聚嘧啶束结合蛋白相关剪接子高表达对糖基化终末产物诱导下人视网膜微血管内皮细胞氧化损伤的作用

The effects of highly expression of polypyramidine tract binding protein-associated splicing factor on advanced glycation end-products-induced human retinal microvascular endothelial cells

目的观察探讨聚嘧啶束结合蛋白相关剪接子(PSF)高表达对糖基化终末产物(AGEs)诱导下人视网微血管内皮细胞(hRMECs)氧化损伤的保护作用及相应分子机制。方法将hRMECs分为正常组、空载组、PSF组、锌原卟啉(ZnPP)组及PSF+ZnPP组进行实验。正常组细胞使用含有10%胎牛血清、青霉素/链霉素的DMEM培养基,置于37°C、95%空气、5%CO2的密闭恒温培养箱中培养。空载组细胞采用空载慢病毒感染。PSF组细胞采用过表达PSF慢病毒感染。ZnPP组细胞采用ZnPP(10 mol/L)处理2 h。PSF+ZnPP组细胞采用过表达PSF慢病毒感染后,再用ZnPP(10 mol/L)预处理2 h。后四组细胞辅以AGEs刺激,HE、Hoechst33258染色法和流式细胞法观察PSF高表达对细胞损伤的保护作用以及ZnPP对PSF的拮抗作用。采用Western blot检测细胞中血红素氧合酶-1(HO-1)、磷酸化(p)细胞外调节蛋白激酶(ERK)、核因子2相关因子2(Nrf2)的蛋白表达。引入ERK通路的特异性拮抗剂U0126,Western blot验证U0126对PSF蛋白所诱导的HO-1表达的逆转作用。结果HE染色和Hoechst33258染色结果显示,PSF组受损细胞核数较正常组明显增加,较PSF+ZnPP组明显减少,差异均有统计学意义(F=27.5、38.7,P<0.05)。流式细胞法结果显示,PSF组细胞产生的ROS较正常组明显增加,较PSF+ZnPP组明显减少,差异有统计学意义(F=126.4,P<0.05)。Western blot检测结果显示,PSF组HO-1蛋白表达量较正常组、空载组明显增加,差异均有统计学意义(F=70.1,P<0.05);AGEs处理30、60、120、240 min时pERK的蛋白表达量与15 min时比较,差异均有统计学意义(F=474.0,P<0.05);PSF+/U0126-组HO-1、Nrf2蛋白表达量较PSF-/U0126-组明显增加,PSF+/U0126+组HO-1、Nrf2蛋白表达量较PSF+/U0126-组明显降低,差异有统计学意义(F=30.2、489.4,P<0.05)。结论PSF高表达可以通过活化ERK通路,促进Nrf2转位入核进而诱导HO-1表达,从而保护hRMECs免受AGEs诱导的氧化损伤。

Objective To investigate the protection and the corresponding molecular mechanisms of polypyramidine tract binding protein-associated splicing factor(PSF)overexpression on human retinal microvascular endothelial cells(hRMECs)induced by advanced glycation end-products(AGEs).Methods The hRMECs were divided into the normal group,the vector group,PSF group,zinc protoporphyrin(ZnPP)group and PSF+ZnPP group for experiment.Cells in the normal group were cultured in a DMEM medium containing 10%fetal calf serum,penicillin/streptomycin,and placed in a closed constant temperature incubator at 37°C,95%air,and 5%CO2.Cells in the vector group were infected with empty lentivirus.The cells in the PSF group were infected with overexpressing PSF lentivirus.Cells in the ZnPP group were treated with ZnPP(10 mol/L)for 2 h.The PSF+ZnPP group cells were infected with overexpressing PSF lentivirus,and then pretreated with ZnPP(10 mol/L)for 2 h.With the last four groups of cells stimulated with AGEs,HE,Hoechst33258 staining and flow cytometry were used to observe the protective effect of high expression of PSF on cell damage and the antagonistic effect of ZnPP on PSF.Western blot was used to detect the protein expression of heme oxygenase-1(HO-1),phosphorylated(p)extracellular regulatory protein kinase(ERK),and Nrf2 in the cells.U0126,a specific antagonist of ERK pathway,was introduced,and Western blot verified the reversal effect of U0126 on the expression of HO-1 induced by PSF protein.Results HE staining and Hoechst33258 staining showed that the number of nuclei of damaged cells of PSF group were significantly increased compared with control group,while decreased compared with PSF+ZnPP group(F=27.5,38.7;P<0.05).The results of flow cytometry showed that the ROS produced by cells in the PSF group was significantly increased compared to the normal group,and significantly decreased compared to the PSF+ZnPP group,the difference was statistically significant(F=126.4,P<0.05).Western blot results showed that HO-1 expression of PSF group was significantly increased compared with control and the vector group(F=70.1,P<0.05).AGEs inducement of 30,60,120 and 240 min could significantly improve pERK expression compared with 15 min(F=474.0,P<0.05).The expression of HO-1 and Nrf2 proteins in the PSF+/U0126-group was significantly more than those in the PSF-/U0126-group,the expression of HO-1 and Nrf2 proteins in the PSF+/U0126+group was significantly lower than that in the PSF+/U0126-group,and the differences were statistically significant(F=30.2,489.4;P<0.05).Conclusion Over expression of PSF can promote the HO-1 expression by activating ERK pathway and promoting the Nrf2 to the nucleus,thus protect hRMECs against AGEs-induced oxidative damage.