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Objective:To observe the effect of the antioxidant N-acetylcysteine (NAC) and selective endoplasmic reticulum stress response inhibitor salubrinal on the apoptosis of retinal pigment epithelial cells induced by all-trans-retinoic acid (ATRA).Methods:Human ARPE-19 cell line was used as the experimental cell line, and was divided into normal control group cultured with complete medium, model control group cultured with complete medium containing 10 μmol/L ATRA, NAC treatment group cultured with complete medium containing 10 μmol/L ATRA+ 5 mmol/L NAC, salubrinal group cultured with complete medium containing 10 μmol/L ATRA+ 40 μmol/L salubrinal, NAC+ salubrinal group cultured with complete medium containing 10 μmol/L ATRA+ 5 mmol/L NAC+ 40 μmol/L salubrinal.After 24-hour culture, apoptosis rate, multicaspase level and reactive oxygen species (ROS) level of ARPE-19 cells were detected by flow cytometry.The expressions of vascular endothelial growth factor A (VEGF-A), C/EBP-homologous protein (CHOP), cleaved-caspase 3 in cells were detected by Western blot.Results:There were significant differences in the apoptosis rate, multicaspase and ROS levels among the five groups ( F=113.23, 602.41, 160.39; all at P<0.001). The apoptosis rate, multicaspase and ROS levels of normal control group, NAC treatment group, salubrinal group and NAC+ salubrinal group were significantly lower than those of model control group (all at P<0.05). There were significant differences in the expression levels of VEGF-A, CHOP and cleaved-caspase 3 among the five groups ( F=24.62, 36.35, 60.25; all at P<0.001). The protein expression levels of VEGF-A, CHOP and cleaved-caspase 3 of normal control group, NAC treatment group, salubrinal group and NAC+ salubrinal group were significantly lower than those of model control group (all at P<0.05). Conclusions:ATRA can induce RPE cells to produce oxidative stress and endoplasmic reticulum stress injury, which leads to apoptosis.NAC and salubrinal can effectively reduce the RPE cell apoptosis by inhibiting stress response.
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Objective To explore the mechanism of the role of Salubrinal in regulating the radiation-induced apoptosis of oral cancer cells.Methods Radioresistant KBR cell line was constructed (4 Gy per fraction,every 7-10 d for 4 times).The radiosensitivity of oral cancer cells after Salubrinal pretreatment was measured by colony formation assay.The expression levels of NF-κB-HIF-1o signaling pathway and apoptosis biomarker cleaved PARP in oral cancer cells were measured by Western blot.The apoptosis rate was detected by Annexin V,PI staining and flow cytometry.Results Colony formation assay demonstrated that Salubrinal increased the radiosensitivity of oral cancer cells.The radiosensitization ratios of KB and KBR cells were 1.19 and 1.24.Western blot revealed that the activation of NF-κB-HIF-1α was time-dependent in the radiation-induced oral cancer cells,whereas Salubrinal inhibited the radiation-induced abnormal activation.In addition,Salubrinal increased the expression of apoptosis biomarker cleaved PARP and apoptosis index in radiation-induced oral cancer cells,whereas TNF-α,an activator of NF-κB,reversed the effect,suggesting that Salubrinal increased the apoptosis of radiation-induced oral cancer cells by suppressing the activation of NF-κB.Pretreatment of NF-κB inhibitor Bay1 1-7082 also increased the cell apoptosis.The expression levels of cleaved PARP of KB and KBR cell lines in the Bay11-7082+IR group were 2.67±0.26 and 1.91±0.17,significantly higher compared with 2.1±0.16 and 1.44±0.15 in the IR group (both P＜0.05).Conclusion Salubrinal can aggravate the apoptosis of radiation-induced oral cancer cells by inhibiting the radiation-induced activation of NF-κB,thereby regulating the radiosensitivity of oral cancer cells.
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Objective@#To explore the mechanism of the role of Salubrinal in regulating the radiation-induced apoptosis of oral cancer cells.@*Methods@#Radioresistant KBR cell line was constructed (4 Gy per fraction, every 7-10 d for 4 times). The radiosensitivity of oral cancer cells after Salubrinal pretreatment was measured by colony formation assay. The expression levels of NF-κB-HIF-1α signaling pathway and apoptosis biomarker cleaved PARP in oral cancer cells were measured by Western blot. The apoptosis rate was detected by Annexin V, PI staining and flow cytometry.@*Results@#Colony formation assay demonstrated that Salubrinal increased the radiosensitivity of oral cancer cells. The radiosensitization ratios of KB and KBR cells were 1.19 and 1.24. Western blot revealed that the activation of NF-κB-HIF-1α was time-dependent in the radiation-induced oral cancer cells, whereas Salubrinal inhibited the radiation-induced abnormal activation. In addition, Salubrinal increased the expression of apoptosis biomarker cleaved PARP and apoptosis index in radiation-induced oral cancer cells, whereas TNF-α, an activator of NF-κB, reversed the effect, suggesting that Salubrinal increased the apoptosis of radiation-induced oral cancer cells by suppressing the activation of NF-κB. Pretreatment of NF-κB inhibitor Bay11-7082 also increased the cell apoptosis. The expression levels of cleaved PARP of KB and KBR cell lines in the Bay11-7082+ IR group were 2.67±0.26 and 1.91±0.17, significantly higher compared with 2.1±0.16 and 1.44±0.15 in the IR group (both P<0.05).@*Conclusion@#Salubrinal can aggravate the apoptosis of radiation-induced oral cancer cells by inhibiting the radiation-induced activation of NF-κB, thereby regulating the radiosensitivity of oral cancer cells.
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AIM:To explore the role of endoplasmic reticulum stress ( ERS) in brain injury following chronic intermittent hypoxia in growing rats and the protective effect of treatment with salubrinal .METHODS: Healthy male SD rats (3~4-week-old, 100~120 g, n=64) were randomly assigned to 8 groups (8 rats in each group):the groups of in-termittent hypoxia for 2 and 4 weeks (2IH and 4IH), the groups of control (C) for 2 and 4 weeks (2C and 4C), the groups of dimethylsulfoxide (DMSO) for 2 and 4 weeks (2DMSO and 4DMSO) and the groups of salubrinal for 2 and 4 weeks (2SAL and 4SAL).The 8-arm radial maze was used to assess the working memory error (WME), reference memory error (RME) and total error (TE) of the rats.The changes of neuronal apoptosis in the hippocampus were observed by ter -minal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) staining.The activity of superoxide dismutase (SOD), and the protein levels of endoplasmic reticulum stress marker compounds , C/EBP homologous protein (CHOP), phosphorylated eukaryotic translation initiation factor 2 alpha ( p-eIF2α) and phosphorylated protein kinase R-like endo-plasmic reticulum kinase (p-PERK), were analyzed.RESULTS: Chronic intermittent hypoxia (CIH) significantly in-creased RME, WME, TE and neuronal apoptotic index (AI) (P<0.01), and decreased the activity of SOD in the hippo-campus and serum (P<0.01).The protein levels of p-PERK and CHOP progressively increased in hippocampus in IH groups (P<0.01), and p-eIF2αwas downregulated (P<0.05).Treatment with salubrinal significantly decreased RME (P<0.05), WME (P<0.05), TE (P<0.01) and AI (P<0.01), and increased the activity of SOD (P<0.01).Sa-lubrinal induced the phosphorylation of eIF 2αsignificantly after CIH in hippocampus and downregulated the level of CHOP (P<0.01).CONCLUSION: Chronic intermittent hypoxia upregulates the protein levels of p-PERK and CHOP in the hippocampus , and decreases p-eIF2αprotein and the activity of SOD .Salubrinal , a selective inhibitor of eIF-2αdephos-phorylation , increases the activity of SOD and prevents CHOP protein activation throughout CIH exposure .Our findings suggest ERS-mediated cell apoptosis is one of the underlying mechanisms of cognitive dysfunction in OSAHS children .Fur-ther, a specific ERS inhibitor salubrinal should be tested for neuroprotection against CIH -induced brain injury .
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Objective To investigate the effect of selective phosphatase inhibitors Salubrinal on autophagy and apoptosis in the lung tissue of rats with acute paraquat (PQ) poisoning,and to explore its mechanism.Methods 200 Wistar rats were randomly divided into four groups by randomized arrangement table formed by computer,with 50 rats in each group.PQ poisoning model was reproduced by one time gastric lavage with 1 mL of 40 mg/kg PQ solution followed by intraperitoneal injection of 1 mL normal saline (NS) once a day.The rats in control group were lavaged once with 1 mL of NS followed by intraperitoneal injection of 1 mL NS twice a day.The rats in Sal 0.5 and Sal 1.0 groups were intraperitoneal injected with 1 mL Salubrinal 0.5 mg/kg or 1.0 mg/kg on the 1st,3rd,and 5th day after PQ poisoning once a day.The lung tissue was harvested on the 7th day after poisoning,and the changes in histomorphology were observed using hematoxylin and eosin (HE) staining.The positive expression of autophagy-related protein LC3-Ⅱ in lung tissue was observed after immunohistochemistry staining,and LC3-Ⅱ and caspase-3 protein expressions were determined by Western Blot.Results HE staining results showed partial abnormal pulmonary structure in the PQ poisoning group:collapse of pulmonary alveoli,enlargement of the cavity,local infiltration of inflammatory cells,increasing thickness in the alveoli wall and obvious bleeding in the local lung tissue.Compared with the PQ poisoning group,the above changes in Sal 0.5 and Sal 1.0 groups were obviously relieved.It was shown by immunohistochemistry staining that compared with control group,the positive expression of LC3-Ⅱ was obviously decreased in the PQ poisoning group,Sal 0.5,and Sal 1.0 groups (A value:78.34 ± 10.71,76.52 ± 8.21,77.48 ± 9.11 vs.117.58 ± 15.26,all P＜0.05).There was no significant difference in positive expression of LC3-Ⅱ between each of the later three groups (all P＞0.05).Western Blot results showed:compared with the control group,the protein expressions of LC3-Ⅱ and caspase-3 were significantly increased in PQ poisoning group [LC3-Ⅱ (A value):0.22 ±0.05 vs.0.14 ±0.03,caspase-3 (A value):0.115 ± 0.013 vs.0.023 ± 0.006,both P＜0.05].Compared with PQ poisoning group,the protein expressions of LC3-Ⅱ and caspase-3 were obviously decreased in the Sal 0.5 and Sal 1.0 groups [LC3-Ⅱ (A value):0.19 ±0.05,0.18 ±0.04 vs.0.22 ±0.05; caspase-3 (A value):0.078 ±0.012,0.076 ±0.010 vs.0.115 ±0.013,all P＜0.05].Conclusions The endoplasmic reticulum stress-autophagy is activated in the pulmonary cell of acute PQ poisoning rats.Salubrinal can decrease the autophagy and apoptosis in the lung of rats with acute PQ poisoning,which play a role in the treatment.