Receptor interacting protein 3-induced RGC-5 cell necroptosis following oxygen glucose deprivation

Abstract

BackgroundNecroptosis is a type of regulated form of cell death that has been implicated in the pathogenesis of various diseases. Receptor-interacting protein 3 (RIP3), a member of the RIP family of proteins, has been reported as an important necroptotic pathway mediator in regulating a variety of human diseases, such as myocardial ischemia, inflammatory bowel disease, and ischemic brain injury. Our previous study showed that RIP3 was expressed in rat retinal ganglion cells (RGCs), where it was significantly upregulated during the early stage of acute high intraocular pressure. Furthermore, RIP3 expression was co-localized with propidium iodide (PI)-positive staining (necrotic cells). These results suggested that RIP3 up-regulation might be involved in the necrosis of injured RGCs. In this study, we aimed to reveal the possible involvement of RIP3 in oxygen glucose deprivation (OGD)-induced retinal ganglion cell-5 (RGC-5) necroptosis.MethodsRGC-5 cells were cultured in Dulbecco’s-modified essential medium and necroptosis was induced by 8 h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. RIP3 expression was detected by western blot and flow cytometry was used to detect the effect of RIP3 on RGC-5 necroptosis following OGD in rip3 knockdown cells. Malondialdehyde (MDA) lipid peroxidation assay was performed to determine the degree of oxidative stress.ResultsPI staining showed that necrosis was present in the early stage of OGD-induced RGC-5 cell death. The presence of RGC-5 necroptosis after OGD was detected by flow cytometry using necrostatin-1, a necroptosis inhibitor. Western blot demonstrated that RIP3 up-regulation may be involved in RGC-5 necroptosis. Flow cytometry revealed that the number of OGD-induced necrotic RGC-5 cells was reduced after rip3 knockdown. Furthermore, MDA levels in the normal RGC-5 cells were much higher than in the rip3-knockdown cells after OGD.ConclusionsOur findings suggest that RGC-5 cell necroptosis following OGD is mediated by a RIP3-induced increase in oxidative stress.

Anisomycin prevents OGD-induced necroptosis by regulating the E3 ligase CHIP

“…Studies have revealed that necroptosis contributed to the selective and delayed death of certain populations of neurons that occurs following an ischemic stroke 8 , which can be caused by a lack of blood supply to the brain 9 . Necroptosis has also been implicated in ischemic necrosis of retinal cells in several studies 10 – 12 . Oxygen-glucose deprivation (OGD), an in vitro model of cerebral ischemia, can also induce the death receptor (DR)-dependent component of necroptotic cell death in cultured neurons 13 – 15 , concomitant with the increase in RIPK3/RIPK1 mRNA and protein levels 16 .…”

Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

“…28 According to the manufacturer's protocol (Roche, Mannheim, Germany), we detected cell apoptosis via TUNEL staining. The brain was removed and frozen in liquid nitrogen and then stored at −80 °C.…”

Pin1 Promotes Regulated Necrosis Induced by Glutamate in Rat Retinal Neurons via CAST/Calpain2 Pathway

“…The RGC-5 cell line was provided by the Department of Ophthalmology, Second Hospital of Jilin University in China ( Ding et al, 2015 ). RGC-5 cells were cultured in DMEM (Thermo Scientific) supplemented with 10% FBS (Thermo Scientific) and 1% Penicillin-Streptomycin (Thermo Scientific).…”