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Objective To study the effect of geniposide(Gen)on the proliferation,migration and angiogenesis of human retinal vascular endothelial cells(hRVECs)induced by high glucose and explore its mechanism.Methods The hRVECs were intervened with different concentrations(0,1,5,10,20,40 and 80 mg·L-1)of Gen for 24 h,and Cell Counting Kit-8(CCK-8)was used to detect the effect of Gen on the proliferation activity of hRVECs.The hRVECs were di-vided into the control group,high glucose(25 mmol·L-1)group,low,middle and high Gen concentration(5,10 and 20 mg·L-1)groups,bevacizumab(BEV,250 μg·L-1)group and high Gen concentration+BEV(250 pg·L-1)group.Cell proliferation activity was detected by CCK-8.The cell migration ability was detected by scratch test.The tube formation ability of cells was detected by the in vitro tube formation assay.The protein expression levels of vascular endothelial growth factor-A(VEGF-A),soluble VEGF receptor-1(sFlt-1),matrix metalloproteinase 2(MMP-2)and matrix metallo-proteinase 9(MMP-9)in cells were detected by Western blot.Results Compared with 0 mg·L-1 Gen,there was no sta-tistically significant difference in the effect of Gen with concentrations of 1,5,10,20,40 and 80 mg·L-1 on the prolifera-tion activity of hRVECs(all P＞0.05).Compared with the control group,the proliferation activity and migration ability of hRVECs in the high glucose group were significantly enhanced(both P＜0.05),the cell circular structure increased,the protein expression levels of VEGF-A,MMP-2 and MMP-9 significantly increased(all P＜0.05),and the protein expression level of sFlt-1 significantly decreased(P＜0.05).Compared with the high glucose group,the proliferative activity and mi-gration ability of cells in all Gen concentration groups and BEV group significantly decreased(all P＜0.05),the circular structure of cells was reduced,the protein expression levels of VEGF-A,MMP-2 and MMP-9 significantly decreased(all P＜0.05),and the protein expression level of sFlt-1 significantly increased(P＜0.05).Compared with the high Gen concentra-tion group,the high Gen concentration+BEV group showed a significant decrease in cell proliferation activity(P＜0.05),a decrease in cell circular structure,a significant decrease in VEGF-A,MMP-2 and MMP-9 protein expression levels(all P＜0.05),and a significant increase in sFlt-1 protein expression level(P＜0.05).Conclusion Gen can inhibit the high glucose-induced proliferation,migration and angiogenesis of hRVECs,and its mechanism may be related to the regulation of VEGF/sFlt-1 axis balance.
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Objective:To investigate the regulatory effect of transient receptor potential cation channel subfamily C member 3 (TRPC3) on the retina in oxygen-induced retinopathy (OIR) mice and biological behavior of human retinal vascular endothelial cells (HREC).Methods:A total of 32 healthy SPF grade 7-day-old C57BL/6 mice were selected and randomly divided into a control group and an OIR group by the random number table method, with 16 mice in each group.The control group received no special treatment, and the OIR model was established in the OIR group.On postnatal day 17 (PN17), the success of the model establishment was verified by immunofluorescence staining of the retinal patch.The in vitro cultured HREC were divided into a normal control group, a transfection reagent group, and a si-TRPC3 group.The normal control group received no special treatment, while the transfection reagent group and the si-TRPC3 group were transfected with transfection reagent or transfection reagent + si-TRPC3.The relative expression of TRPC3 mRNA was detected by real-time quantitative fluorescence PCR.The relative expressions of TRPC3, transcription factor NF-E2 related factor (Nrf2), and superoxide dismutase (SOD) proteins were determined by Western blot.HREC were further divided into a normal control group, a vascular endothelial growth factor (VEGF) group, a si-TRPC3 group, and a Pyr3 (TRPC3 channel inhibitor) group, which were cultured in complete medium, medium containing 20 ng/ml VEGF recombinant protein, medium containing 20 ng/ml VEGF recombinant protein (si-TRPC3 transfection for 72 hours), and medium containing 20 ng/ml VEGF recombinant protein+ 1 μmol/L Pyr3 for 48 hours, respectively.The proliferation ability of HREC was detected using cell counting kit 8 (CCK-8). The horizontal and vertical migration ability of cells were detected by cell scratch assay and transwell assay, respectively.This study followed the 3R principles of animal welfare and was approved by the Ethics Committee of Hebei Eye Hospital (No.2023LW04). Results:Pathological neovascular clusters with strong fluorescent staining appeared in the retina of OIR mice on PN17.The relative expressions of TRPC3 mRNA and protein in the retina of OIR mice were 2.057±0.244 and 1.517±0.290, respectively, significantly higher than 0.983±0.033 and 0.874±0.052 of control group ( t=6.165, 3.094; both at P<0.05). The relative expression levels of TRPC3 mRNA and protein were significantly lower, and the relative expression levels of Nrf2 and SOD proteins were higher in the si-TRPC3 group than in the normal control and transfection reagent groups, and the differences were statistically significant (all at P<0.05). The CCK-8 experiment results showed that the cell absorbance value was higher in the VEGF group than in the normal control group, and lower in the si-TRPC3 and Pyr3 groups than in the VEGF group, with statistically significant differences (all at P<0.05). The results of the cell scratch experiment showed that the lateral migration rate of VEGF group cells was higher than that of normal control group, while the lateral migration rate of si-TRPC3 group and Pyr3 group cells was lower than that of VEGF group, and the differences were statistically significant (all at P<0.05). The transwell experiment results showed that the number of stained cells in the VEGF group was higher than that in the normal control group, and the number of stained cells in the si-TRPC3 group and Pyr3 group was lower than that in the VEGF group, with statistically significant differences (all at P<0.05). Conclusions:Hypoxia induces increased TRPC3 expression in OIR mouse retina, and downregulation of TRPC3 inhibits HREC proliferation and migration.The mechanism is related to the activation of the Nrf2-related oxidative stress pathway.
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Objective:To observe the effect of Nodal on the biological behavior of retinal vascular endothelial cells (RF/6A cells) in monkeys with high glucose.Methods:RF/6A cells were divided into normal group, mannitol group, high glucose group, high glucose combined with non-specific small interfering RNA treatment group (HG+NC group), high glucose combined with small interfering Nodal treatment group (HG+siNodal group). The transfection efficiency of siNodal was observed by real-time fluorescence quantitative PCR and western blot protein immunoblotting. The effect of Nodal on the proliferation of RF/6A cells was detected by thiazole blue colorimetry. The effect of Nodal on migration ability of RF/6A cells was detected by cell scratch assay. The effect of Nodal on the formation of RF/6A cell lumen was measured by Matrigel three-dimensional in vitro. The expression of extracellular signal phosphorylated regulated kinase 1/2 (pERK1/2) in RF/6A cells was detected by western blot protein immunoblotting. One-way analysis of variance was used to compare groups.Results:Compared with HG+NC group, Nodal protein ( F=33.469) and mRNA relative expression levels ( F=38.191) in HG+siNodal group were significantly decreased, cell proliferation was significantly decreased ( F=28.548), and cell migration ability was significantly decreased ( F=24.182). The number of cell lumen formation was significantly decreased ( F=52.643), and the differences were statistically significant ( P<0.05). Compared with HG+NC group, the relative expression of pERK1/2 protein in HG+siNodal group was significantly decreased, and the difference was statistically significant ( F=44.462, P<0.01). Conclusions:Silencing Nodal expression can inhibit proliferation, migration and tube formation of RF/6A cells induced by high glucose. It may act by inhibiting pERK1/2 expression.
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Objective:To observe the effect of interleukin-8 (IL-8) on the adhesion and migration of retinal vascular endothelial cells (RCEC).Methods:A cell experiment. Human RCEC (hRCEC) was divided into normal control group (N group), advanced glycation end product (AGE) treatment group (AGE group), and AGE-induced combined IL-8 antagonist SB225002 treatment group (AGE+SB group). The effect of AGE on IL-8 expression in hRCEC was observed by Western blot. The effect of SB225002 on hRCEC migration was observed by cell scratch assay. The effects of SB225002 on leukocyte adhesion and reactive oxygen species (ROS) on hRCEC were detected by flow cytometry. Student- t test was performed between the two groups. Oneway analysis of variance was performed among the three groups. Results:Compared with group N, the expression level of IL-8 in cells of AGE group was significantly increased, with statistical significance ( t=25.661, P<0.001). Compared with N group and AGE+SB group, cell mobility in AGE group was significantly increased ( F=29.776), leukocyte adhesion number was significantly increased ( F=38.159, 38.556), ROS expression level was significantly increased ( F=22.336), and the differences were statistically significant ( P<0.05). Conclusion:IL-8 antagonist SB225002 may down-regulate hRCEC adhesion and migration by inhibiting ROS expression.
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Objective:To investigate the effect of Nodal protein on retinal neovascularization under hypoxia.Methods:In 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.Results:In 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). Conclusion:SB431542 can inhibit the proliferation, migration and the ability to form lumens, reduce the level of glycolysis of hRMECs cells induced by hypoxia.
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Objective:To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism.Methods:The HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. Results:The number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups ( t=12.311, 15.643, 17.346; P<0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups ( t=16.311, 14.833, 18.442; P<0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1 proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences ( t=17.342, 16.813, 18.794; P<0.001). Conclusion:PSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.
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Objective:To observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods:The experimental study was divided into two parts: in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group, with 6 mice in each group. Diabetes mice were induced by streptozotocin to establish diabetes model. Eight weeks after modeling, quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas. In vitro cell experiments: the human retinal microvascular endothelial cells (hRMEC) were divided into three groups: conventional cultured cells group (N group), empty vector transfected (Vector group); pcDNA-PAK4 eukaryotic expression plasmid transfected group (PAK4 group). WB and qPCR were used to detect transfection efficiency, while scratching assay, cell scratch test was used to detect cell migration in hRMEC of each group. In vitro white blood cell adhesion experiment combined with 4 ', 6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group. The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration, adenosine triphosphate (ATP) production, maximum respiration, and reserve respiration capacity. The t-test was used for comparison between the two groups. Single factor analysis of variance was used for comparison among the three groups. Results:In vivo animal experiments: compared with normal control group, the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased, with statistical significance ( t=25.372, 22.419, 25.372; P<0.05). In vitro cell experiment: compared with the N group and Vector group, the PAK4 protein, mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased, with statistical significance ( F=36.821, 38.692, 29.421; P<0.05). Flow cytometry showed that the adhesion number of leukocytes on hRMEC in PAK4 group was significantly increased, and the difference was statistically significant ( F=39.649, P<0.01). Mitochondrial pressure measurement results showed that the capacity of mitochondrial basic respiration, ATP production, maximum respiration and reserve respiration in hRMEC in PAK4 group was significantly decreased, with statistical significance ( F=27.472, 22.315, 31.147, 27.472; P<0.05). Conclusion:Over-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.
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Objective:To observe the effect of metformin (Met) on inflammatory bodies and focal death in human retinal microvascular endothelial cells (hRMEC) in diabetes mellitus (DM) microenvironment.Methods:Experimental research was divided into in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 9 healthy C57BL/6J male mice were randomly divided into DM group, normal control group, and DM+Met group, with 3 mice in each group. DM group and DM+Met group mice were induced by streptozotocin to establish DM model, and DM+Met group was given Met 400 mg/ (kg · d) intervention. Eight weeks after modeling, the expression of NLRP3, cleaved-membrane perforating protein D (GSDMD) and cleaved-Caspase-1 in the retina of mice in the normal control group, DM group and DM+Met group were observed by immunohistochemical staining. In vitro cell experiments: hRMEC was divided into conventional culture cell group (N group), advanced glycation end products (AGE) group, and AGE+Met group. Joining the AGE, AGE+Met groups cells were induced by 150 μg/ml of glycation end products, and 2.0 mmol/L Met was added to the AGE+Met group. Pyroptosis was detected by flow cytometry; 2' ,7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect the expression of reactive oxygen species (ROS) in cells of each group. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the relative mRNA and protein expression levels of NLRP3, cleaved-GSDMD, cleaved-Caspase-1 in each group of cells. Single factor analysis of variance was used for comparison among the three groups.Results:In vivo animal experiments: compared with the DM group, the expression of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in the retina of normal control group and DM+Met group mice was significantly reduced, with significant difference among the 3 groups ( F=43.478, 36.643, 24.464; P<0.01). In vitro cell experiment and flow cytometry showed that the pyroptosis rate of AGE group was significantly higher than that of N group and AGE+Met group ( F=32.598, P<0.01). The DCFH-DA detection results showed that the intracellular ROS levels in the N group and AGE+Met group were significantly lower than those in the AGE group, with the significant difference ( F=47.267, P<0.01). The mRNA ( F=51.563, 32.192, 44.473; P<0.01) and protein levels ( F=63.372, 54.463, 48.412; P<0.01) of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in hRMEC of the AGE+Met group were significantly reduced compared to the N group. Conclusion:Met can down regulate the expression of NLRP3 inflammatory body related factors in hRMEC and inhibit pyroptosis.
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Objective:To investigate the effect of polypeptide N-acetylgalactosaminaminyltransferase 2 (GALNT2) on the proliferation and apoptosis of human retinal vascular endothelial cells (HRCECs) cultured in high glucose and its possible mechanism.Methods:The small hairpin RNA (shRNA) targeting GALNT2 gene was constructed to interfere with the lentiviral vector and infect HRCECs.HRCECs were divided into blank control group, model group, NC-shGALNT2 group and shGALNT2 group, which were cultured in medium containing 5.5 mmol/L glucose, 25 mmol/L glucose, shGALNT2 negative control virus 25 mmol/L glucose and shGALNT2 knockdown virus 25 mmol/L glucose for 24 hours, respectively.The relative expression of GALNT2 mRNA in the four groups was detected by real-time fluorescence quantitative PCR.The relative expression levels of GALNT2, epidermal growth factor (EGF), EGF receptor (EGFR) and phosphorylated EGFR (p-EGFR) were detected by Western blot.The proliferative values of HRCECs were detected by cell counting kit-8 method.The apoptosis rate of different groups was detected by flow cytometry. Results:The relative expression levels of GALNT2 mRNA and protein were significantly higher in model group than in blank control group, and were significantly lower in shGALNT2 group than in blank control group (all at P<0.05). The cell proliferation value was significantly lower in model group than in blank control group, and was significantly higher in shGALNT2 than in model group and NC-shGALNT2 group (all at P<0.05). The apoptosis rates of blank control group, model group, NC-shGALNT2 group and shGALNT2 group were (4.73±0.26)%, (8.66±0.25)%, (9.26±1.12)% and (5.47±0.18)%, respectively, with a significant overall difference ( F=342.921, P<0.001). The apoptosis rate was significantly higher in model group than in blank control group, and was significantly lower in shGALNT2 group than in model group and NC-shGALNT2 group (all at P<0.05). The relative expression level of EGFR protein was significantly higher and the relative expression level of p-EGFR protein was significantly lower in model group than in blank control group (all at P<0.05). The relative expression of p-EGFR protein was significantly higher in shGALNT2 group than in model group (all at P<0.05). Conclusions:Knocking down GALNT2 can improve the proliferative ability of HRCECs under high glucose culture and reduce apoptosis, which may be related to the activation of EGFR signaling pathway.
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@#AIM: To explore the effect of dapagliflozin on the apoptosis and oxidative stress of high glucose-induced human retinal vascular endothelial cells and its regulatory effect on forkhead FOXO4. <p>METHODS: High glucose-induced human retinal vascular endothelial cells(HRVECs)were used to establish a cell injury model(high glucose group). Experimental groups include high glucose+dapagliflozin low-dose group(1ng/L dapagliflozin), high glucose+dapagliflozin medium-dose group(5ng/L dapagliflozin), high glucose+dapagliflozin high-dose group(10ng/L dapagliflozin), high glucose+dapagliflozin high-dose+pcDNA group, high glucose+dapagliflozin high-dose+pcDNA-FOXO4 group, and normal sugar group(5.5mmol/L D-glucose). Flow cytometry was used to detect the apoptosis rate. The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were tested with corresponding kits. Western blot assay was used to detect the protein level of FOXO4. <p>RESULTS: Compared with the normal sugar group, the apoptosis rate(<i>P</i><0.05), the level of MDA(<i>P</i><0.05)and FOXO4(<i>P</i><0.05)were increased, but the level of SOD was decreased(<i>P</i><0.05)in high-glucose group. Compared with the high glucose group, cell apoptosis rate(<i>P</i><0.05), the level of MDA(<i>P</i><0.05)and the protein level of FOXO4 were decreased(<i>P</i><0.05), but the level of SOD was increased(<i>P</i><0.05)in high glucose+medium-dose dapagliflozin group and high glucose+high-dose dapagliflozin group. Compared with high glucose+dapagliflozin high-dose+pcDNA group, the apoptosis rate(<i>P</i><0.05)and the level of MDA(<i>P</i><0.05)were increased, but the level of SOD was decreased(<i>P</i><0.05)in high glucose+dapagliflozin high-dose+pcDNA-FOXO4 group(<i>P</i><0.05). <p>CONCLUSION: Dapagliflozin could inhibit oxidative stress and cell apoptosis in high glucose-induced HRVECs by down-regulating FOXO4, thereby reducing cell damage.
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@#miRNA-15a(miR-15a)is a non-coding small molecule RNA located on 13q14 gene. It affects the growth, development, differentiation and apoptosis of all organs and cells of the whole body. As the study progressively deepened, it was found that the role of miR-15a in different tissues and cells was not entirely consistent. Sometimes it plays a role in suppressing cancer, and sometimes it promotes cancer. The signal pathways it affects are complex and diverse. With the deepening of biological research into cell signaling pathways, miRNA-15a has become a miRNA more extensively studied. But in the ophthalmology, the corresponding research is not much. In this article, we mainly focus on the mechanism of miR-15a and its current research situation in ophthalmic diseases, so as to provide a reference for further study and their treatment.
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@#AIM: To investigate the effect of microRNA-96-5p(miR-96-5p)on proliferation and apoptosis of rat retinal vascular endothelial cells induced by high glucose and to explore its mechanism. <p>METHODS: SD rat retinal vascular endothelial cells(RRVEC)were cultured and the RRVEC was divided into control group(NG)and high glucose group(HG). The high glucose-induced RRVECs were harvested separately or co-transfected with miR-96-5p mimic, miR-NC, si-FOXO4, si-NC. The expression of miR-96-5p and FOXO4 was detected by qRT-PCR and Western blotting, respectively. MTT assay was used to detect the proliferation activity. Flow cytometry was used to detect the apoptosis rate. The dual luciferase reporter assay validated the target gene of miR-96-5p. Western blotting was used to detect the expression of CyclinD1, p21, p27, Bcl-2, Bax and cleaved-caspased-3. <p>RESULTS:The expression levels of miR-96-5p, CyclinD1 and Bcl-2 in RRVEC were significantly decreased after high glucose treatment, and the expression levels of FOXO4, p21, p27, Bax and cleaved-caspased-3 were significantly increased, inhibiting cell proliferation activity, but promoting apoptosis. Overexpression of miR-96-5p and inhibition of FOXO4 expression increased the expression levels of CyclinD1 and Bcl-2, inhibited the expression of p21, p27, Bax, cleaved-caspased-3, enhanced cell proliferation and inhibited apoptosis. Dual luciferase reporter assay demonstrated that FOXO4 was a target gene for miR-96-5p. Overexpression of FOXO4 reversed the effect of miR-96-5p overexpression on high glucose-induced proliferation and apoptosis of RRVEC. <p>CONCLUSION:miR-96-5p inhibits high glucose-induced apoptosis of rat retinal vascular endothelial cells and promotes cell proliferation by targeting FOXO4.
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@#miRNA-15a(miR-15a)is a non-coding small molecule RNA located on 13q14 gene. It affects the growth, development, differentiation and apoptosis of all organs and cells of the whole body. As the study progressively deepened, it was found that the role of miR-15a in different tissues and cells was not entirely consistent. Sometimes it plays a role in suppressing cancer, and sometimes it promotes cancer. The signal pathways it affects are complex and diverse. With the deepening of biological research into cell signaling pathways, miRNA-15a has become a miRNA more extensively studied. But in the ophthalmology, the corresponding research is not much. In this article, we mainly focus on the mechanism of miR-15a and its current research situation in ophthalmic diseases, so as to provide a reference for further study and their treatment.
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@#AIM: To observe the effect of adiponectin(APN)on proliferation, migration and tube formation of RF/6A(monkey choroid and retinal endothelial cell line)cells cultured in the conditions of high glucose.<p>METHODS: Well-cultured RF/6A cells <i>in vitro</i> were randomly divided into four groups: blank control group, mannitol control group, high glucose group and high glucose+10μg/mL APN group. Cell proliferation, migration and tube formation was detected by CCK-8 assay, transwell chamber and matrigel assay, respectively.<p>RESULTS: There was no significant difference in the cell proliferation rate(100.00%±0.00% <i>vs</i> 99.09%±0.46%), the number of cell migration(121.60±6.02 <i>vs</i> 119.60±9.39)and the number of tube formation(12.11±0.84 <i>vs</i> 12.22±0.96)between the blank control group and mannitol control group(all <i>P</i>>0.05). The cell proliferation rate of the high glucose group(71.42%±2.29%)was significantly lower than that of the blank control group and the mannitol control group, and the number of cell migration(144.20±9.65)and tube formation(16.00±2.90)of the high glucose group were significantly higher than that of the blank control group and the mannitol control group(all <i>P</i><0.05). The proliferation rate of cells in the high glucose+APN group(90.87%±1.68%)was significantly lower than that of the blank control group and the mannitol control group, while higher than that in the high glucose group(all <i>P</i><0.05). The number of cell migration(73.00±6.04)and tube formation(7.89±0.38)in the high glucose+APN group was significantly lower than that of the blank control group, the mannitol control group and the high glucose group(all <i>P</i><0.05).<p>CONCLUSION: APN can promote the survival and proliferation of RF/6A cells, and inhibit the migration and tube formation of RF/6A cells under high glucose conditions, suggesting that APN has a protective effect on the damage of RF/6A cells caused by high glucose and inhibits the angiogenesis process under pathological stimulations.
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Objective To investigate the effects of HMGB1 small interference RNA (siRNA) on retinal vascular endothelial cells.Methods siRNA was used to inhibit the expression of HMGB1,followed by the application of CCK8 assay,Hochest33342 staining and flow cytometry to observe the effects of HMGB1 siRNA on retinal vascular endothelial cells in high glucose environment.Meanwhile,the expression of proteins related to apoptosis was detected by Western blot.Results The transfection of HMGB1 siRNA down-regulated the protein expression level of HMGB1 by 73％ in siRNA group compared with normal control (NC) group (P ＜ 0.05),and the protein expression level of HMGB1 in siRNA group was decreased by 75％ compared with scr-siRNA group (P ＜ 0.05).There was no significant difference between NC group and scrsiRNA group (P ＞ 0.05).The total apoptotic rate of NC group,high-glucose group,scrsiRNA group and siRNA group was (0.40 ± 0.03)％,(49.80 ± 3.50)％,(47.60 ±1.98) ％ and (23.60 ± 2.40) ％ by flow cytometry.Compared with NC group,the apoptotic rates of high-glucose group,scr-siRNA group and siRNA group were increased (all P ＜ 0.05).Compared with scr-siRNA group,the apoptotic rate of HRECs in siRNA group was reduced,with significant statistical difference (P ＜ 0.05).There was no significant difference in the rate of cell apoptosis between scr-siRNA group and high-glucose group (P ＞ 0.05).Compared with the NC group,the protein expression level of cleavedcaspase3 protein in high-glucose group and scr-siRNA group were increased by (233 ±10) ％ and (266 ± 22) ％,respectively (both P ＜ 0.05);compared with scr-siRNA group,the protein expression level of cleaved-caspase 3 in siRNA group was reduced by (43 ±3) ％ (P ＜ 0.05);and there was no significant difference in the protein expression of cleaved-caspase 3 in high-glucose group and scr-siRNA group (P ＞ 0.05).Compared with the NC group,the protein expression of B-cell lymphoma-2 (Bcl-2) in high-glucose group and scr-siRNA group was decreased by (32 ± 2) ％ and (29 ± 3) ％,accordingly (both P ＜ 0.05);compared with scr-siRNA group,the protein expression level of Bcl-2 in siRNA group was increased by (42 ± 2) ％ (P ＜ 0.05);and there was no significant difference in the protein expression of Bcl-2 in high-glucose group and scr-siRNA group (P ＞ 0.05).Conclusion HMGB1 siRNA can reduce the apoptosis of retinal vascular endothelial cells in high glucose environment by inhibiting the activation of cleavedcaspase3 and increasing the expression of Bcl-2.
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@#AIM: To study the effects of FOXO4 on oxidative stress and apoptosis of retinal vascular endothelial cells under high glucose environmental conditions. <p>METHODS: Human retinal vascular endothelial cells were cultured with high glucose medium. Real-time PCR and Western blot were used to detect the expressions of FOXO4 within cells; meanwhile, both FOXO4 RNAi lentivirus and control vector lentivirus were infected the retinal vascular endothelial cells cultured in high sugar culture medium. Real-time PCR and Western blot techniques were used to detect the interference efficiency. After collection of supernatant and cells treated with various interferences, the SOD activity, MDA content in the supernatants and ROS level within cells were detected. Flow cytometry was used to determine the changes of cell apoptosis. Western blot was used to detect the expressions of apoptotic protein cleaved Caspase-3 and cleaved Caspase-9 within cells. <p>RESULTS: The expression of FOXO4 was increased in retinal vascular endothelial cells after treatment with high glucose medium. FOXO4 RNAi lentivirus infection reduced the expression level of FOXO4 in retinal vascular endothelial cells under high glucose environmental conditions. By contrast, control vector lentivirus had no effect on FOXO4 expression in cells. High glucose induced elevated levels of ROS in retinal vascular endothelial cells, reduced the activity of SOD in cell culture medium, increased the content of MDA, elevated the rate of apoptosis, and promoted the expressions of cleaved Caspase-3 and cleaved Caspase-9 proteins in cells. After down-regulation of FOXO4 expression, retinal endothelial cells were induced by high glucose, the activity of SOD in the cell culture medium increased, the levels of MDA, ROS, apoptosis, and the levels of cleaved Caspase-3 and cleaved Caspase-9 proteins decreased in cells as compared with those of retinal vascular endothelial cells. Moreover, compared with those did not interfere with FOXO4 expression, there was statistically significant differences(<i>P</i><0.05). <p>CONCLUSION: High glucose induces the expression of FOXO4 in retinal vascular endothelial cells. Knocking-down of FOXO4 expression reduces oxidative stress and apoptosis induced by high glucose medium.
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AIM:To investigate effects of resveratrol on mitochondrial DNA copy in retinal vascular endothelial cells cultured under high glucose conditions and its mechanism.METHODS:Human retinal vascular endothelial cells were cultured in low glucose or high glucose medium,the genomic DNA was extracted and copy number of mitochondrial DNA was detected by real-time PCR.Effects of resveratrol on the mitochondrial DNA copy in retinal vascular endothelial cells cultured under high glucose medium were studied.The expression of mitochondrial transcription factor A (TFAM) and acetylated proliferator-activated receptor coactivator-1 alpha (PGC-1α) were analyzed by Western blot and coimmunoprecipitation.RESULTS:High glucose inhibited the copy number of mitochondrial DNA in retinal vascular endothelial cells.However,resveratrol decreased the level of acetylated PGC-1α in retinal vascular endothelial cells,increased the expression of TFAM and the copy number of mitochondrial DNA.CONCLUSION:Resveratrol may improve mitochondrial function of retinal vascular endothelial cells exposed to a high glucose environment via activation of the PCG-1α-TFAM signaling pathway.
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Objective To observe the effects of Ligustrazine on proliferation of human retinal vascular endothelial cells (HRCECs) induced by high glucose.Methods HRCECs cells were divided into control (saline) group,model (25 mmol/L glucose) group,ligustrazine low,medium,high concentration (50,100 and 200 mol/L) group,cultured for 48 h.Cell proliferation was detected by MTT cytotoxicity assay.Flow cytometry was used to detect cell cycle,and the expression of VEGF was detected by ELISA analysis.Results Compared with control group,HRCECs cell proliferation,cell division (M) phase ratio and supematant of VEGF concentration in model group increased significantly (P ＜ 0.05,0.01).Compared with model group,cell proliferation,M phase ratio and supematant of VEGF in Ligustrazine low,medium and high concentration groups decreased significantly (P ＜ 0.05,0.01),and showed a concentration dependence.Conclusion Ligustrazine can inhibit the expression of VEGF in HRCECs cells induced by high glucose to block cell cycle and inhibit the proliferation of HRCECs cells.
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Background The suppression of retinal angiogenesis is one of primary treatment targets for retinal vascular diseases,so seeking the intervention targets of retinal neovascularization is a hot research.Studies showed that insulin-like growth factor 1 (IGF-1) can promote the growth and restrain the apoptosis of vascular endothelial cells.However,whether IGF-1 is an intervention target for the treatment of retinal vascular diseases is unelucidated.Objective This study was to address the effects of IGF-1 on the migration,apoptosis and capillary tube formation of human retinal vascular endothelial cells (HRECs) and mechanism.Methods HRECs were cultured in vitro,and the cells in the exponential phase were prepared for subsequent experiments.The expression of IGF-1R mRNA in the cells was examined using reverse transcriptase PCR assay.Different concentrations of IGF-1 were added in the medium based on the difference of tests.The relative free-cell area difference (△S) after test was measured by Photoshop CS4 software and compared among 0,10 and 200 ng/ml IGF-1 groups 12 and 24 hours after cell scratching,respectively.The cell apoptotic rate was assayed by flow cytometry and compared between 0 ng/ml IGF-1 group and 1 000 ng/ml IGF-1 group,and the number of capillary tubes was examined by Matrigel test and assessed among 0,10,100 and 200 ng/ml IGF-1 groups 24 hours after addition of IGF-1.The expressions of platelet derived growth factor (PDGF)-BB mRNA and caspase-3 mRNA in the cells of the 0,500 and 1 000 ng/ml IGF-1 groups were detected by real-time fluorescence quantitative PCR after adding IGF-1 for 6 hours.Results Cultured cells grew well and attached 90％ confluence 2-3 days after incubation,and IGF-1R mRNA was positively expressed in the cells.In 12 and 24 hours after scratching,the relative migrating area of the cells was gradually reduced with the increase of IGF-1 contents.The △S was (4.83 ± 0.61) × 105 μm2 in the 200 ng/ml IGF-1 group,which was significantly larger than (3.28±0.64) ×105 μm2 in the 0 ng/ml IGF-1 group 24 hours after stretching (t=-3.707,P=0.021).The apoptotic rate in the 0 ng/ml IGF-1 group and 1 000 ng/ml IGF-1 group was (18.77±2.37) ％ and (12.05 ±0.88) ％,with a significant difference between them (t =2.869,P =0.046).The number of intact tubes was significantly increased in the 200 ng/ml IGF-1 group compared with the 0 ng/ml IGF-1 group ([20.33±2.83]/well vs.[17.94± 1.96]/well;t =-2.940,P =0.042).Compared with 0 ng/ml IGF-1 group,the relative expression level of PDGF-BB mRNA was elevated and that caspase-3 mRNA was evidently reduced in the 1 000 ng/ml IGF-1 group (t=-3.489,P =0.025;t =7.287,P =0.002).Conclusions IGF-1 can promote the migration and angiogenesis of HRECs and inhibit the apoptosis of HRECs.These effects of IGF-1 probably are associated with the up-regulation of PDGF-BB and down-regulation of caspase-3 in the cells.
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Background Oxidative damage may cause the functional dysfunction and death of retinal vascular endothelial cells(RVECs),and further leads to the development of retinal vascular diseases.Fufang xueshuantong has a therapeutic effect on retinal vascular diseases,but little is known about its molecular mechanism.Objective The goal of this study was to investigate the protective effects and mechanism of fufang xueshuantong on injury of human RVECs induced by tert-butyl hydroperoxide(t-BHP).Methods Human RVECs were isolated from healthy donor eyes and primarily cultured and then identified by flow cytometry.The third to fifth generations of cells were used in this experiments.The fufang xueshuantong solution of 0.0625,0.1250,0.2500,0.5000 and 1.0000 g/L were added in the cuhure plate with 5 × 104/L cells respectively in the experimental groups,and t-BHP of 75,100,200 and 300 μ.mol/L were added in the model control groups.MTT was used to detect the A490and survival rate of RVECs.The apoptotic rate and death rate of the cells were evaluated by double staining of Annexin V-FITC/PI.Morphology of human RVECs were examined using invert microscopy and Hoechst33258 staining.The expressions of nitro tyrosine (a marker of oxidative damage of protein)and 8-OHdG(a marker of oxidative damage of DNA)in human RVECs were assessed by the immunofluorescence staining.Western blot was used to detect the expressions of nuclear factorkappa B(NF-KB),p53,bcl-2 and bax after 6,12,24 hours t-BHP action.This study was approved by the Ethic Committee of Zhongshan Ophthalmic Center.Results No significant difference was found in A490value among the normal control group,0.0625,0.1250,0.2500,0.5000 and 1.0000 g/L fufang xueshuantong groups(F =1.989,P＞0.05).The survival rates of the cells were lower in 75,100,200 and 300 μmol/L t-BHP groups compared with corresponding fufang xueshuantong groups(t =14.57,13.82,21.51,32.64,P＜ 0.01).The percentages of normal cells were evidently lower in 75,100,200 and 300 μmol/L t-BHP compared with corresponding fufang xueshuantong groups(t=14.908,5.495,17.165,26.330,P＜0.01).The numbers of deformation and death of the human RVECs increased as the elevated concentration of t-BHP,but those in fufang xueshuantong groups were less than the t-BHP groups under the invert microscopy.Compared with t-BHP groups,the expressions of nitro tyrosine,8-OHdG,NF-KB,p53 and bax were lower but the expression of bcl-2 was higher in human RVECs with the statistically significant differences(P＜0.05).Fufang xueshuantong at the concentration of 0.2500 g/L showed maximally protective effect on human RVECs.Conclusions Fufang xueshuantong protects human RVECs against the t-BHP-induced injury through downregulating the expression of NF-kB,p53,bax and up-regulating the express of the bcl-2 protein.