title: Receptor interacting protein 3-induced RGC-5 cell necroptosis following oxygen glucose deprivation.
abstract: BACKGROUND
  Necroptosis 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.
  
  
  METHODS
  RGC-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.
  
  
  RESULTS
  PI 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.
  
  
  CONCLUSIONS
  Our findings suggest that RGC-5 cell necroptosis following OGD is mediated by a RIP3-induced increase in oxidative stress.
authors.affiliation: Department of Anatomy and Neurobiology, Morphological Sciences Building, School of Basic Medical Sciences, Central South University, 172 Tongzi Po Road, Changsha, 410013, Hunan, China. ldd2007200@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Ding
authors.affiliation: Department of Anatomy and Neurobiology, Morphological Sciences Building, School of Basic Medical Sciences, Central South University, 172 Tongzi Po Road, Changsha, 410013, Hunan, China. shanglei1986@163.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Shang
authors.affiliation: Department of Anatomy and Neurobiology, Morphological Sciences Building, School of Basic Medical Sciences, Central South University, 172 Tongzi Po Road, Changsha, 410013, Hunan, China. huangjufang@csu.edu.cn.
authors.forename: Ju-Fang
authors.initials: JF
authors.lastname: Huang
authors.affiliation: Department of Anatomy and Neurobiology, Morphological Sciences Building, School of Basic Medical Sciences, Central South University, 172 Tongzi Po Road, Changsha, 410013, Hunan, China. 769049085@qq.com.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Anatomy and Neurobiology, Morphological Sciences Building, School of Basic Medical Sciences, Central South University, 172 Tongzi Po Road, Changsha, 410013, Hunan, China. 57460119@qq.com.
authors.forename: Dan
authors.initials: D
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biology, Health Science Center, Peking University, Beijing, 100191, China. lixiangxue@hsc.pku.edu.cn.
authors.forename: Li-Xiang
authors.initials: LX
authors.lastname: Xue
authors.affiliation: Department of Anatomy and Neurobiology, Morphological Sciences Building, School of Basic Medical Sciences, Central South University, 172 Tongzi Po Road, Changsha, 410013, Hunan, China. xiongkun2001@163.com.
authors.forename: Kun
authors.initials: K
authors.lastname: Xiong
mesh_terms: D000818:Animals; D016923:Cell Death / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000172:deficiency*; D007093:Imidazoles / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D015227:Lipid Peroxidation; D008315:Malondialdehyde / Q000378:metabolism; D051379:Mice; D018384:Oxidative Stress; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000502:physiology*; D015854:Up-Regulation
chemical_list: D007093:Imidazoles; D007211:Indoles; C507699:necrostatin-1; D008315:Malondialdehyde; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse; D005947:Glucose