The effects of highly expression of polypyramidine tract binding protein-associated splicing factor on advanced glycation end-products-induced human retinal microvascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Xu Manhong , Qi Chen , Liu Xun , Lin Tingting , Wang Qiong , Hong Yaru , Li Xiaorong ,  Dong Lijie

Tianjin International Joint Research and Development Centre of Ophthalmology and Vision Science, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Polypyrimidine tract-binding protein; Glycosylation end products, advanced; Retinal vessels/cytology; Endothelial cells/physiology; Cells, cultured

DOI：

10.3760/cma.j.cn511434-20181012-00344

Video：

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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.

Citation： Xu Manhong, Qi Chen, Liu Xun, Lin Tingting, Wang Qiong, Hong Yaru, Li Xiaorong, Dong Lijie. The effects of highly expression of polypyramidine tract binding protein-associated splicing factor on advanced glycation end-products-induced human retinal microvascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2020, 36(8): 633-640. doi: 10.3760/cma.j.cn511434-20181012-00344 Copy

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1. Shao Y, Dong LJ, Takahashi Y, et al. miRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy[J]. Am J Physiol Endocrinol Metab, 2019, 316(3): 443-452. DOI: 10.1152/ajpendo.00360.2018.
2. Dong L, Nian H, Shao Y, et al. PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy[J]. Cell Tissue Res, 2015, 360(2): 233-243. DOI: 10.1007/s00441-014-2104-5.
3. Tian F, Zhao J, Bu S, et al. KLF6 induces apoptosis in human lens epithelial cells through the ATF4-ATF3-CHOP axis[J]. Drug Des Devel Ther, 2020, 14: 1041-1055. DOI: 10.2147/DDDT.S218467.
4. Dong L, Zhang X, Fu X, et al. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig epsilon gene transcription by recruitment of HDAC1[J]. J Biol Chem, 2011, 286(5): 3451-3459. DOI: 10.1074/jbc.M110.168377.
5. Xing X, Huang L, Lv Y, et al. DL-3-n-butylphthalide protected retinal müller cells dysfunction from oxidative stress[J]. Curr Eye Res, 2019, 44(10): 1112-1120. DOI: 10.1080/02713683.2019.1624777.
6. 黄亮瑜, 柯屹峰, 林婷婷, 等. 慢病毒介导聚嘧啶束结合蛋白相关剪接因子对氧诱导视网膜病变小鼠视网膜新生血管的抑制作用[J]. 中华眼底病杂志, 2020, 36(1): 53-56. DOI: 10.3760/cma.j.issn.1005-1015.2020.01.012.Huang LY, Ke YF, Lin TT, et al. Effects of polypyrimidine bundle binding protein-related splicing factors on apoptosis of retinal pigment epithelial cells induced by hydrogen peroxide[J]. Chin J Ocul Fundus Dis, 2020, 36(1): 53-56. DOI: 10.3760/cma.j.issn.1005-1015.2020.01.012.
7. 田芳, 东莉洁, 周玉, 等. 重组腺相关病毒-多聚嘧啶序列结合蛋白相关剪接因子对氧诱导视网膜新生血管形成的抑制作用[J]. 中华眼底病杂志, 2014, 30(5): 504-508. DOI: 10.3760/cma.j.issn.1005-1015.2014.05.019.Tian F, Dong LJ, Zhou Y, et al. Inhibitory effect of recombinant adeno-associated virus-polypyrimidine sequence binding protein-related splicing factor on oxygen-induced retinal neovascularization[J]. Chin J Ocul Fundus Dis, 2014, 30(5): 504-508. DOI: 10.3760/cma.j.issn.1005-1015.2014.05.019.
8. 田芳, 东莉洁, 吉洁, 等. 多聚嘧啶序列结合蛋白相关剪接因子对视网膜血管内皮细胞IGF-1/VEGF信号通路的抑制作用[J]. 中华实验眼科杂志, 2016, 34(1): 11-16. DOI: 10.3760/cma.j.issn.2095-0160.2016.01.003.Tian F, Dong LJ, Ji J, et al. Inhibitory effect of polypyrimidine sequence binding protein-related splicing factor on IGF-1/VEGF signal pathway in retinal vascular endothelial cells[J]. Chin J Exp Ophthalmol, 2016, 34(1): 11-16. DOI: 10.3760/cma.j.issn.2095-0160.2016.01.003.
9. 田芳, 胡博杰, 李文博, 等. 高表达多聚嘧啶序列结合蛋白相关剪接因子对糖基化终产物诱导下视网膜Müller细胞凋亡的影响[J]. 中华眼底病杂志, 2019, 35(1): 70-75. DOI: 10.3760/cma.j.issn.1005-1015.2019.01.015.Tian F, Hu BJ, Li WB, et al. Effect of high expression of polypyrimidine sequence binding protein-related splicing factors on apoptosis of retinal Müller cells induced by advanced glycation end products[J]. Chin J Ocul Fundus Dis, 2019, 35(1): 70-75. DOI: 10.3760/cma.j.issn.1005-1015.2019.01.015.
10. Li RL, Zhao WW, Gao BY. Advanced glycation end products induce neural tube defects through elevating oxidative stress in mice[J]. Neural Regen Res, 2018, 13(8): 1368-1374. DOI: 10.4103/1673-5374.235249.
11. Youn JY, Siu KL, Lob HE, et al. Role of vascular oxidative stress in obesity and metabolic syndrome[J]. Diabetes, 2014, 63(7): 2344-2355. DOI: 10.2337/db13-0719.
12. Joseph D, Kimar C, Symington B, et al. The detrimental effects of acute hyperglycemia on myocardial glucose uptake[J]. Life Sci, 2014, 105(1-2): 31-42. DOI: 10.1016/j.lfs.2014.04.009.
13. Giacco F, Brownlee M. Oxidative stress and diabetic complications[J]. Circ Res, 2010, 107(9): 1058-1070. DOI: 10.1161/CIRCRESAHA.110.223545.
14. Ott C, Jacobs K, Haucke E, et al. Role of advanced glycation end products in cellular signaling[J]. Redox Biol, 2014, 2: 411-429. DOI: 10.1016/j.redox.2013.12.016.
15. 漆晨, 郭如如, 东莉洁, 等. PSF蛋白及其在眼科的应用研究[J]. 国际眼科纵览, 2015, 39(3): 195-199. DOI: 10.3706/cma.j.issn.1673-5803.Qi C, Guo RR, Dong LJ, et al. Study on PSF protein and its application in ophthalmology[J]. Int Rev Ophthalmol, 2015, 39(3): 195-199. DOI: 10.3706/cma.j.issn.1673-5803.
16. 漆晨, 东莉洁, 乐毅, 等. 多聚嘧啶序列结合蛋白相关剪接因子对体外培养的视网膜色素上皮细胞磷脂酰肌醇3激酶/丝氨酸-苏氨酸蛋白激酶信号通路的调控作用[J]. 中华眼底病杂志, 2015, 31(4): 363-367. DOI: 10.3760/cma.j.issn.1005-1015.2015.04.013.Qi C, Dong LJ, Yue Y, et al. Regulation of polypyrimidine sequence binding protein-related splicing factor on phosphatidylinositol 3 kinase/serine-threonine protein kinase signal pathway in cultured retinal pigment epithelial cells[J]. Chin J Ocul Fundus Dis, 2015, 31(4): 363-367. DOI: 10.3760/cma.j.issn.1005-1015.2015.04.013.
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Chinese Journal of Ocular Fundus Diseases

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

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