ObjectiveTo explore the effect of cathepsin L (CTSL) inhibitor on apoptosis of retinal pigment epithelial (RPE) cells and mitochondrial oxidative stress. MethodsRPE cells were cultured in vitro and divided into control group, hydrogen peroxide (H2O2) group, and H2O2+CTSL inhibitor group. The cells of H2O2 group and H2O2+CTSL inhibitor group were incubated in the medium containing 400 μmol/L H2O2 for 24 hours and 10 μmol/L CTSL inhibitor was added in H2O2+CTSL inhibitor group at the same time. The cells of normal group were routinely cultured cells. The follow-up experiment was carried out 24 hours after modeling. The rate of apoptosis was detected by flow cytometry. The expression of CTSL was detected by immunofluorescence staining, Western blot and real time-polymerase chain reaction. The level of mitochondrial super oxide was detected by MitoSOX fluorescent probe, and the mitochondrial structure was observed after MitoTracker staining, the average area, form factors, and branch of mitochondria were quantitatively analyzed. The two groups were compared using two-tailed Student t test, while numerous groups were compared using one-way ANOVA. ResultsCompared with control group, the rate of apoptosis in H2O2 group was significantly higher (t=3.307, P=0.029 7), the expression level of CTSL was significantly increased (t=19.950, 6.916, 14.220; P＜0.05). Compared with H2O2 group, the expression level of CTSL, the rate of apoptosis and the mitochondrial ROS level in H2O2+CTSL inhibitor group were significantly lower (t=11.940, 4.718, 16.680; P＜0.05). The mitochondria of H2O2+CTSL inhibitor group were elongated, oval-shaped or rod-shaped, while the mitochondria of H2O2 group lost their continuous contour shape and complete structure. The differences of the average area, form factors, and brach of mitochondria among 4 groups were statistically significant (F=251.700, 34.010, 60.500; P＜0.000 1). ConclusionsH2O2 can significantly induce apoptosis in RPE cells and increase CTSL expression. CTSL inhibitor can inhibit the H2O2-induced apoptosis of RPE cells, lower the mitochondrial super oxide level, and successfully repair the mitochondrial structure.
With the advancement of molecular biology technology and the development of genetics, the viral vector system has been continuously improved and optimized. The viral vector system has gradually become one of the best carriers in ophthalmic gene therapy. Adenovirus vector has the characteristics of transient expression and plays an important role in reducing corneal immune response. Lentiviral vector has the characteristics of stable and high efficiency and can be expressed slowly in the body for a long time.Adeno-associated virus vector has the characteristics of low immunogenicity, high efficiency and precision and can infect a variety of retinal cells. The combined use of adeno-associated virus vector and CRISPR-Cas9 provides new methods for precise treatment of ophthalmic genetic diseases. The advent of viral vectors has significantly increased the potential of gene therapy and has unparalleled advantages over traditional therapies. We have reason to believe that virus-based gene transduction technology will soon achieve clinical application in the near future, and a large number of difficult ophthalmic problems will be solved by then.
Objective To explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs). MethodsA experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by real-time quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups. ResultsThe ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant (F=53.40, 50.30; P＜0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant (F=54.35, 52.84, 84.35; P＜0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant (F=53.66, 83.54; P＜0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant (F=24.87, 53.84; P＜0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P＜0.001; BMP4 group: F=56.87, 59.35, 58.35; P＜0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group (F=48.32, 56.33, 55.01; P＞0.05). ConclusionBMP4 induces the proliferation and migration of hRMECs through glycolysis.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.
With the development of life sciences and informatics, bioinformatics is developing as an interdisciplinary subject. Its main application is the relationship between genes and proteins and their expression. With the help of genomics, proteomics, transcriptomics, and metabolomics, researchers introduce bioinformatics research methods into fundus disease research. A series of gratifying research results have been achieved including the screening of genetic susceptibility genes, the screening of diagnostic markers, and the exploration of pathogenesis. Genomics has the characteristics of high efficiency and accuracy. It has been used to detect new mutation sites in retinoblastoma and retinal pigment degeneration research, which helps to further improve the pathogenesis of retinal genetic diseases. Transcriptomics, proteomics, and metabolomics have high throughput characteristics. They are used to analyze changes in the expression profiles of RNA, proteins, and metabolites in intraocular fluid or isolated cells in disease states, which help to screen biomarkers and further elucidate the pathogenesis. With the advancement of technology, bioinformatics will provide new ideas for the study of ocular fundus diseases.
Objective To explore the effect of SB431542 on monkey choroidal-retinal endothelial (RF/6A) cells in high glucose state and its mechanism of regulating mitochondrial autophagy by mediating the PINK1/Parkin pathway. MethodsCell experiments. The minimum effective drug concentration of SB431542 was determined by using the Cell Counting Kit-8 (CCK-8). RF/6A cells cultured in vitro were divided into normal group (NC group), mannitol group, high glucose group (HG group), high glucose with dimethyl sulfoxide group (HG+DMSO group), and high glucose+SB431542 group (HG+SB431542 group). CCK-8 and cell scratch assay were used to detect the proliferation and migration of RF/6A cells induced by high glucose. The expression of autophagosomes was detected by autophagy staining kit; the expression level of reactive oxygen species was detected by reactive oxygen species kit; the expression level of mitochondrial superoxide in cells was detected by MitoSOX fluorescent probe; the mitochondrial membrane potential level in cells was detected by JC-10 staining; the morphology of mitochondria was observed by MitoTracker staining, and the total area of mitochondria, average shape factor and branch length were quantitatively analyzed.Cellular immunofluorescence (IF) staining was used to detect the fluorescence expression of EndMT markers vimentin and VE-cadherin; Western blotting (WB) was used to detect the protein expression of vimentin, VE-cadherin, and mitochondrial autophagy-related proteins TOMM20, LC3, P62, PINK1, and Parkin. One-way analysis of variance was used for comparisons among multiple groups. ResultsThe minimum effective drug concentration of SB431542 was 5 μmol/L. SB431542 significantly inhibited the proliferation and migration of RF/6A cells induced by high glucose (F= 81.92, 87.84; P＜0.000 1). SB431542 suppressed the expression of reactive oxygen species and mitochondrial superoxide induced by high glucose (F= 429.50, 450.20; P＜0.000 1), restored the mitochondrial membrane potential level (F=315.3, P＜0.000 1), and restored the mitochondrial morphology (F=209.50, P＜0.000 1). IF and WB confirmed that SB431542 inhibited the expression of Vimentin induced by high glucose (F=117.30、51.11; P＜0.000 1) and upregulated the expression of VE-cadherin (F=136.80, 27.54; P＜0.000 1). WB further confirmed that SB431542 upregulated the protein expression of LC3, PINK1, and Parkin (F = 16.64, 37.72, 32.63; P＜0.05) and inhibited the protein expression of TOMM20 and P62 (F=33.87, 67.77; P＜0.01). ConclusionSB431542 upregulates mitochondrial autophagy expression through activation of the PINK1/Parkin pathway, effectively restores mitochondria-related functions to maintain homeostasis, and inhibits high glucose-induced RF/6A cell proliferation, migration, and EndMT formation.
ObjectiveTo observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.MethodshRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.ResultsDifferent concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant (t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant (t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1285-3p, hsa-miR-30d-3p, hsa-miR-3170, hsa-miR-7976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions.ConclusionFA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.
ObjectiveTo investigate the effects of interferon gene stimulating protein (STING) inhibitor (C176) on human retinal microvascular endothelial cells (hRMEC) under oxidative stress. MethodsAn animal experimental study. In vivo experiment: 48 healthy male C57BL/6J mice were randomly divided into wild type mice group (WT group) and diabetes (DM) group, with 24 mice in each group. DM mice were induced by streptozotocin to establish DM model. After successful modeling, DM group was divided into DM+dimethyl sulfoxide (DMSO) group and DM+C176 group, with 12 mice in each group. The mice in the DM+DMSO group were intraperitoneally injected with DMSO at the dose of 50 mg/kg. Mice in DM+C176 group were intraperitoneally injected with STING inhibitor C176 750 nmol at the dose of 50 mg/kg. Four weeks after modeling, immunohistochemical staining, Western blot and real-time fluorescence quantitative polymerase chain reaction were used to detect the expression of STING in the retina of WT and DM mice. The leukocyte adhesion test was used to detect the number of leukocytes adhering to hRMEC in mice with WT, DM+DMSO and DM+C176 groups. In vitro experiment: hRMEC was randomly divided into conventional culture cell group (N group), dimethyl sulfoxide (DMSO) group (with DMSO intervention) and C176 group (with C176 intervention). The cells were induced by 150 μg/ml glycation end products for each group. In vitro leukocyte adhesion test combined with 4', 6-diamino-2-phenylindole staining was used to detect the number of leukocytes adhering to hRMEC. The adherent leukocytes were quantitatively analyzed by flow cytometry; H2DCFDA/reactive oxygen species (ROS) fluorescence probe was used to detect ROS expression in cells; Seahorse XFe96 cell energy metabolism analyzer was used to measure the level of intracellular glycolysis. t-test was used to compare the two groups; single factor analysis of variance was used to compare the three groups. ResultsIn vivo experiment: compared with WT group, the expression level of STING (t=73.248) and the relative expression amount of mRNA (t=67.385) in the retina of DM group mice increased significantly (P＜0.05). Compared with WT group, the number of leukocytes adhering to the retinal vessels of mice in DM+DMSO group was significantly increased, while that in DM+C176 group was significantly decreased (F=84.352, P＜0.01). In vitro: compared with N group and DMSO group, the number of leukocyte adhesion on hRMEC in C176 group decreased significantly (F=35.251, P＜0.01). Compared with N group, the number of leukocytes adhering to hRMEC in DMSO group and C176 group decreased significantly (F=26.374, P＜0.01). The ROS level in hRMEC in C176 group was significantly lower than that in N group and C176 group (F=41.362, P＜0.01). Compared with N group and DMSO group, the glycolysis level of hRMEC in C176 group was significantly reduced, with a statistically significant difference (F=68.741, P＜0.01). ConclusionInhibiting the expression of STING in retinal vascular endothelial cells can improve the progress of DM by inhibiting leukocyte adhesion, ROS production and glycolysis level.
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 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.