Role Of Neuronal Amide - Endoplasmic Reticulum Stress - AMPK In Apoptosis Of Retinal Pigment Epithelial Cells And Its Mechanism

Objective:To investigate the role of ceramide, endoplasmic reticulum stress and AMPK in the apoptosis of retinal pigment epithelium induced by oxidative stress.Methods:(1) The apoptotic model of cultured RPE cells was build up with H2O2(200μM). We tested the effects of H2O2/UV on AMPK signaling changes in cultured RPE cells by western blot. The apoptosis was detected by TUNEL in the RPE cells which were pretreated with AMPK activator AICAR(1mM) and inhibitor Compound C (CC)(10μM).(2) We tested the effects of H2O2/UV on endoplasmic reticulum stress changes in cultured RPE cells by western blot. The apoptosis was detected by TUNEL in the RPE cells which were pretreated with endoplasmic reticulum stress inhibitor Salubrinal (Sal)(10μM).(3) In order to confirmed the role of ceramide on apoptosis of oxidation-induced RPE cells, we firstly detected the ceramide content of H2O2/UV treated RPE cells by autoradiography, and then the apoptosis was conducted by TUNEL after adding C6ceramide(1μg/mL), de novo synthesis of neuraminidase inhibitors fumonisin B1(FB-1)(10μM) to RPE cells.(4) The activation of AMPK was evaluated by Western blot in the RPE cells which were pretreated with endoplasmic reticulum stress inhibitors Sal. The endoplasmic reticulum stress and the activation of AMPK were both tested by Western blot after the RPE cells were added the C6ceramide pretreatment. Results:(1) UV and H2O2induced an early ceramide production, profound ER stress and AMPK activation. Inhibitors against ER stress (salubrinal), ceramide production (FB-1) and AMPK activation (compound C) suppressed UV/H2O2induced RPE cell apoptosis. C6ceramide and AMPK activator AICAR promoted RPE cell apoptosis.(2) C6profound ER stress and AMPK activation, Inhibitors against ER stress (salubrinal) suppressed AMPK activation.Conclusion:UV/H2O2activates ceramide-ER stress-AMPK signaling axis to promote RPE cell apoptosis.