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Objective:To observe the effects of four prostaglandin E2 (PGE2) receptors (EP 1-4R) on the activation of inflammasomes and cell damage in human retinal microvascular endothelial cells (hRMEC) in a high glucose environment. Methods:The hRMEC were divided into normal group and high glucose group, and they were cultured in Dulbecco modified Eagle medium containing 5.5 and 30.0 mmol/L glucose, respectively. Flow cytometry was used to observe the apoptosis rate of the high glucose group and the normal group; enzyme chain immunosorbent assay (ELISA) was used to detect the level of PGE2 in the culture supernatant of hRMEC cells. Western blot was used to detect the protein expression of cyclooxyganese (COX2) and EP 1-4R in hRMEC. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of EP 1-4R mRNA in hRMEC. After 72 h of culture, the cells in the high glucose group were divided into control group, PGE2 group, EP 1-4R agonist group, PGE2+EP 1-4R inhibitor group, and dimethylsulfoxide group. According to the group, each group was given the corresponding agonist or inhibitor to continue the culture for 24 h. QRT-PCR was used to detect the expression of nucleotide-binding oligomerization structure-like receptor protein (NLRP3) and pro-interleukin (IL)-1β mRNA in each group of cells. ELISA was used to detect the content of IL-1β and lactic dehydrogenase (LDH) in the cell culture supernatant. Western blot was used to detect the expression of cleaved Caspase-1 in each group of cells. At the same time, hRMEC in a high glucose environment was given IL-1β stimulation for 24 h, and the activity of LDH in the supernatant of the cell culture medium was detected. Results:The apoptotic rate, COX2 protein expression, and PGE2 protein content in hRMEC in the high glucose group were significantly higher than those in the normal group, and they were time-dependent. Compared with the normal group, the expression levels of EP 1R, EP 2R, EP 4R protein and mRNA in hRMEC in the high glucose group were higher than those in the normal group ( P<0.05). Compared with the control group, PGE2 group ( t=4.627, P<0.01), EP 1-4R agonist group ( t=3.889, 3.583, 2.445, 3.216; P<0.05) hRMEC NLRP3 mRNA expression level was significantly increased; the expression level of pro-IL-1β mRNA increased, however the difference was not statistically significant (PGE2 group: t=1.807, P>0.05; EP 1-4R agonist group: t=1.807, 1.477, 0.302, 1.926, P>0.05). Compared with the PGE2 group, the expression of NLRP3 mRNA in hRMEC in the PGE2+EP 2R inhibitor group was significantly reduced ( t=2.812, P<0.05); the expression of pro-IL-1β mRNA in hRMEC in the PGE2+EP 3R inhibitor group was significantly increased ( t=4.113, P<0.01). The protein content of IL-1β in the cell culture supernatant of the PGE2 group, EP 1R agonist group and EP 2R agonist group was significantly higher than that of the control group ( t=5.155, 4.136, 4.817; P<0.01). Compared with PGE2 group, the protein content of IL-1β in the cell culture supernatant of the PGE2+EP 2R inhibitor group and the PGE2+EP 4R inhibitor group were significantly lower than that of the PGE2 group ( t=1.964, 4.765; P<0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2 group and EP 2R agonist group was significantly higher than that in the control group ( t=5.332, 4.889; P<0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2+EP 2R inhibitor group was significantly lower than that of the PGE2 group ( t=6.699, P<0.01). The LDH activity in the cell culture supernatant of the PGE2 group and the EP 2R agonist group was significantly higher than that of the control group ( t=4.908, 4.225; P<0.05). The activity of LDH in the cell culture supernatant of the PGE2+EP 2R inhibitor group was significantly lower than that of the PGE2 group ( t=5.301, P<0.01). Compared with the control group, the LDH activity in the culture supernatant of hRMEC cells in the high glucose environment was significantly increased ( t=3.499, P<0.05). Conclusions:The four receptors of PGE2 can activate NLRP3 and its effector molecules to varying degrees. EP 2R mainly mediates hRMEC damage under high glucose environment.
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Objective:To observe the effects of miR-146a on human retinal endothelial cell (HREC) under high glucose condition.Methods:Total of 57 cases diagnosed as diabetic mellitus and 40 cases with diabetic retinopathy (DR) in Wuxi People's Hospital Affiliated to Nanjing Medical University from October to December 2013.Forty-one healthy volunteers were enrolled and served as control group.The clinical data and venous blood samples of subjects were collected.HRECs were cultured in normal glucose (5.5 mmol/L) or high glucose medium (30 mmol/L). Real-time PCR was used to detect the expression of miR-146a.The cultured HRECs were transfected with miR-146a mimic, mimic negative control, inhibitor and inhibitor negative control by lipofectamine2000, respectively.The expression of miR-146a and intercellular cell adhesion molecule-1 (ICAM-1) mRNA was examined by real-time PCR and the expression of nuclear factor-кB (NF-кB) p65 and NF-кB p65 Ser536 was detected by Western blot assay. Results:The relative expression of miR-146a mRNA in the diabetic mellitus group and DR group was 0.36±0.08 and 0.27±0.08, respectively, which were significantly lower than 1.00±0.16 in the control group (both at P<0.01). The expression of miR-146a mRNA was 0.37±0.11 in the high glucose group, which was lower than 1.00±0.18 in the normal control group ( t=5.57, P<0.01). The relative expression of miR-146a mRNA in the miR-146a mimic group was 2 540.00±105.00, which was significantly higher than 61.00±17.90 in the miR-146a mimic control group; The relative expression of miR-146a mRNA in the miR-146a inhibitor group was 0.04±0.01, which was significantly lower than 0.88±0.04 in the miR-146a inhibitor control group ( t=23.23, 17.12; both at P<0.01). The relative expression of ICAM-1 mRNA in the miR-146a mimic group was 0.35±0.12, which was significantly lower than 1.00±0.13 in the miR-146a mimic control group; The relative expression of ICAM-1 mRNA in the miR-146a inhibitor group was 2.74±0.48, which was significantly higher than 1.00±0.16 in the miR-146a inhibitor control group ( t=3.58, 3.37; both at P<0.05). The relative expression of NF-кB p65 Ser536 in the miR-146a mimic group was 0.43±0.03, which was significantly lower than 1.07±0.09 in the miR-146a mimic control group ( t=6.74, P<0.01). The relative expression of NF-кB p65 Ser536 in the miR-146a inhibitor group was 2.08±0.12, which was significantly higher than 1.00±0.01 in the miR-146a inhibitor control group ( t=8.76; P<0.01). Conclusions:miR-146a can reduce inflammation of HREC in high glucose condition through inhibiting ICAM-1 expression and NF-кB phosphorylation.
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Objective To investigate the expression and mechanism of miR-1470 in plasma of diabetic retinopathy (DR) patients.Methods Thirty patients with DR (DR group),30 patients with diabetes (DM group) and 30 normal healthy subjects (normal group) were enrolled in the study.Three groups of subjects were taken 5 ml of venous blood,and total plasma RNA was extracted and purified.The differentially expressed miRNAs in the plasma of DR patients were screened by gene chip,and the results of gene chip detection were verified by reverse transcription polymerase chain reaction (RT-PCR).Bioinformatics was used to predict potential target genes for miRNA regulation,and miR-1470 and its target gene epidermal growth factor receptor (EGFR) were screened.Human retinal microvascular endothelial cells (hREC) were divided into normal group (sugar concentration 5.5 mmol/L) and high glucose group (sugar concentration 25.0 mmol/L).hREC was transfected into miR-1470 mimics to establish a miR-1470 high expression cell model,which was divided into blank control group,high expression group and negative control group.The expression of miR-1470 was detected by RT-PCR.The expression of EGFR protein was detected by Western blot.The measurement data of the two groups were compared using the independent sample t test.The comparison of the measurement data between the two groups was analyzed by ANOVA.The comparison between the measurement data of the groups was compared by multiple comparisons.Results The results of RT-PCR were consistent with those of the gene chip.The expression of miR-1470 in the plasma of the DR group,the DM group and the normal group was statistically significant (F=63.486,P=0.049).Compared with the DM group and the normal group,the expression of miR-1470 in the DR group was significantly decreased,and the difference was statistically significant (q=l 11.2,73.9;P＜0.05).The expression of miR-1470 in hREC in the high glucose group was significantly lower than that in the normal group (t=42.082,P=0.015).The expression of EGFR protein in hREC of high glucose group was significantly higher than that of normal group (t=-39.939,P=0.016).The expression of miR-1470 (F=637.069,P=0.000) and EGFR (F=122.908,P=0.000) protein expression in hREC of blank control group,negative control group and high expression group were statistically significant.Compared with the blank control group and the negative control group,the expression of miR-1470 in hREC of high expression group was significantly increased (q=329.7,328.8;P＜0.05),and the expression of EGFR protein was significantly decreased (q=242.5,234.6;P＜0.05).There was no significant difference in the expression of miR-1470 and EGFR protein in hREC between the negative control group and the blank control group (q=1.5,7.9;P＞0.05).Conclusion The expression ofmiR-1470 in the plasma of patients with DR is significantly down-regulated,and the increase of EGFR expression may be related to it.
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Objective To measure the concentration of serum transthyretin (TTR) of patients with different stages of diabetic retinopathy (DR).Methods A total of 176 patients with diabetes mellitus were included in this study.There were 104 males and 72 females.The patients aged from 21 to 74 years,with the mean age of(56± 11) years.The diabetes duration raged from 1 to 30 years,with the mean diabetes duration of (10 ± 7) years.The HbA 1C was 5.2％-14.1％,with the mean HbA 1C of (8.6 ± 2.0)％.According to the fundus examination,58 patients had DR (33.0％),but the other 118 patients not (67.0％).For these DR patients,10 patients were in stage Ⅰ (5.7％),26 patients in stage Ⅱ (14.8％),8 patients in stage Ⅲ (4.5％),and 14 patients in stage Ⅳ (8.0％).The concentration of serum TTR was measured by enzyme-linked immunosorbentassay kit.The differences in the concentration of serum TTR between different DR stages were compared.Bivariate analysis was used to analyze the influencing factors of TTR.Results The concentrations of serum TTR of the patients without DR or with DR of stage Ⅰ to Ⅳ were (224.96±65.47),(383.68± 102.99),(247.44±63.21),(228.2 ± 45.89),(189.34± 70.12) mg/L,respectively.The difference between different DR stages was statistically significant (F=14.690,P＜ 0.001).Bivariate analysis showed that the concentration of TTR was correlation to DR (r=0.179,P=0.017).There was no correlation between the concentration of TTR and diabetes duration (r=-0.027,P=0.727),hypertension (r=0.018,P=0.810),hyperlipoidemia (r=0.101,P=0.182),and the use of insulin (r=-0.032,P=0.675).Conclusion The concentration of serum TTR was increased in early DR patients,and gradually decreased with the progression of DR.The concentration of TTR is correlated to DR.
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Background Receptor for advanced glycation end products (RAGE) play an important role in the process of type 2 diabetes and its microvascular complications.RAGE gene Gly82Ser exists polymorphism,but the correlation of gene polymorphism and diabetic retinopathy (DR) needs further research.Objective This study was to investigate the association of Gly82Ser polymorphism of RAGE gene with DR in Han people of Wuxi area with type 2 diabetic mellitus.Methods One hundred and eighty-five patiens with type 2 diabetes were included in Wuxi district from March 2013 to February 2014.The patients were divided into non DR (NDR) group (93 cases) and DR group (92 cases) according to the DR International Clinical Classification Criteria in 2002,and 120 healthy subjects were included at the same time as the control.All of the subjects received eye examinations,body mass index (BMI) and blood pressure measurement as well as laboratory tests,including blood biochemical indexes,blood lipids and fasting blood glucose levels.The peripheral blood of 3 ml was collected from each subject,and the genotype and allelic frequencies were assayed by PCR-direct sequencing.This study protocol was approved by Ethic Committee of Nanjing Medical University.Written informed consent was obtained from each subject prior to any medical examination.Results The course was significantly longer in the DR group than that in the NDR group (t =2.25,P =0.01).There were two alleles of G and A in the RAGE gene Gly82Ser locus in all the subjects and the distribution of single nucleotide polymorphism was in accordance with Hardy-Weinberg equilibrium (DR group:x2 =0.51,P =0.48;NDR group:x2 =1.38,P =0.24;healthy control group:x2 =0.20,P =0.24).The AA genotype frequency of the subjects in DR group,NDR group and healthy control group were respectively 6.5％,3.2％ and 2.5％,and AA genotype frequency in DR group was higher than that of the NDR group and healthy control group,showing significant intergroup differences (x2 =5.146,P =0.023;x2 =5.039,P =0.037).The distribution of A allele frequency in the DR group was significantly higher than that of NDR group and healthy control group (x2=5.494,P=0.019;x2 =5.235,P =0.023),and the frequencies of G allele and GG genotype in the DR group were lower than those of the NDR and the healthy control group (GG:x2 =4.260,P =0.039;x2 =4.794,P =0.027;G:x2 =5.309,P =0.021;x2 =5.476,P=0.032).No significant differences were seen in the frequencies of genotype and allele of subjects between the NDR group and the healthy control group (AA:x2 =5.346,P=0.127;GG:x2 =6.981,P=0.137;A:x2 =5.618,P =0.082;G:x2 =4.860,P =0.088).Conclusions The Gly82Ser polymorphism of RAGE gene is associated with the pathogenesis of DR in Han population with type 2 diabetes and A allele may be a risk factor of DR.