Effects of prostaglandin E2 receptor on the activation of inflammasomes and cell damage in human retinal microvascular endothelial cells in a high-glucose environment
10.3760/cma.j.cn511434-20201028-00514
- VernacularTitle:前列腺素E2受体对高糖环境中人视网膜微血管内皮细胞炎症小体活化和细胞损伤作用
- Author:
Zhonghong ZHANG
1
;
Yong YAO
;
Tianhua XIE
;
Meili WU
;
Jian ZOU
;
Xiaolu WANG
Author Information
1. 南京医科大学附属无锡人民医院眼科 214023
- Keywords:
Receptors, prostaglandin E;
Retinal microvascular endothelial cells;
NLRP3 inflammasome
- From:
Chinese Journal of Ocular Fundus Diseases
2021;37(8):623-631
- CountryChina
- Language:Chinese
-
Abstract:
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.
