Effects of Brimonidine, Omidenepag Isopropyl, and Ripasudil Ophthalmic Solutions to Protect against H₂O₂-Induced Oxidative Stress in Human Trabecular Meshwork Cells

Abstract

Purpose

We investigated whether hydrogen peroxide (H₂O₂)-induced oxidative stress causes human trabecular meshwork (HTM) cell dysfunction observed in open angle glaucoma (OAG) in vitro, and the effects of topical glaucoma medications on oxidative stress in HTM cells.

Methods

We used commercially available ophthalmic solutions of brimonidine, omidenepag isopropyl, and ripasudil in the study. HTM cells were exposed to H₂O₂ for 1 h, with or without glaucoma medications. We assessed cell viability and senescence via WST-1 and senescence-associated-β-galactosidase (SA-β-Gal) activity assays. After exposure to H₂O₂ and glaucoma medications, we evaluated changes in markers of fibrosis and stress by using real-time quantitative polymerase chain reaction (qPCR) to measure the mRNA levels of collagen type I alpha 1 chain (COL1A1), fibronectin, alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), endoplasmic reticulum stress markers of C/EBP homologous protein (CHOP), 78-kDa glucose-regulated protein (GRP78), and splicing X-box binding protein-1 (sXBP-1).

Results

HTM cell viability decreased and SA-β-Gal activity increased significantly after exposure to H₂O₂. Treatment with three ophthalmic solutions attenuated these changes. Real-time qPCR revealed that H₂O₂ upregulated the mRNA levels of COL1A1, fibronectin, α-SMA, CHOP, GRP78, and sXBP-1, whereas it downregulated MMP-2 mRNA expression significantly. Brimonidine suppressed the upregulation of stress markers CHOP and GRP78. Additionally, omidenepag isopropyl and ripasudil decreased the upregulation of COL1A1 and sXBP-1. Furthermore, ripasudil significantly suppressed fibrotic markers fibronectin and α-SMA, compared with the other two medications.

Conclusion

In vitro, H₂O₂ treatment of HTM cells induced characteristic changes of OAG, such as fibrosis changes and the upregulation of stress markers. These glaucomatous changes were attenuated by additional treatments with brimonidine, omidenepag isopropyl, and ripasudil ophthalmic solutions.

Keywords:

• Human trabecular meshwork cells
• ophthalmic solutions
• oxidative stress
• endoplasmic reticulum stress
• hydrogen peroxide

Acknowledgements

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Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 22K09807 and JSPS KAKENHI Grant Number 20H03839.