The effects of highly expression of polypyramidine tract binding protein-associated splicing factor on advanced glycation end-products-induced human retinal microvascular endothelial cells
10.3760/cma.j.cn511434-20181012-00344
- VernacularTitle: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
- Author:
Manhong XU
1
;
Chen QI
;
Xun LIU
;
Tingting LIN
;
Qiong WANG
;
Yaru HONG
;
Xiaorong LI
;
Lijie DONG
Author Information
1. 天津医科大学眼科医院、眼视光学院、眼科研究所 天津市眼科学与视觉科学国际联合研究中心 300384
- From:
Chinese Journal of Ocular Fundus Diseases
2020;36(8):633-640
- CountryChina
- Language:Chinese
-
Abstract:
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% CO 2. 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.
