The effects of Receptor Interacting Protein 3 on necroptosis in RGC‐5 following elevated hydrostatic pressure

Abstract

ObjectiveRIP3 has been found to induce necroptosis. Previous study have demonstrate necroptosis is an important neuronal death mode in retinal ischemia, and RIP3 may relate to necrosis in our observation. The lack of RIP3 inhibitor limited our study in vivo, so we first identify necroptosis in RGC‐5 following EHP, and then detect the protein level of RIP3, the percentage of necrosis using the pressure chamber via rip3 RNAi. Finally, we try to elaborate the mechanism of necroptosis.MethodsAfter 6 hr, 12 hr, 24 hr of recovery, we execute PI‐staining with different pressure values insults, then detected the level of RIP3 expression and the number of necrosis cells using western‐blotting, fluorescence‐staining. We analyzed the percentage of necrosis in 24 hr to identify the existence of necroptosis within necrostatin‐1. Finally rip3‐RNAi were carried out to get the converse evidence on necroptosis.ResultsPI‐staining in different pressure levels showed there was little necrosis cell under 60 mmHg and the percentage of cell necrosis increased following the pressure elevation. As pressure level elevated, the number of necrosis cells increased too. RIP3 expression was up‐regulated and there exist double‐labeling of some RIP3 enhance‐labeled and PI‐positives after 6 hr. The results of flow cytometry indicated the ratio of necrosis cells were decreased from 13% to 5% within necrostatin‐1, the percentage of necrosis cells decreased to nearly 7% using rip3‐RNAi.ConclusionRIP3 may participate in necroptosis following EHP.This work was supported by National Natural Science Foundation of China (81371011),Wu Jie‐Ping Medical Foundation of the Minister of Health of China (320.6750.14118), Natural Science Foundation of Hunan Province (2015JJ2187)