title: [Salubrinal protects human lens epithelial cells against endoplasmic reticulum stress-associated apoptosis].
abstract: OBJECTIVE
  To study the protective effect of Salubrinal on human lens epithelial cells and its mechanism in endoplasmic reticulum stress (ERS).
  
  
  METHODS
  Hydrogen peroxide (H2O2 200 μmol/L) was used to intervene in the cultured human lens epithelial cells B3 (HLE-B3) so as to create an oxidative stress model and induce ERS in the model. Different concentration of Salubrinal (10, 15, 20, 25, 30 and 35 μmol/L) were added to the cultured HLE-B3 with or without H2O2 intervention. Then the cells were cultured for 24 hours. The cell counting kit (CCK-8) assay was used to test the viability of HLE-B3. The HLE-B3 cells were divided into three groups: Group A (normal control group), Group B (H2O2 200 μmol/L group), and Group C (H2O2 200 μmol/L+ Salubrinal 25 μmol/L group). After 48 h, TUNEL and flow cytometry assay (FCM) were used to examine the effect of Salubrinal on HLE-B3 apoptosis. The expression of glucose-regulated protein 78(GRP78), C/EBP homologous protein (CHOP), cysteinyl aspartate specific proteinase 12 (Caspase-12) and phosphorylation eukaryotic translation initiation factor 2α (p-elF2α) were tested by western blot at different points in time. Data from different groups was analyzed by one-way analysis of variance (ANOVA) while Dunnett t test was used under an equal condition, Dunnett's T3 for the unequal variances.
  
  
  RESULTS
  CCK-8 results showed that without the intervention of H2O2, different concentrations of Salubrinal had no inhibitive effect on HLE-B3 viability, and that survival rates were (98.6±3.3) %, (98.7±2.6) %, (99.4±3.2) %, (98.6±1.9) %, (98.8±2.5) %, (99.3±3.2) % and (99.5±2.4) %. There was no statistically significant difference between them (F=0.09, P=0.10). With the increasing of Salubrinal concentration, the survival rates of HLE-B3 in the presence of H2O2 intervention were (52.9±4.7) %, (65.0±3.6) %, (72.9±3.8) %, (84.5±3.6) %, (91.6±2.1) %, (93.1±2.9) %, (92.0±3.3) %. There was statistically significant difference from the control group (all P<0.01). However, the survival rates no longer increased (P=0.56, 0.88) if the Salubrinal concentration was greater than 25 μmol/L. FCM results indicated that apoptosis rates of Group A, B and C were (1.9±0.7) %, (8.8±0.5) %, (4.3±0.3) %, respectively and the differences were statistically significant (F=396.26, P<0.01, comparing with Group A, all P<0.01). TUNEL results showed that apoptosis indexes of Group A, B, and C were (7.7±1.0) %, (36.9±1.0) %, (16.7±2.2) %, respectively and the differences were statistically significant. (F=618.39, P<0.01, comparing with Group A, all P<0.01). RESULTS of western blotting in group B at different points in time (0, 12, 24, 36, 48 h) showed that p-elF2α had increased by (2.16±0.38) times at 6 h; GRP78 had increased by (2.56±0.15) times at 12 h; CHOP started to rise after 12 h until it dropped after 24 h, and its amount had increased by (2.49±0.23) times at 48 h; Caspase-12 had increased significantly by (3.53±0.30) times at 48 h. The expression of GRP78, CHOP and p-elF2α in group C was greater than that in Group B, but the expression of Caspase-12 in Group C was lower than that in Group B (GRP78: F=37.85, P<0.01; CHOP: F=61.09, P<0.01; Caspse-12: F=22.27, P<0.01; p-eIF2α: F=15.11, P<0.01).
  
  
  CONCLUSIONS
  Salubrinal might protect HLE-B3 against H2O2-induced apoptosis by inhibiting ERS related apoptosis pathways.(Chin J Ophthalmol, 2016, 52: 437-443).
authors.affiliation: The Key Discipline Open Laboratory of Clinical Medicine for Institutions of Higher Learning in Henan Province, Department of Ophthalmology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: G Y
authors.initials: GY
authors.lastname: Zheng
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053454:Caspase 12 / Q000378:metabolism; D002934:Cinnamates / Q000494:pharmacology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects*; D015852:Eukaryotic Initiation Factor-2; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide; D020287:In Situ Nick-End Labeling; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects*; D018384:Oxidative Stress; D010766:Phosphorylation; D015398:Signal Transduction; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D051742:Transcription Factor CHOP / Q000378:metabolism
chemical_list: D002934:Cinnamates; D000091342:Endoplasmic Reticulum Chaperone BiP; D015852:Eukaryotic Initiation Factor-2; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C496827:salubrinal; D051742:Transcription Factor CHOP; D006861:Hydrogen Peroxide; D053454:Caspase 12; D013890:Thiourea