Objective To observe the effects of overexpression of polypyrimidine tract binding protein-associated splicing factor (PSF) on the endoplasmic reticulum (ER) oxidative stress damage of human retinal microvascular endothelial cells (hRMEC) under high concentration of 4-hydroxynonenal (4-HNE). MethodsThe logarithmic growth phase hRMEC cultured in vitro was divided into normal group, simple 4-HNE treatment group (simple 4-HNE group), empty plasmid combined with 4-HNE treatment group (Vec+4-HNE group), and PSF high expression combined with 4-HNE treatment group (PSF+4-HNE group). In 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group cell culture medium, 10 μmol/L 4-HNE was added and stimulated for 12 hours. Subsequently, the Vec+4-HNE group and PSF+4-HNE group were transfected with transfection reagent liposome 2000 into pcDNA empty bodies and pcDNA-PSF eukaryotic expression plasmids, respectively, for 24 hours. Flow cytometry was used to detect the effects of 4-HNE and PSF on cell apoptosis. The effect of PSF overexpression on the expression of reactive oxygen species (ROS) in hRMEC was detected by 2', 7'-dichlorodihydrofluorescein double Acetate probe. Western blot was used to detect ER oxide protein 1 (Ero-1), protein disulfide isomerase (PDI), C/EBP homologous transcription factor (CHOP), glucose regulatory protein (GRP) 78, protein kinase R-like ER kinase (PERK)/phosphorylated PERK (p-PERK), and Eukaryotic initiation factor (eIF) 2α/the relative expression levels of phosphorylated eIF (peIF) and activated transcription factor 4 (ATF4) proteins in hRMEC of normal group, 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group. Single factor analysis of variance was performed for inter group comparison. ResultsThe apoptosis rates of the simple 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group were (22.50±0.58)%, (26.93±0.55)%, and (11.70±0.17)%, respectively. The intracellular ROS expression levels were 0.23±0.03, 1.60±0.06, and 0.50±0.06, respectively. The difference in cell apoptosis rate among the three groups was statistically significant (F=24.531, P＜0.05). The expression level of ROS in the Vec+4-HNE group was significantly higher than that in the simple 4-HNE group and the PSF+4-HNE group, with a statistically significant difference (F=37.274, P＜0.05). The relative expression levels of ER Ero-1 and PDI proteins in the normal group, simple 4-HNE group, Vec+4-HNE group, and PSF+4-HNE group were 1.25±0.03, 0.45±0.03, 0.63±0.03, 1.13±0.09, and 1.00±0.10, 0.27±0.10, 0.31±0.05, and 0.80±0.06, respectively. The relative expression levels of CHOP and GRP78 proteins were 0.55±0.06, 1.13±0.09, 0.90±0.06, 0.48±0.04 and 0.48±0.04, 1.25±0.03, 1.03±0.09, 0.50±0.06, respectively. The relative expression levels of Ero-1 (F=43.164), PDI (F=36.643), CHOP (F=42.855), and GRP78 (F=45.275) proteins in four groups were compared, and the differences were statistically significant (P＜0.05). Four groups of cells ER p-pERK/pERK (F=35.755), peIF2 α/ The relative expression levels of eIF (F=38.643) and ATF4 (F=31.275) proteins were compared, and the differences were statistically significant (P＜0.05). ConclusionPSF can inhibit cell apoptosis and ROS production induced by high concentration of 4-HNE, and its mechanism is closely related to restoring the homeostasis of ER and down-regulating the activation level of PERK/eIF2α/ATF4 pathway.
ObjectiveTo investigate the effects of targeted regulation of SMAD9 expression by bone morphogenetic protein 4 (BMP4) on Müller cell migration, reactive oxygen species (ROS) generation and vascular endothelial growth factor (VEGF) expression. MethodsMüller cells cultured in vitro were divided into normal control group, BMP4 group, BMP4+ no-load plasmid group (BMP4+NC group) and BMP4+SMAD9 small interference plasmid group (BMP4+siSMAD9). Cells in BMP4 group, BMP4+NC group and BMP4+siSMAD9 group were induced by adding 100 ng/ml BMP4 into cell medium for 24 h. Subsequently, BMP4+NC group was transfected with empty plasmid. BMP4+siSMAD9 group was transfected with SMAD9 small interference plasmid for 48 h. The effect of BMP4 on Müller cell migration was determined by cell scratch test. The effect of BMP4 on the production of ROS in Müller cells was detected by flow cytometry. Western blots and real-time quantitative fluorescence polymerase chain reaction (qPCR) were used to detect the relative mRNA expression levels of glutamine synthetase (GS) and glial fibrinoacidic protein (GFAP) in Müller cells. VEGF expression in Müller cells was detected by immunofluorescence. One-way analysis of variance was used to compare groups. ResultsThe results of cell scratch test showed that the cell mobility of BMP4+siSMAD9 group was significantly lower than that of BMP4 and BMP4+NC group, and the difference was statistically significant (F=68.319, P＜0.001). Flow cytomethods showed that the level of ROS in BMP4+siSMAD9 group was significantly lower than that in BMP4 and BMP4+NC group, and the difference was statistically significant (F=52.158, P＜0.001). Western blot and qPCR results showed that the protein levels of GS and GFAP (F=42.715, 36.618) and mRNA relative expression levels (F=45.164, 43.165) in BMP4+siSMAD9 group were significantly lower than those in BMP4 and BMP4+NC group. The difference was statistically significant (P＜0.01). The results of immunofluorescence detection showed that the intracellular VEGF fluorescence intensity in BMP4 group and BMP4+NC group was significantly higher than that in BMP4+siSMAD9 group, and the difference was statistically significant (F=46.384, P＜0.05). ConclusionTargeted regulation of SMAD9 expression by BMP4 can up-regulate VEGF expression and promote the migration and ROS production of Müller cells.
Objective To investigate the effect of Nodal protein on retinal neovascularization under hypoxia. MethodsIn vivo animal experiment: 48 healthy C57BL/6J mice were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+dimethyl sulfoxide (DMSO) group and OIR+SB431542 group, with 12 mice in each group. Retinal neovascularization was observed in mice at 17 days of age by retina flat mount. Counts exceeded the number of vascular endothelial nuclei in the retinal inner boundary membrane (ILM) by hematoxylin eosin staining. In vivo cell experiment: human retinal microvascular endothelial cells (hRMEC) were divided into normal group, hypoxia group, hypoxia+DMSO group and hypoxia +SB431542 group. The cell proliferation was detected by thiazolyl blue colorimetry (MTT). The effect of SB431542 on hRMEC lumen formation was detected by Matrigel three-dimensional in vitro molding method. Cell migration in hRMEC was detected by cell scratch assay. The Seahorse XFe96 Cell Energy Metabolism analyzer measured extracellular acidification rate (ECAR) of intracellular glycolysis, glycolysis reserve, and glycolysis capacity. One-way analysis of variance was used to compare groups. ResultsIn vivo animal experiment: compared with normal group, the neovascularization increased in OIR group (t=41.621, P＜0.001). Compared with OIR group, the number of vascular endothelial nuclei breaking through ILM in OIR+SB431542 group was significantly reduced, and the difference was statistically significant (F=36.183, P＜0.001). MTT test results showed that compared with normal group and hypoxia+SB431542 group, the cell proliferation of hypoxia group and hypoxia+DMSO group was significantly increased, and the difference was statistically significant (F=39.316, P＜0.01). The cell proliferation of hypoxia+SB431542 group was significantly lower than that of hypoxia+DMSO group, and the difference was statistically significant (t=26.182, P＜0.001). The number of intact lumen formation and migration cells in normal group, hypoxia group, hypoxia+DMSO group and hypoxia+SB431542 group were statistically significant (F=34.513, 41.862; P＜0.001, ＜0.01). Compared with the hypoxia+DMSO group, the number of intact lumen formation and migrating cells in the hypoxia+SB431542 group decreased significantly, and the differences were statistically significant (t=44.723, 31.178; P＜0.001, ＜0.01). The results of cell energy metabolism showed that compared with the hypoxia +DMSO group, the ECAR of intracellular glycolysis and glycolysis reserve in the hypoxia +SB431542 group was decreased, and the ECAR of glycolysis capacity was increased, with statistical significance (t=26.175, 33.623, 37.276; P＜0.05). ConclusionSB431542 can inhibit the proliferation, migration and the ability to form lumens, reduce the level of glycolysis of hRMECs cells induced by hypoxia.