ObjectiveTo investigate the influence of Ataxia-telangiectasia mutated (ATM) activation on cellular oxidative stress induced by high glucose in bovine retinal capillary endothelial cells(BRECs). Methods The BRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 30 mmol/L glucose, 30 mmol/L glucose+10 μmol/L KU55933) as normal glucose group, high glucose group and treatment group respectively.After the cells incubated for 48 hours, the protein expression of ATM, P-ATM, Mitogen-Activated Protein Kinase P38(P38), P-P38, Extracellular signal-regulated kinases(ERKs), P-ERKs was detected by Western blot; cellular ROS level was detected by Reactive Oxygen Species Assay Kit; propidium iodide/Hoechst staining was used for analysis of apoptosis; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by Enzyme-Linked Immunosorbent Assay (ELISA); the paracellular permeability between endothelium cells was detected by FITC-dextran. ResultsCompared with the protein level of P-ATM, P-P38 and P-ERKs in high glucose group increased. Especially, P-P38, P-ERKs expressed much more than in high glucose group. The secretion of VEGF in high glucose group was higher than that in the normal glucose group but less than that in treatment group. The same tendency existed in ROS assay, apoptosis assay and paracellular permeability measuring. ConclusionsHigh glucose induced altered activation of ATM which might play a protective role in cellular oxidative stress. Deficiency of ATM might lead to ROS explosion, cell apoptosis and dysfunction of endothelial barrier. The mechanism might be associated with P38, ERKs and VEGF.
Objective To investigate the effects of exosomes from cultured human retinal pigment epithelium (ARPE-19) cells affected by oxidative stress on the proliferation and expression of vascular endothelial growth factor-A (VEGF-A) and Akt of ARPE-19 cells. Methods Culture ARPE-19 cells. The concentration of 2.5 μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome. Exosomes were isolated by ExoQuick exosome precipitation solution. Transmission electron microscopy was used to identify the morphology of exosomes. Western blot was used to detect exosomes’ surface-specific maker protein CD63. ARPE-19 cells affected by oxidative stress were cultured with exosome as experimental group, normal ARPE-19 cells were cultured with exosome as control group. The cell proliferation was examined by methyl thiazolyl tetrazolium assay. Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF-A mRNA and Akt mRNA. Results The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm. The isolated exosomes expressed CD63. AREP-19 cells were cultured with ARPE-19 (affected by rotenone)-exosome, the cell viability in experimental group was significantly reduced than in the control group. Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the control group, VEGF-A was up-regulated expressed and Akt was down-regulated expressed. Western blot results showed that, VEGF-A protein expression in the experimental group were higher than the control group. Akt protein expression in the experimental group were less than the control group. The difference was statically significant (t=3.822, 6.527;P＜0.05). RT-PCR results showed that VEGF-A mRNA expression levels was higher in the experimental group than the control group. Akt mRNA expression levels was lower in the experimental group than the control group. The difference was statically significant (t=8.805, −7.823;P＜0.05). Conclusions Exosomes from ARPE-19 cells affected by oxidative stress inhibit the proliferation of normal ARPE-19 cells, increase the expression of VEGF-A and reduce the expression of Akt.
Metabolic memory means if the hyperglycemia can't be controlled at early stage of diabetes, chronic complications such as diabetic retinopathy (DR) will continue to develop even if the blood glucose level maintains normal level at later stage. Oxidative stress plays an important role in the "metabolic memory" of DR, which interacts with the nitrative stress, advanced glycation end products, genetic modification and endoplasmic reticulum stress in the pathogenesis of DR. Further elucidation of the relationship between oxidative stress and "metabolic memory" of DR can open the way for the discovery of novel therapeutic targets to prevent DR progression.
MiRNAs are stable small RNAs that are expressed abundantly in animals and plants. They can bind to the 3'-untranslated region of the target mRNA, and regulate its expression at the post-transcriptional level. The miRNAs’ abnormal expression and its following abnormal biological regulation are closely related to the occurrence and development of age-related macular degeneration (AMD), including inflammatory response, oxidative stress injury, phagocytosis dysfunction and abnormal angiogenesis. Since the dysregulation of miR-155, miR-125b and miR-34a seems to play a more important role in AMD, these microRNAs may be expected to become the new biomarkers and therapeutic targets for AMD.
Age-related macular degeneration (AMD) is a multifactorial disease affected by environmental factors and genetic variation, which is a major cause of irreversible vision loss in the elderly. miRNA is a kind of endogenous non-coding RNA, which plays an important role in the pathogenesis of AMD, such as oxidative stress, pathological neovascularization and inflammation, by inhibiting or silencing the expression of transcription genes. miRNA has unique advantages in terms of ease synthesis, targeting and additive effect, a large number of experiments have proved the therapeutic potential of miRNA in AMD, which is expected to become a new method for the treatment of AMD in the future. Since the pathogenesis of AMD has not been fully elucidated, it is still necessary to continue to study the pathogenesis of AMD, the biological effects and mechanisms of various miRNA in the occurrence and development of AMD, and observe its therapeutic effects in AMD, so as to provide more effective options for the precise prevention and treatment of AMD.