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.
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 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.
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 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.
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.
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.
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. MethodsExperimental 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. ResultsIn 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. ConclusionMet can down regulate the expression of NLRP3 inflammatory body related factors in hRMEC and inhibit pyroptosis.
ObjectiveTo observe the effects of p21 activated kinase 4 (PAK4) on the mitochondrial function and biological behavior in retinal vascular endothelial cells. MethodsThe 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. ResultsIn 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). ConclusionOver-expression of PAK4 impairs mitochondrial function and significantly promotes leukocyte adhesion and migration in retinal vascular endothelial cells.
ObjectiveTo construct the connective tissue growth factor (CTGF) recombinant interference vector (shRNA) and observe its inhibitory effect on the expression of endogenous CTGF in retinal vascular endothelial cells. Methods The human CTGF shRNA was constructed and the high-titer CTGF shRNA lentivirus particles was acquired via three-plasmid lentivirus packaging system to infect retinal vascular endothelial cells. The optimal multiplicity and onset time of lentivirus infection were identified by tracing down the red florescent protein in interference vector. The cells were classified into three groups: blank control group, infection control group and CTGF knockdown group. The differences in cells migrating ability was observed through Transwell allay. The mRNA and protein expression of CTGF, fibronectin, α-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were quantified through real-time PCR testing and Western blot system. Data between the three groups were examined via one-way analysis of variance. ResultsThe result showed that an optimal multiplicity of 20 and onset time of 72 hours were the requirements to optimize lentivirus infection. Transwell allay result showed a contrast in the number of migrated cells in the CTGF knockdown group and that in the blank control group and infection control group (F=20.64, P=0.002). Real-time PCR testing showed a contrast in related gene expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the CTGF knocked-down group and that in the blank control group and infection control group (F=128.83, 124.44, 144.76, 1 374.44; P=0.000, 0.000, 0.000, 0.000). Western blot system showed the statistical significance of the contrasted number of related protein expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the knockdown group and that in the blank control group (F=22.55, 41.60, 25.73, 161.68; P=0.002, 0.000, 0.001, 0.000). ConclusionThe success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF expression.