The effects and regulatory mechanism of RIP3 on RGC-5 necroptosis following elevated hydrostatic pressure

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 ana- lysis revealed that the number of EHP-induced necrotic retinal ganglion cell 5 （RGC-5） cells was reduced after RIP3-knockdown. Furthermore, malondialdehyde （MDA） levels and glycogen phos- phorylase （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 buty- lated 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. Western 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 EHP- induced 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.