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

Effects of prostaglandin E2 receptor on the activation of inflammasomes and cell damage in human retinal microvascular endothelial cells in a high-glucose environment

目的观察前列腺素E2(PGE2)4种受体(EP1-4R)对高糖环境中人视网膜微血管内皮细胞(hRMEC)中炎症小体活化和细胞损伤作用。方法将hRMEC分为正常组、高糖组,分别置于含5.5、30.0mmol/L葡萄糖的Dulbecco改良Eagle培养基中培养。采用流式细胞仪观察高糖组、正常组的细胞凋亡率;酶链免疫吸附试验(ELISA)检测hRMEC细胞培养上清液中PGE2水平;蛋白免疫印迹法(Western blot)检测细胞环氧化酶2(COX2)、的蛋白表达;实时荧光定量聚合酶链反应(qRT-PCR)检测hRMEC中HP1-4RmRNA的表达。高糖组细胞培养后72h后,将其再分为对照组、PGE2组、EP1-4激动剂组、PGE2+EPMR1-4抑制剂组、二甲基亚砜组。根据组别,各组给予相应的激动剂或抑制剂继续培养24 h。采用qRT-PCR检测各组细胞核苷酸结合寡聚化结构样受体蛋白(NLRP3)、白细胞介素(IL)-1P前体(pro-IL-1β)mRNA的表达;ELISA检测细胞培养上清液中IL-1β、乳酸脱氢酶(LDH)的含量;Western blot检测各组细胞活化的半胱氨酸天冬氨酸酶(Caspase)-1蛋白表达。同时对高糖环境的hRMEC给予IL-1β刺激24h,检测其细胞培养上清液中LDH的活性。结果高糖组ЫШЕС细胞凋亡率、C0X2蛋白表达、PGE2蛋白含量较正常组明显升高,并呈时间依赖性。与正常组比较,高糖组hRMEC中EP1R、ЕР2R、ЕР4蛋R白及mRNA表达水平较正常组升高(P<0.05)。与对照组比较,PG2R组(t=4.627,P<0.01)、ЕР2R激动剂组(3.889、3.583、2.445、3.216,P<0.05);pGE2+EP2R中NLRP3mRNA表达水平明显升高,差异有统计学意义;рго-IL-1β mKNA表达水平有所升高,但差异无统计学意义(PGE2组:t=1.807,P>0.05);EPMR激动剂组:(t=1.807、1.477、0.302、1.926,P>0.05)。与PGE2组比较,PGE2+Ep2R抑制剂组hRMEC中NLRP3 mRNA表达水平明显降低,差异有统计学意义(t=2.812,P<0.05);PGE2+EP3R抑制剂组hRMEC中pro-IL-ipmRNA表达水平明显升高,差异有统计学意义(t=4.113,Р<0.01)。PGE2组、ЕР2R激动剂组、ЕР2R激动剂组细胞培养上清液中IL-1β的蛋白含量较对照组明显升高,差异有统计学意义(t=5.155、4.136、4.817,P<0.01);PGE2+EP2R抑制剂组和PGE2+EP4R抑制剂组细胞培养上清液中IL-1β的蛋白含量较PGE2组明显降低,差异有统计学意义(t=1.964、4.765,P<0.05)。PGE2组和EP2R激动剂组hRMEC中活化的Caspase-1蛋白表达较对照组明显增多,差异有统计学意义(P=5.332、4.889,P<0.05);PGE2+EP2R抑制剂组hRMEC中活化的Caspase-1蛋白表达较PGE2组明显降低,差异有统计学意义(t=6.699,P<0.01)。PGE2组和EP2R激动剂组细胞培养上清液中LDH活性较对照组明显升高,差异有统计学意义(t=4.908、4.225,P<0.05);PGE2+EP2R抑制剂组细胞培养上清液中LDH活性较PGE2组明显降低,差异有统计学意义(t=5.301,P<0.01)。与对照组比较,高糖环境hRMEC细胞培养上清液中LDH活性明显升高,差异有统计学意义(t=3.499,P<0.05)。结论PGE2的4种受体对NLRP3及其效应分子均有不同程度活化作用,其中EP2R主要介导了高糖环境下hRMEC的损伤。

Objective To observe the effects of four prostaglandin E2(PGE2)receptors(EP^R)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 imrmmosorbent 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 EPMR in hRMEC.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of EPWR mRNA in hRMEC.After 72 h of culture,the cells in the high glucose group were divided into control group,PGE2 group,EPN4R agonist group,PGE2+EPN4R 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)-lp mRNA in each group of cells.ELISA was used to detect the content of IL-ip 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 enviromnent was given IL-ip stimulation for 24 h,and the tivity of LDH in the supernatant of the cell culture medium was detected.Results The apoptotic rate,COX2 protein expression,and PGE2 protein content in hEMEC 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 ofЕР1,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,Р<0.01),EP2R agonist group(/=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-lp mRNA increased,however the difference was not statistically significant(PGE2 group:t=1.807,P>0.05;EPMR 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 ofрго-IL-ip mRNA in hRMEC in the PGE2+EP3R inhibitor group was significantly increased(^=4.113,P<0.01).The protein content of IL-ip in the cell culture supernatant of the PGE2 group,ЕРДagonist group and EP2R agonist group was significantly higher than that of the control group(/=5.155,4.136,4.817;P<0.01).Compared with PGE2 group,the protein content of IL-ip 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).Conclusions The four receptors of PGE2 can activate NLRP3 and its effector molecules to varying degrees.EP2R mainly mediates hRMEC damage irnder high glucose environment.