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

Ding W, Shang L, Huang JF, Li N, Chen D, Xue LX, Xiong K - BMC Neurosci (2015)

Bottom Line: 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.Flow cytometry revealed that the number of OGD-induced necrotic RGC-5 cells was reduced after rip3 knockdown.Our findings suggest that RGC-5 cell necroptosis following OGD is mediated by a RIP3-induced increase in oxidative stress.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Ratio of necrotic cells is reduced following Nec-1 pre-treatment by OGD. a Normal control cells; b RGC-5 cell necrosis after OGD; c RGC-5 cells were pre-treated with Nec-1 (10 μM) to block necroptosis for 24 h before OGD and analysis of necroptotic cells. Cells were stained with Annexin fluorescein isothiocyanate and PI, and analyzed by FACS using FL1 (Annexin V) and FL3 (PI) channels. d The statistical analysis of RGC-5 necrosis, * vs CTL, P < 0.05; # vs Nec-1 pre-treatment, P < 0.05.
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Fig2: Ratio of necrotic cells is reduced following Nec-1 pre-treatment by OGD. a Normal control cells; b RGC-5 cell necrosis after OGD; c RGC-5 cells were pre-treated with Nec-1 (10 μM) to block necroptosis for 24 h before OGD and analysis of necroptotic cells. Cells were stained with Annexin fluorescein isothiocyanate and PI, and analyzed by FACS using FL1 (Annexin V) and FL3 (PI) channels. d The statistical analysis of RGC-5 necrosis, * vs CTL, P < 0.05; # vs Nec-1 pre-treatment, P < 0.05.

Mentions: The immunofluorescence results indicated that there was a large number of necrotic RGC-5 cells after 6 h re-oxygenation following OGD; thus, we chose this time point to analyze cellular necroptosis by flow cytometry with PI/Annexin V double staining following pretreatment with Nec-1 (RGC-5 cells were incubated with 10 µM Nec-1 for 24 h prior to OGD). The results showed that necrosis occurred after OGD (Fig. 2b), but the number of necrotic (PI-positive) cells decreased significantly with Nec-1 pretreatment (Fig. 2c, d, P < 0.05). These results indicate that RGC-5 cell necrosis can be inhibited by Nec-1 and that necroptosis occurred at the early stage of OGD.Fig. 2


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

Ding W, Shang L, Huang JF, Li N, Chen D, Xue LX, Xiong K - BMC Neurosci (2015)

Ratio of necrotic cells is reduced following Nec-1 pre-treatment by OGD. a Normal control cells; b RGC-5 cell necrosis after OGD; c RGC-5 cells were pre-treated with Nec-1 (10 μM) to block necroptosis for 24 h before OGD and analysis of necroptotic cells. Cells were stained with Annexin fluorescein isothiocyanate and PI, and analyzed by FACS using FL1 (Annexin V) and FL3 (PI) channels. d The statistical analysis of RGC-5 necrosis, * vs CTL, P < 0.05; # vs Nec-1 pre-treatment, P < 0.05.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4524047&req=5

Fig2: Ratio of necrotic cells is reduced following Nec-1 pre-treatment by OGD. a Normal control cells; b RGC-5 cell necrosis after OGD; c RGC-5 cells were pre-treated with Nec-1 (10 μM) to block necroptosis for 24 h before OGD and analysis of necroptotic cells. Cells were stained with Annexin fluorescein isothiocyanate and PI, and analyzed by FACS using FL1 (Annexin V) and FL3 (PI) channels. d The statistical analysis of RGC-5 necrosis, * vs CTL, P < 0.05; # vs Nec-1 pre-treatment, P < 0.05.
Mentions: The immunofluorescence results indicated that there was a large number of necrotic RGC-5 cells after 6 h re-oxygenation following OGD; thus, we chose this time point to analyze cellular necroptosis by flow cytometry with PI/Annexin V double staining following pretreatment with Nec-1 (RGC-5 cells were incubated with 10 µM Nec-1 for 24 h prior to OGD). The results showed that necrosis occurred after OGD (Fig. 2b), but the number of necrotic (PI-positive) cells decreased significantly with Nec-1 pretreatment (Fig. 2c, d, P < 0.05). These results indicate that RGC-5 cell necrosis can be inhibited by Nec-1 and that necroptosis occurred at the early stage of OGD.Fig. 2

Bottom Line: 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.Flow cytometry revealed that the number of OGD-induced necrotic RGC-5 cells was reduced after rip3 knockdown.Our findings suggest that RGC-5 cell necroptosis following OGD is mediated by a RIP3-induced increase in oxidative stress.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus