Necroptosis is a type of regulated cell death that has been implicated in various diseases.Receptor-interacting protein 3 (RIP3),a member of the RIP family,is an important mediator of the necroptotic pathway.Cleavage of RIP3 at Asp328 by caspase-8 abolishes the kinase activity of RIP3,which is critical for necroptosis.Moreover,RIP3 is significantly upregulated during the early stages of acute high intra-ocular pressure and oxygen glucose deprivation.In this study,the effects of RIP3 during elevated hydrostatic pressure (EHP) were investigated and the possible mechanism through which caspase-8 regulated RIP3 cleavage was explored.Flow cytometry analysis revealed that the number of EHP-induced necrotic retinal ganglion cell 5 (RGC-5) cells wasreduced after RIP3-knockdown.Furthermore,malondialdehyde (MDA) levels and glycogen phosphorylase (PYGL) activity in normal RGC-5 cells were much higher than those in RIP3-knockdown cells after EHP.EHP-induced RGC-5 necrosis was significantly reduced after treatment with butylated hydroxyanisole (BHA),a reactive oxygen species (ROS) scavenger.MDA levels and PYGL activity were lower in normal RGC-5 cells than those in cells with caspase-8 inhibition after EHP.Weste blot analysis demonstrated that the RIP3 cleavage product was upregulated in cells with caspase-8 inhibition.Additionally,flow cytometry analysis revealed that the number of EHPinduced necrotic RGC-5 cells was increased after caspase-8 inhibition.Our results suggested that RGC-5 necroptosis following EHP was mediated by RIP3 through induction of PYGL activity and subsequent ROS accumulation.Thus,caspase-8 may participate in the regulation of RGC-5 necroptosis via RIP3 cleavage.