Etomidate protects retinal ganglion cells from H 2 O 2 - induced injured via Nrf2/HO-1 pathway

Abstract Background Our previous research provided an experimental basis for etomidate (ET) to treat optic nerve (ON) injuries. However, the mechanism of ET action in anti-oxidative stress remains unclear and requires further investigation. This study aimed to determine whether ET has a protective effect on retinal ganglion cells (RGCs) injured by H2O2 and to explore ET's possible anti-oxidative stress mechanism. Methods Cultured RGCs were identified using the double immunofluorescent labeling of Thy1.1 and Microtubule association protein-2. An H2O2-induced RGCs oxidative stress injury model was established in vitro. Cells were pretreated with different concentrations of ET (1, 5, and 10 µmol/L) for 4 hours, followed by further exposure to H2O2 at 1000 µmol/L. CCK-8 and Annexin V/PI assay were applied to detect the RGCs viability and apoptosis rate at 12, 24, and 48 hours after H2O2 stimulation. The levels of nitric oxide (NO), malondialdehyde (MDA), and glutathione (GSH) in culture media were measured at these time points. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to observe the effects of ET on the mRNA and protein expressions of nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase1 (GPX1) and acrolein in RGCs at 12, 24, and 48 hours after H2O2 stimulation and in the retina at 12 hours after optic nerve transection (ONT). Results The viability of RGCs was increased significantly with 5 and 10 µM of ET. The qRT-PCR showed that iNOS expression was decreased and Nrf2 and HO-1 expressions were increased in RGCs and retinas with ET. The Western blot showed that the expressions of iNOS and acrolein were decreased, and the expressions of Nrf2 and HO-1 were increased in RGCs with ET at 12, 24, and 48 hours after H2O2 stimulation and in the retina with ET at 12 hours after ONT. Conclusion ET has a neuroprotective effect in primary cultured RGCs injured by H2O2. The effect of ET was dose-dependent being greatest at 10 µM. ET plays an antioxidant role by inhibiting iNOS, up-regulating Nrf2/HO-1, decreasing the production of acrolein, and increasing acrolein scavenging.