ABSTRACT
Objective@#To determine the changes of protein expressions in human retinal microvascular pericytes (HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy (DR).@*Methods@#HRMPCs were divided into two groups.The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose, while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose.The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin.Peptides of 2 μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA.The results were further analyzed using bioinformatics software.@*Results@#CCK-8 results showed that the absorbance (A450) of HRMPCs in high glucose group was 0.75±0.04, which was significantly lower than 0.91±0.05 in control group (t=5.784, P=0.000 2). In total, 1 972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1.5). Among them, 13 proteins were up-regulated, including CTNNB1 and CTBP2; while 41 proteins were down-regulated, including SQSTM1 and HMGCS1.The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration.@*Conclusions@#The expressions of many proteins in HRMPCs change under the stimulation of high glucose, which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.
ABSTRACT
Objective To detect the protein expression change in the proliferation of human retinal microvascular endothelial cells (hRMECs) stimulated with 4-Hydroxynonenal (4-HNE).Methods hRMECs were in a logarithmic growth phase, and then were separated into 4-HNE-stimulated group and negative control group. The concentration of 4-HNE included 5, 10, 20 and 50 μmol/L in 4-HNE-stimulated group, while the negative control group was added in the same volume of ethanol (the solvent of 4-HNE). Then the cells were stimulated with 4-HNE for 24 hours following by CCK-8 kits incubating for 4 hours to detect absorbance. It was found that 10 μmol/L 4-HNE had the most obvious effect on the proliferation of hRMECs. Therefore, the cellular proteins from 10 μmol/L 4-HNE-stimulated group and negative control group were acquired and prepared by FASP sample preparation method. Data independent acquisition was used for data acquisition, and the GO analysis and pathway enrichment were performed for analysis of differentially expressed proteins. Results CCK-8 kits detection results showed that theA value of the 10 and 20 μmol/L 4-HNE-stimulated groups were significantly higher than negative control group and 5 μmol/L 4-HNE-stimulated group (F=25.42, P<0.01), while there were no differences between 10 and 20 μmol/L 4-HNE-stimulated groups, and theA value of 50 μmol/L 4-HNE-stimulated groups was lower than negative control. A total of 2710 quantifiable proteins were identified by peoteomics,and 118 proteins were differentially expressed (fold change>1.5,P<0.05). Seventy-two proteins were up-regulated after 4-HNE stimulation, whereas 46 proteins were down-regulated. Particularly, the expressions of Heme oxygenase-1, Sulfoxdoxin-1, Heat shock protein A1B, Thioredoxin reductase-1, Glutathione reductase, ATPase and prothrombin were increased when cells were added in 4-HNE, whereas the expressions of apolipoprotein A1 and programmed cell death protein 4 were decreased. These differentially expressed proteins were mainly involved in the biological processes such as oxidative stress, cell detoxification, and ATPase-coupled membrane transport.Conclusion After stimulated with 4-HNE, the oxidative stress, cell detoxification, and ATPase-coupled membrane transport protein expression may change in hRMECs in order to regulate oxidative stress and growth situation.
ABSTRACT
Objective To determine the changes of protein expressions in human retinal microvascular pericytes ( HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy ( DR) . Methods HRMPCs were divided into two groups. The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose,while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose. The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin. Peptides of 2μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA. The results were further analyzed using bioinformatics software. Results CCK-8 results showed that the absorbance ( A450 ) of HRMPCs in high glucose group was 0. 75±0. 04,which was significantly lower than 0. 91±0. 05 in control group (t=5. 784,P=0. 0002). In total,1972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1. 5). Among them,13 proteins were up-regulated,including CTNNB1 and CTBP2;while 41 proteins were down-regulated,including SQSTM1 and HMGCS1. The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration. Conclusions The expressions of many proteins in HRMPCs change under the stimulation of high glucose,which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.