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NFE2L2 activator RS9 protects against corneal epithelial cell damage in dry eye models
Yuka Matsuda1, Mamiko Machida1, Yasuhiro Nakagami2
1Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
Abstract:
Oxidative stress may cause ocular surface damage during the development of dry eye. Mammalian cells have defense systems against oxidative stress. A central regulator of the stress response is nuclear factor-erythroid 2-related factor 2 (NFE2L2). NFE2L2 is activated by the novel triterpenoid RS9 (a biotransformation compound of RTA 402). The purpose of this study was to assess the efficacy of RS9 against dry eye using in vitro and in vivo models. Bioactivity was estimated by the induction of mRNAs for two NFE2L2-targeted genes: NQO1 (prevents radical species) and GCLC (glutathione synthesis), using a corneal epithelial cell line (HCE-T). Protection against oxidation and cell damage was tested in vitro by culturing cells under hyperosmotic stress or by the addition of menadione, a generator of reactive oxygen species (ROS). Dry eye in vivo was induced by the injection of scopolamine into rats. Then, 930 nM of RS9 was applied to both eyes for 2 weeks. Oxidative stress was measured by the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Corneal wound healing was measured by scoring for superficial punctate keratitis (SPK). Corneal epithelial cell densities were evaluated histologically. RS9 and RTA 402 induced the expression of NQO1 and GCLC mRNAs in HCE-T cells. And both compounds suppressed hyperosmotic-ROS generation and menadione induced cellular damage. However RS9 had a stronger protective effect than RTA 402. Ocular instillation of RS9 also significantly upregulated the expression of Nqo1 mRNA in the corneal epithelium. Accumulation of 8-OHdG, increase of SPK scores and decrement of basal cell density were observed in corneal epithelium from scopolamine-injected rats. These changes were significantly ameliorated by the topical administration of RS9. RS9 induced Nfe2l2 activation and Nfe2l2-targeted genes, reduced oxidation, and ameliorated symptoms of dry eye using in vitro and in vivo models. Thus, RS9 might be a potent candidate agent against dry eye disease.
Insights
The novel compound RS9 effectively combats dry eye disease by activating cellular defenses against oxidative stress. This study demonstrates RS9
Area of Science:
- Ophthalmology
- Cell Biology
- Pharmacology
Background:
- Oxidative stress contributes to ocular surface damage in dry eye disease.
- Mammalian cells possess defense mechanisms against oxidative stress, regulated by nuclear factor-erythroid 2-related factor 2 (NFE2L2).
- The triterpenoid RS9, derived from RTA 402, activates NFE2L2.
Purpose of the Study:
- To evaluate the efficacy of RS9 in treating dry eye using in vitro and in vivo models.
- To assess RS9's ability to activate NFE2L2 and its downstream genes.
- To determine RS9's protective effects against oxidative stress and cellular damage in ocular models.
Main Methods:
- Assessed NFE2L2 bioactivity by measuring mRNA induction of NQO1 and GCLC in corneal epithelial cells (HCE-T).
- Tested in vitro protection against hyperosmotic stress and menadione-induced reactive oxygen species (ROS) and cell damage.
- Induced dry eye in rats via scopolamine injection and administered topical RS9 for 2 weeks, measuring oxidative stress (8-OHdG), corneal wound healing (SPK), and cell density.
Main Results:
- RS9 and RTA 402 induced NQO1 and GCLC mRNA expression in HCE-T cells.
- Both compounds reduced hyperosmotic-induced ROS generation and menadione-induced cellular damage, with RS9 showing a stronger effect.
- Topical RS9 upregulated Nqo1 mRNA in rat corneal epithelium, ameliorated 8-OHdG accumulation, reduced SPK scores, and improved cell density in a scopolamine-induced dry eye model.
Conclusions:
- RS9 activates NFE2L2 and its target genes, reducing oxidative stress in ocular tissues.
- RS9 demonstrates significant protective effects against dry eye symptoms and pathology in both in vitro and in vivo models.
- RS9 shows potential as a therapeutic agent for dry eye disease due to its antioxidant and NFE2L2-activating properties.
