1.Effect of Heat Shock Protein 72 Expression on Etoposide-induced Cell Death of Rat Retinal Ganglion Cells.
Seongsoo SOHN ; Ji Eun IM ; Tae Eun KIM ; Changwon KEE
Korean Journal of Ophthalmology 2013;27(1):48-51
PURPOSE: To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. METHODS: Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. RESULTS: Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. CONCLUSIONS: Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease.
Animals
;
Blotting, Western
;
Cell Death/*genetics
;
Cell Survival
;
Cells, Cultured
;
DNA/*genetics
;
Disease Models, Animal
;
Etoposide/toxicity
;
*Gene Expression Regulation
;
HSP72 Heat-Shock Proteins/biosynthesis/*genetics
;
Immunohistochemistry
;
Rats
;
Retinal Degeneration/*genetics/metabolism/pathology
;
Retinal Ganglion Cells/drug effects/*metabolism/pathology
2.Inhibition of calpain on oxygen glucose deprivation-induced RGC-5 necroptosis.
Shuang CHEN ; Jie YAN ; Hai-Xiao DENG ; Ling-Ling LONG ; Yong-Jun HU ; Mi WANG ; Lei SHANG ; Dan CHEN ; Ju-Fang HUANG ; Kun XIONG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2016;36(5):639-645
The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). 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. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.
Animals
;
Apoptosis Inducing Factor
;
biosynthesis
;
genetics
;
Calpain
;
biosynthesis
;
genetics
;
Gene Expression Regulation
;
drug effects
;
Glucose
;
metabolism
;
Humans
;
Leupeptins
;
administration & dosage
;
Mice
;
Oxygen
;
metabolism
;
Retinal Ganglion Cells
;
metabolism
;
pathology
;
Retinal Necrosis Syndrome, Acute
;
genetics
;
pathology
3.The Neuroprotective Effect of Maltol against Oxidative Stress on Rat Retinal Neuronal Cells.
Yookyung SONG ; Samin HONG ; Yoko IIZUKA ; Chan Yun KIM ; Gong Je SEONG
Korean Journal of Ophthalmology 2015;29(1):58-65
PURPOSE: Maltol (3-hydroxy-2-methyl-4-pyrone), formed by the thermal degradation of starch, is found in coffee, caramelized foods, and Korean ginseng root. This study investigated whether maltol could rescue neuroretinal cells from oxidative injury in vitro. METHODS: R28 cells, which are rat embryonic precursor neuroretinal cells, were exposed to hydrogen peroxide (H2O2, 0.0 to 1.5 mM) as an oxidative stress with or without maltol (0.0 to 1.0 mM). Cell viability was monitored with the lactate dehydrogenase assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. To investigate the neuroprotective mechanism of maltol, the expression and phosphorylation of nuclear factor-kappa B (NF-kappaB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were evaluated by Western immunoblot analysis. RESULTS: R28 cells exposed to H2O2 were found to have decreased viability in a dose- and time-dependent manner. However, H2O2-induced cytotoxicity was decreased with the addition of maltol. When R28 cells were exposed to 1.0 mM H2O2 for 24 hours, the cytotoxicity was 60.69 ± 5.71%. However, the cytotoxicity was reduced in the presence of 1.0 mM maltol. This H2O2-induced cytotoxicity caused apoptosis of R28 cells, characterized by DNA fragmentation. Apoptosis of oxidatively-stressed R28 cells with 1.0 mM H2O2 was decreased with 1.0 mM maltol, as determined by the TUNEL method. Western blot analysis showed that treatment with maltol reduced phosphorylation of NF-kappaB, ERK, and JNK, but not p38. The neuroprotective effects of maltol seemed to be related to attenuated expression of NF-kappaB, ERK, and JNK. CONCLUSIONS: Maltol not only increased cell viability but also attenuated DNA fragmentation. The results obtained here show that maltol has neuroprotective effects against hypoxia-induced neuroretinal cell damage in R28 cells, and its effects may act through the NF-kappaB and mitogen-activated protein kinase signaling pathways.
Animals
;
*Apoptosis
;
Blotting, Western
;
Cell Survival
;
Cells, Cultured
;
Disease Models, Animal
;
Flavoring Agents/pharmacology
;
In Situ Nick-End Labeling
;
Oxidative Stress/*drug effects
;
Pyrones/*pharmacology
;
Rats
;
Retinal Ganglion Cells/drug effects/metabolism/*pathology