Tert-butyl hydroperoxide induces trabecular meshwork cells injury through ferroptotic cell death

Abstract

Trabecular meshwork (TM) tissue has a crucial role in regulating aqueous humor circulation in the eye, thus maintaining normal intraocular pressure (IOP). TM dysfunction causes IOP elevation, which leads to glaucoma. To investigate biological changes in TM tissue in patients with glaucoma, we analyzed the mRNA expression microarray dataset, GSE27276. Gene ontology analysis indicated that redox microenvironment imbalance is among the main changes of TM tissue in patients with glaucoma. Subsequently, we induced oxidative stress in TM cells using the tert-butyl hydroperoxide (tBHP) treatment, to generate in vivo and in vitro models, and conducted mRNA sequencing to identify genes with critical roles in maintaining the redox microenvironment balance. We found that the tBHP caused TM dysfunction in vivo, characterized by aqueous humor circulation resistance, IOP elevation, and TM cell death. Further, Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that ferroptosis signaling was enriched in tBHP-treated TM cells. Consistently, in vitro analyses showed that levels of reactive oxygen species, ferric ion, and malondialdehyde were increased after the tBHP treatment, indicating TM cell ferroptosis. Furthermore, inhibiting ferroptosis alleviated tBHP-induced TM cell injury. This study provides new insights suggesting that inhibition of ferroptosis has potential as a treatment for glaucoma.

Keywords: ferroptosis; glaucoma; oxidative stress; trabecular meshwork.

FIGURE 1

Oxidative stress correlates with trabecular meshwork injury. (A). Volcano plot of differentially expressed genes (DEGs) between POAG and control samples in the GSE27276 dataset. (B). Gene ontology analysis of DEGs from GSE27276. (C). Gadolinium‐enhanced magnetic resonance images from vehicle (n = 4) and tBHP‐treated mice (n = 6) showing resistance of aqueous humor circulation. (D). Gd signal was significantly increased in tBHP‐treated mice by pixel intensity evaluation in the anterior chamber angle of (C) using Image J software. E. Intraocular pressure measurement in vehicle (n = 8) and tBHP‐treated mice (n = 8). Statistical analyses were determined using Student's t‐test. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Tert‐butyl hydroperoxide (tBHP) has no effect on anterior chamber angle structure but induces trabecular meshwork (TM) stiffness, fibrosis and apoptosis. (A). Slit lamp examination (up) and Hematoxylin and Eosin (HE) staining (down) of vehicle (n = 3) and tBHP‐treated mice (n = 3), showing normal anterior chamber structure but TM stiffness. (B and C), Representative images of α‐SMA (B) and TUNEL (C) staining in TM sections from vehicle (n = 3) and tBHP‐treated mice (n = 3), indicating TM fibrosis and apoptosis.

FIGURE 3

Tert‐butyl hydroperoxide (tBHP) induces trabecular meshwork (TM) cell injury. (A). Cell viability analysis of TM cells with (n = 5) or without tBHP treatment (n = 5) by CCK8 assay. (B). Reactive oxygen species detection in TM cells with (n = 3) or without tBHP treatment (n = 3) by flow cytometry. (C). Detection of TM cell apoptosis with (n = 3) or without tBHP treatment (n = 3) by flow cytometry. Statistical analyses was performed using Student's t‐test. ***p < 0.001.

FIGURE 4

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) in trabecular meshwork (TM) cells after tert‐butyl hydroperoxide (tBHP) treatment. (A). Heatmap of DEGs in TM cells with or without tBHP treatment. B–C, GO analysis showing the top 25 pathways enriched for genes up‐regulated (B) and down‐regulated (C) in tBHP‐treated TM cells. D–E, KEGG pathway enrichment analysis of genes up‐regulated (D) and down‐regulated (E) in tBHP‐treated TM cells. F. Heatmap of ferroptosis‐related genes in TM cells with or without tBHP treatment.

FIGURE 5

Deferiprone (DFP) effectively inhibits trabecular meshwork (TM) cell ferroptosis induced by tert‐butyl hydroperoxide (tBHP). (A). Measurement of ferric ion concentrations in TM cells with (n = 3) or without tBHP (n = 3) treatment. (B). Measurement of malondialdehyde concentrations in TM cells with (n = 3) or without tBHP (n = 3) treatment. (C). mRNA levels of SLC7A11, TFRC, HMOX1, and GPX4 analyzed by quantitative real‐time polymerase chain reaction in TM cells with (n = 3) or without tBHP (n = 3) treatment. (D). Protein levels of SLC7A11, TFRC, HMOX1, and GPX4 analyzed by western blot in TM cells with (n = 3) or without tBHP (n = 3) treatment. (E). Cell viability analysis of vehicle (n = 4), tBHP (n = 4) and tBHP + DFP (n = 4) group determined by CCK8 assay. (F). ROS levels of vehicle (n = 3), tBHP (n = 3) and tBHP + DFP (n = 3) group determined by flow cytometry. The Student's t‐test was used to compare two groups and one‐way ANOVA analysis was applied for comparisons of more than two groups. p < 0.05 was considered significant. **p < 0.01; ***p < 0.001.