Etomidate protects retinal ganglion cells from hydrogen peroxide-induced injury via Nrf2/HO-1 pathway.

Abstract

Aim: To determine whether etomidate (ET) has a protective effect on retinal ganglion cells (RGCs) injured with hydrogen peroxide (H₂O₂) and to explore the potential mechanism underlying the antioxidative stress effect of ET.

Methods: Cultured RGCs were identified by double immunofluorescent labeling of microtubule-associated protein 2 and Thy1.1. An injury model of H₂O₂-induced RGCs oxidative stress was established in vitro. Cells were pretreated with different concentrations of ET (1, 5, and 10 µmol/L) for 4h, followed by further exposure to H₂O₂ at 1000 µmol/L. Cell counting kit 8 and Annexin V/propidium iodide assays were applied to detect the viabilities and apoptosis rates of the RGCs at 12, 24, and 48h after H₂O₂ stimulation. The levels of nitric oxide, malondialdehyde, and glutathione in culture media were measured at these time points. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were performed to observe the effects of ET on the messenger RNA and protein expression of inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), glutathione peroxidase 1 and the level of conjugated acrolein in RGCs at 12, 24, and 48h after H₂O₂ stimulation and in the retina at 12h after optic nerve transection (ONT).

Results: The applications of 5 and 10 µmol/L of ET significantly increased the viability of RGCs. Results from qRT-PCR indicated a decrease in the expression of iNOS and an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs at 12, 24 and 48h after H₂O₂ stimulation, as well as in ET-treated retinas at 12h after ONT. Western blot analysis revealed a decrease in the expression of iNOS and levels of conjugated acrolein, along with an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs in vitro and ET-treated retinas in vivo.

Conclusion: ET is a neuroprotective agent in primary cultured RGCs injured by H₂O₂. The effect of ET is dose-dependent with the greatest effect being at 10 µmol/L. ET plays an antioxidant role by inhibiting iNOS, up-regulating Nrf2/HO-1, decreasing the production of acrolein, and increasing the scavenge of acrolein.