Objective To investigate the effects of docosahexenoic acid (DHA) on large conductance Ca2+-activated K+ (BK) channels in normal retinal artery smooth muscle cells (RASMCs). Methods Cultured human RASMCs (6 th-8 th generations) were used to patch clamp experiment. The open probabihties (NP0) in BK channels with different concentrations (0.0, 1.0, 3.0, 5.0, 7.5, 10.0 μmol/L) of DHA were recorded by patch clamp technique in single channel configuration. RASMCs were intervened by different concentrations (0.0, 1.0, 5.0 μmol/L) of DHA as control group, low and high doses of DHA groups, respectively. The protein expressions of β subunit of BK channels in RASMCs from three groups were measured by Western blot. Results The NP0 of BK channels were 0.044 4±0.001 2, 0.081 2±0.004 2, 0.209 0±0.006 1, 0.310 5±0.005 3, 0.465 0±0.007 8 and 0.497 7±0.014 5 with perfusate of 0.0, 1.0, 3.0, 5.0, 7.5, 10.0 μmol/L DHA. DHA activated BK channels in a dose-dependent manner (F=2.621,P＜0.05). There was no significant difference in the protein expression of control group, low and high doses of DHA groups (F=11.657,P＞0.05). Conclusion DHA can directly activate BK channels, no increasing in subunit expression of BK channels.
ObjectiveTo investigate the expression and mechanism of miR-1470 in plasma of diabetic retinopathy (DR) patients.MethodsThirty 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.ResultsThe 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=111.2, 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).ConclusionThe expression of miR-1470 in the plasma of patients with DR is significantly down-regulated, and the increase of EGFR expression may be related to it.
ObjectiveTo observe the effects of four prostaglandin E2 (PGE2) receptors (EP1-4R) on the activation of inflammasomes and cell damage in human retinal microvascular endothelial cells (hRMEC) in a high glucose environment.MethodsThe 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 EP1-4R in hRMEC. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of EP1-4R mRNA in hRMEC. After 72 h of culture, the cells in the high glucose group were divided into control group, PGE2 group, EP1-4R agonist group, PGE2+EP1-4R 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.ResultsThe 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 EP1R, EP2R, EP4R 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), EP1-4R 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; EP1-4R 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+EP2R inhibitor group was significantly reduced (t=2.812, P＜0.05); the expression of pro-IL-1β mRNA in hRMEC in the PGE2+EP3R 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, EP1R agonist group and EP2R 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+EP2R inhibitor group and the PGE2+EP4R 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 EP2R 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+EP2R 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 EP2R 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+EP2R 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).ConclusionsThe four receptors of PGE2 can activate NLRP3 and its effector molecules to varying degrees. EP2R mainly mediates hRMEC damage under high glucose environment.
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 HbA1C was 5.2%−14.1%, with the mean HbA1C 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.
ObjectiveTo explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment.MethodshRECs were divided into 8 groups, including normal glucose group (5.5 mmol/L glucose), hypoxia group, high glucose group (25.0 mmol/L glucose), high glucose and hypoxia group, normal glucose group+TTR, normal glucose and hypoxia group+TTR, high glucose group+TTR, high glucose and hypoxia group+TTR. Flow cytometry was used to analyze cellular apoptosis. The expression level of Akt, p-Akt, eNOS, Bcl-2 and Bax protein were measured by Western blot.ResultsHypoxia could induce apoptosis as the apoptosis rate of normal and hypoxia group was higher than normal group (χ2=25.360, P＜0.05), high glucose and hypoxia group was higher that high glucose group (χ2=17.400, P＜0.05). The cell apoptosis rate of high glucose and hypoxia group+TTR were increased significantly as compared with high glucose and hypoxia group (χ2=9.900, P＜0.05). There was no statistically significant difference on the cell apoptosis rate between normal group and high glucose group, normal group+TTR and normal group, high glucose group+TTR and high glucose group, normal and hypoxia group+TTR and normal and hypoxia group (P＞0.05). Western blot showed that the expression of Akt did not change significantly in all eight groups（F=2.450, P＞0.05). Compared to normal group, the expression of p-Akt, eNOS, Bcl-2 in normal and hypoxia group were decreased (t=9.406, 5.306, 4.819), and the expression of Bax (t=−4.503) was increased (P＜0.05). Compared to high glucose group, same trend was found in high glucose and hypoxia group (t=8.877, 7.723, 6.500, −14.646; P＜0.05). The expression of p-Akt in normal and hypoxia group+TTR was higher than normal and hypoxia group (t=−5.024, P＜0.05) , but there was no difference on the expression of eNOS, Bcl-2, Bax between these two groups (t=−2.235, −2.656, −0.272; P＞0.05). Compared to high glucose and hypoxia group, the expression of p-Akt and Bcl-2 in high glucose and hypoxia group+TTR were decreased (t=4.355, 4.308; P＜0.05), the expression of Bax was increased (t=−4.311, P＜0.05), and there was no difference on the expression of eNOS between these two groups (t=−1.590, P＞0.05). There was no statistically significant difference in the expression of p-Akt, eNOS, Bcl-2, Bax between high glucose group and normal group (t=−3.407, −4.228, −4.302, −2.076; P＞0.05), normal group+TTR and normal group (t=−4.245, −4.298, −2.816, −1.326; P＞0.05), high glucose group+TTR and high glucose group (t=4.016, −0.784, 0.707, −0.328; P＞0.05).ConclusionUnder high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.