The Effect Of Receptor Interacting Protein3on Oxygen Glucose Deprivation (OGD)-induced RGC-5Necroptosis

OBJECTIVE:To investigate the expression of Receptor interacting protein3(RIP3) in retinal ganglion cell-5(RGC-5) in the process of necroptosis after oxygen glucose deprivation. Preliminarily, we intend to explore the possible effect and mechanism of RIP3in oxygen glucose deprivation (OGD)-induced RGC-5necroptosis.METHOD:(1) RGC-5were revived from being frozen in liquid nitrogen, after the cells were cultivated to80%density in culture dish, cells were chosen from the third to eighth passages.(2) All the cells were grouped and cultivated in thermostat to80%density in culture dish, to induce OGD, cells were washed with glucose-free DMEM, then placed in the same medium in a hypoxic incubator (95%N2,5%CO2) for8h. At the end of the OGD period, cells were washed with high-glucose DMEM, and the cultures were put back in the incubator at the regular atmospheric oxygen level.(3) Necroptosis indueced by OGD was characterized by PI staining and double staining with Annexin V and PI (analysis through flow cytometry).(4) The protein level of RIP3after OGD was detected by western blot.(5) Morpholino oligo and endoporter were added into the medium to prepare RIP3knock-down RGC-5cell line (RIP3i-RGC-5). Double staining with Annexin V and PI, amounts of malondialdehyde (MDA) in RGC-5were used to explore the possible effect and down-stream mechanism of RIP3in OGD-induced RGC-5necroptosis.RESULTS:(1) PI staining showed that there was more necrotic cell death following OGD. Flow cytometry exhibited that increased ratio of necrotic cells (6hours after the duration of OGD for8hours), however, pretreatment with10μmol/L nec-1(specific inhibitor of necroptosis) for24hours, the ratio of necrotic cells remarkable decreased.(2) Western blot demonstrated that RIP3protein levels increased following OGD.(3) Flow cytometry exhibited that the ratio of necrotic RIP3i-RGC-5was lower than normal model following OGD.(4) MDA concentration of normal model group and RIP3i-RGC-5group significantly increase following OGD compare with normal control group, meanwhile, MDA concentration of normal model group was much higher than RIP3i-RGC-5group.CONCLUSION:There existed necroptosis of RGC-5following OGD, and mediated by RIP3via the increase of oxidative stress.