analysis revealed that ER stress and terminal-UPR induction contribute to the pathogenesis of DED. This relationship is confirmed in both the desiccating mouse model and the hyperosmotic in vitro model, demonstrating that induction of UPR by ER stress increases reactive ROS production. Importantly, TXNIP was identified as a mediator involved in PERK-CHOP activation, which is associated with ER stress induced by desiccating conditions in DED. These findings suggest that the PERK-CHOP axis and TXNIP may play a role in the pathogenesis of DED and warrant further research to explore their potential as therapeutic targets. 
The signaling pathways were identified through which ER-induced UPR activation mediates DED. The induction of an increase in the PERK-CHOP pathway induced by activation of UPR-linked signaling was confirmed by demonstrating that the changes induced by ER stress mirrored those induced by TM, an established UPR activator. These results make sense, because the ocular surface epithelial cells are often stressed by different external stimulation conditions,3841 enabling them to serve as primary sensors responding to ER stress. In addition, ER stress is activated under various pathological changes in the cornea and conjunctiva.4244 Notably, administration of TM in mice without DED can cause dry eye-like ocular surface damage, including corneal epithelial injury and reduced tear secretion.45 These results suggest that drug targeting a mediator of UPR activation may improve therapeutic management of DED development. 
Our results suggest that ER stress-induced UPR activation mediates hyperosmotic-induced rises in ROS production in corneal epithelial cells. Hyperosmolarity is a well-recognized clinical hallmark of DED.46 The use of scopolamine to induce dry eye conditions has been well-documented, demonstrating significant alterations in tear film stability and increased tear osmolality due to reduced tear production and increased evaporation.47 Previous research has demonstrated that exposing limbal stem cell lines to a hyperosmotic condition of 450 mOsm for 24 hours effectively induces ER stress, whereas another study showed a significant increase in ROS production and inflammation in corneal epithelial cells under 500 mOsm condition for 24 hours.48,49 Such control involves complex crosstalk between pathways controlling inflammation and oxidative stress.50,51 ROS is a byproduct of cellular metabolism, mainly derived from various organelles of mitochondria and others.52 Oxidative stress-mediated inflammation is a crucial factor in triggering the development of DED, and mediating the “inflammatory vicious cycle.”41,5355 Consistent with previous studies,10,53 we found ROS production rose, which accompanied NLRP3 inflammasome activation in DED mouse models and hyperosmotic cell models. ER stress regulates both NLRP3 expression and activation , through different mechanisms, including oxidative stress, ER calcium leakage, and CHOP-dependent NF-κB activation.5658 In multiple studies, 4-PBA was shown to effectively suppress ER stress.59,60 We found that 4-PBA reduces ROS production and NLRP3 inflammasome activation, along with a decline in IL-1β expression. This suggests that the increase in ROS production caused by high osmotic pressure may be related to ER stress. These results indicate that 4-PBA, a United States Food and Drug Administration (FDA)-approved drug,61,62 may serve as a potential therapeutic agent for treating DED by targeting ER stress inhibition. 
TXNIP is a crucial mediator linking the activation of the PERK-CHOP signaling pathway to oxidative stress and inflammation in DED. We showed that PERK knockdown suppressed the PERK-CHOP pathway, which in turn inhibits TXNIP/NLRP3 inflammasome-associated inflammation in DED. Moreover, silencing TXNIP not only impedes its interaction with NLRP3 and subsequent inflammasome assembly, but it also suppresses the upregulation of NLRP3 expression.63,64 These observations suggest that TXNIP is a point of convergence of different types of triggers, and its response in turn modulates the effects of oxidative stress and ER stress on the signaling pathways, that regulate inflammatory processes underlying DED. 
In conclusion, characterizing the role of the PERK-CHOP branch in the terminal-UPR pathway unravels the contribution of ER to both UPR stress and DED progression. Furthermore, insights are provided into how the terminal-UPR controls oxidative stress and NLRP3-IL-1β mediated inflammation in DED (Fig. 7). Additional studies are warranted to determine if drug targeting the PERK-CHOP signaling pathway is a viable approach to improving the therapeutic management of DED. 
Acknowledgments
The authors thank the financial support provided by the National Natural Science Foundation of China (82371035 and 82171021); Zhejiang Province Medical Science and Technology Project (2024KY1595); Ningbo Top Medical and Health Research Program (No. 2023030716). The authors would like to thank Wenwen Zeng for the language polishing. Moreover, the funder had no role in the study design, data collection, analysis, interpretation, or the decision to submit this manuscript for publication. 
Availability of Data and Materials: The single-cell dataset presented in this study can be found in the GEO online database under the accession number GSE182582. The original contributions are included in the article. Further inquiries may be made to the corresponding author. 
Disclosure: Z. Zha, None; D. Xiao, None; Z. Liu, None; F. Peng, None; X. Shang, None; Z. Sun, None; Y. Liu, None; W. Chen, None 
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Figure 1.
 
Augmentation and characterization of the UPR in a DED mice model. (A) UMAP plot of mice corneal epithelial cells showing major cell types in control (Ctrl) and DED groups. LSC, limbal stem cell; TAC, transit amplifying cell; Squam, squamous; Conj, conjunctiva. (B) Differences in pathway activities determined by Gene set enrichment analysis (GSEA) between DED and Ctrl groups based on the HALLMARK database. The pathway scores are normalized, with a threshold established at padj < 0.5 and absolute NES > 1.2. (C) Heatmap of the expression level of terminal-UPR signature genes across each subtype of stem/progenitor cells. Corresponding box plots for gene scoring analysis based on terminal-UPR signature genes for different cell types are plotted above the heatmap. The P values were determined by the Wilcoxon test. ****P ≤ 0.0001. (D) UMAP plot of terminal-UPR signature scores calculated by the AddModuleScore function.
Figure 1.
 
Augmentation and characterization of the UPR in a DED mice model. (A) UMAP plot of mice corneal epithelial cells showing major cell types in control (Ctrl) and DED groups. LSC, limbal stem cell; TAC, transit amplifying cell; Squam, squamous; Conj, conjunctiva. (B) Differences in pathway activities determined by Gene set enrichment analysis (GSEA) between DED and Ctrl groups based on the HALLMARK database. The pathway scores are normalized, with a threshold established at padj < 0.5 and absolute NES > 1.2. (C) Heatmap of the expression level of terminal-UPR signature genes across each subtype of stem/progenitor cells. Corresponding box plots for gene scoring analysis based on terminal-UPR signature genes for different cell types are plotted above the heatmap. The P values were determined by the Wilcoxon test. ****P ≤ 0.0001. (D) UMAP plot of terminal-UPR signature scores calculated by the AddModuleScore function.
Figure 2.
 
The protein level of ER stress and terminal-UPR increased in the cornea of DED mouse. (A) A mouse DED model was successfully established, with representative pictures of CFS of control group mice and Scop group mice and statistical results of CFS. (B) Tear secretion of the control group mice and Scop group mice was measured by phenol red cotton. The color of the cotton thread changes from yellow to red on absorbing tears. (C) Continuous subcutaneous injection of Scop for 5 days increased the expression of GRP78, PERK, and ATF6 among ER stress markers in mouse corneal tissue. (D) Representative immunofluorescence images of GRP78 in corneal tissue. Scale bar = 50 µm. (E) The expression of p-eIF2α, ATF4, and CHOP among the terminal-UPR markers in the cornea of mice in the Scop group was increased. (F) Representative immunofluorescence images of CHOP. Scale bar = 50 µm. Results are presented as mean ± SD (n = 4–6). *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 2.
 
The protein level of ER stress and terminal-UPR increased in the cornea of DED mouse. (A) A mouse DED model was successfully established, with representative pictures of CFS of control group mice and Scop group mice and statistical results of CFS. (B) Tear secretion of the control group mice and Scop group mice was measured by phenol red cotton. The color of the cotton thread changes from yellow to red on absorbing tears. (C) Continuous subcutaneous injection of Scop for 5 days increased the expression of GRP78, PERK, and ATF6 among ER stress markers in mouse corneal tissue. (D) Representative immunofluorescence images of GRP78 in corneal tissue. Scale bar = 50 µm. (E) The expression of p-eIF2α, ATF4, and CHOP among the terminal-UPR markers in the cornea of mice in the Scop group was increased. (F) Representative immunofluorescence images of CHOP. Scale bar = 50 µm. Results are presented as mean ± SD (n = 4–6). *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 3.
 
Hyperosmotic 500 mOsm stress induces ER stress and upregulates terminal-UPR in HCE-2 cells and primary HCECs. (A) Western blot analysis of ER stress and terminal-UPR in HCE-2 cells with or without NaCl (312 or 500 mOsm) treatment. (B) Statistical analysis of the data shown in A. (C) Representative images showing immunofluorescent staining of GRP78 (green) in HCE-2 cells with or without NaCl (312 or 500 mOsm) treatment. Scale bar = 50 µm. (D) Western blot analysis of ER stress and terminal-UPR in primary HCECs with or without NaCl (312 or 500 mOsm) treatment. (E) Statistical analysis of the data shown in D. Results are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, **P < 0.001.
Figure 3.
 
Hyperosmotic 500 mOsm stress induces ER stress and upregulates terminal-UPR in HCE-2 cells and primary HCECs. (A) Western blot analysis of ER stress and terminal-UPR in HCE-2 cells with or without NaCl (312 or 500 mOsm) treatment. (B) Statistical analysis of the data shown in A. (C) Representative images showing immunofluorescent staining of GRP78 (green) in HCE-2 cells with or without NaCl (312 or 500 mOsm) treatment. Scale bar = 50 µm. (D) Western blot analysis of ER stress and terminal-UPR in primary HCECs with or without NaCl (312 or 500 mOsm) treatment. (E) Statistical analysis of the data shown in D. Results are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, **P < 0.001.
Figure 4.
 
Hyperosmotic-induced ER stress activates the ROS-NLRP3-IL - 1β signaling pathway in HCE-2. (A) Representative images of the staining of ROS in HCECs: HCE-2 cells treated with 4-PBA under hyperosmolarity conditions and HCE-2 treated with TM under normal culture conditions. (B) Statistical analysis of the mean fluorescence intensity data presented in A. (C) Treatment with 4-PBA reversed the increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β induced by hyperosmolarity in HCE-2. (D) Statistical analysis of the data shown in C. (E) Treatment of HCE-2 cells with TM resulted in increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β. (F) Statistical analysis of the data shown in E. Results are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 4.
 
Hyperosmotic-induced ER stress activates the ROS-NLRP3-IL - 1β signaling pathway in HCE-2. (A) Representative images of the staining of ROS in HCECs: HCE-2 cells treated with 4-PBA under hyperosmolarity conditions and HCE-2 treated with TM under normal culture conditions. (B) Statistical analysis of the mean fluorescence intensity data presented in A. (C) Treatment with 4-PBA reversed the increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β induced by hyperosmolarity in HCE-2. (D) Statistical analysis of the data shown in C. (E) Treatment of HCE-2 cells with TM resulted in increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β. (F) Statistical analysis of the data shown in E. Results are presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 5.
 
TXNIP knockdown suppresses hyperosmolarity-induced activation of NLRP3 pathway in HCE-2 cells. (A–C) Western blot analysis of TXNIP in DED mice cornea (n = 4) and immortalized and primary HCECs with or without NaCl (312 or 500 mOsm) treatment (n = 3). (D) Co-immunofluorescent staining of NLRP3 (red) and TXNIP (green) in HCE-2 cells treated with hyperosmolarity is shown in representative images. Scale bar = 50 µm (n = 3). (E) TXNIP knockdown in HCE-2 resulted in decreased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β upon hyperosmolarity treatment (n = 3). (F) Statistical analysis of the data presented in B. (G) Overexpression of TXNIP in HCE-2 cells led to increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β (n = 3). (H) Statistical analysis of the data shown in G. Results are presented as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 5.
 
TXNIP knockdown suppresses hyperosmolarity-induced activation of NLRP3 pathway in HCE-2 cells. (A–C) Western blot analysis of TXNIP in DED mice cornea (n = 4) and immortalized and primary HCECs with or without NaCl (312 or 500 mOsm) treatment (n = 3). (D) Co-immunofluorescent staining of NLRP3 (red) and TXNIP (green) in HCE-2 cells treated with hyperosmolarity is shown in representative images. Scale bar = 50 µm (n = 3). (E) TXNIP knockdown in HCE-2 resulted in decreased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β upon hyperosmolarity treatment (n = 3). (F) Statistical analysis of the data presented in B. (G) Overexpression of TXNIP in HCE-2 cells led to increased expression of NLRP3, pro-IL-1β, and cleaved-IL-1β (n = 3). (H) Statistical analysis of the data shown in G. Results are presented as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001.
Figure 6.
 
PERK-CHOP signaling pathway in the terminal-UPR upregulates TXNIP expression. (A) Treatment with 4-PBA reversed the increased expression of TXNIP induced by hyperosmolarity in HCE-2 cells. (C) Statistical analysis of the data shown in A. (B) Treatment of HCE-2 cells with TM resulted in increased expression of TXNIP. (D) Statistical analysis of the data shown in B. (E) Suppression of PERK in HCE-2 cells resulted in inhibition of hyperosmolarity-induced increase in TXNIP expression. (F) Statistical analysis of the data are presented as mean ± SD (n = 3). *P < 0.05, ***P < 0.001, ****P < 0.0001.
Figure 6.
 
PERK-CHOP signaling pathway in the terminal-UPR upregulates TXNIP expression. (A) Treatment with 4-PBA reversed the increased expression of TXNIP induced by hyperosmolarity in HCE-2 cells. (C) Statistical analysis of the data shown in A. (B) Treatment of HCE-2 cells with TM resulted in increased expression of TXNIP. (D) Statistical analysis of the data shown in B. (E) Suppression of PERK in HCE-2 cells resulted in inhibition of hyperosmolarity-induced increase in TXNIP expression. (F) Statistical analysis of the data are presented as mean ± SD (n = 3). *P < 0.05, ***P < 0.001, ****P < 0.0001.
Figure 7.
 
Hypertonic-induced ER stress increases ROS production, augments the PERK-CHOP pathway of terminal-UPR and ultimately triggers activation of TXNIP/NLRP3 in corneal epithelial cells.
Figure 7.
 
Hypertonic-induced ER stress increases ROS production, augments the PERK-CHOP pathway of terminal-UPR and ultimately triggers activation of TXNIP/NLRP3 in corneal epithelial cells.
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