Effects of polypyramidine tract binding protein-associated splicing factor overexpression on apoptosis of human Müller cells under advanced glycation end products treatment_Chinese Journal of Ocular Fundus Diseases

Authors：

Tian Fang , Hu Bojie , Li Wenbo , Huang Liangyu , Gao Meizi , Su Ruihong , Zhang Xiaomin , Li Xiaorong ,  Dong Lijie

Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, College of Optometry and Ophthalmology, Tianjin 300384, China;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Polypyrimidine tract-binding protein; Glycosylation end products, advanced; Apoptosis; Müller cells

DOI：

10.3760/cma.j.issn.1005-1015.2019.01.015

Video：

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ObjectiveTo observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.MethodsExperimental study. Müller cells were cultured and divided into groups according to the project design, plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro, then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay. The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit. Meanwhile, 2′,7′-dichlorofluorescin diacetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.ResultsThe morphology of cells in normal group was full and the cytoplasm staining was uniform. In N+AGEs group and Vec+AGEs group, cell volume decreased, cytoplasm was dense and concentrated, and eosinophilic staining was enhanced. The cell morphology of PSF+AGEs group was still full, with uniform cytoplasm staining and uniform nucleus staining. The viability of N+AGEs group, Vec+AGEs group and PSF+AGEs group were 0.42±0.11, 0.35±0.12 and 0.68±0.12. The apoptosis values were 1.08±0.16, 0.96±0.20 and 0.44±0.08. The intracellular ROS levels were 28 833.67±3 550.06, 28 356.67±4 854.81, 186 163.00±382.54. Compared with N+AGEs group and Vec+AGEs group, the cell viability of PSF+AGEs group was significantly improved (F=20.65, P=0.000), cell apoptosis value (F=43.43, P=0.000) and intracellular ROS level (F=18.86, P=0.000).ConclusionPSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müller cells.

Citation： Tian Fang, Hu Bojie, Li Wenbo, Huang Liangyu, Gao Meizi, Su Ruihong, Zhang Xiaomin, Li Xiaorong, Dong Lijie. Effects of polypyramidine tract binding protein-associated splicing factor overexpression on apoptosis of human Müller cells under advanced glycation end products treatment. Chinese Journal of Ocular Fundus Diseases, 2019, 35(1): 70-75. doi: 10.3760/cma.j.issn.1005-1015.2019.01.015 Copy

1.	Paaon JG, Mayer SA, Tempst P, et al. Characterization and molecularcloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing[J]. Genes Dev, 1991, 5(7): 1237-1251. DOI: 10.1101/gad.5.7.1237.
2.	Patton JG, Porto EB, Galceran J, et al. Cloning and characterization of PSF, a novel pre-mRNA splicing factor[J]. Genes Dev, 1993, 7(3): 393-406. DOI: 10.1101/gad.7.3.393.
3.	Dye BT, Patton JG. An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles[J]. Exp Cell Res, 2001, 1263(1): 131-144. DOI: 10.1006/excr.2000.5097.
4.	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.
5.	Lowery LA, Rubin J, Sive H. Whitesnake/sfpq is required for cell survival and neuronal development in the zebrafish[J]. Dev Dyn, 2007, 236(5): 1347-1357. DOI: 10.1002/dvdy.21132.
6.	Elsaeidi F, Bemben MA, Zhao XF, et al. Jak/Stat signaling stimulates zebra fish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq[J]. Neurosci, 2014, 34(7): 2632-2644. DOI: 10.1523/JNEUROSCI.3898-13.2014.
7.	Smith PD, Sun F, Park KK, et al. SOCS3 deletion promotes optic nerve regeneration in vivo[J]. Neuron, 2009, 64(5): 617-623. DOI: 10.1016/j.neuron.2009.11.021.
8.	Kowluru RA. Mitochondria damage in the pathogenesis of diabetic retinopathy and in the metabolic memory associated with its continued progression[J]. Curr Med Chem, 2013, 20(26): 3226-3233. DOI: 10.2174/09298673113209990029.
9.	Tam J, Dhamdhere KP, Tiruveedhula P, et al. Subclinical capillary changes in non-proliferative diabetic retinopathy[J]. Optom Vis Sci, 2012, 89(5): 692-703. DOI: 10.1097/OPX.0b013e3182548b07.
10.	Berweck S, Thieme H, Lepple-Wienhues A, et al. Insulin-induced hyperpolarization in retinal capillary pericytes[J]. Invest Ophthalmol Vis Sci, 1993, 34(12): 3402-3407.
11.	Lecleire-Collet A, Tessier LH, Massin P. et al Advanced glycation end products can induce glial reaction and neuronal degeneration in retinal explants[J]. Br J Ophthalmol, 2005, 89(12): 1631-1633. DOI: 10.1136/bjo.2005.079491.
12.	Hirata C, Nakano K, Nakamura N, et al. Advanced glycation end products induce expression of vascular endothelial growth factor by retinal Müller cells[J]. Biochem Biophys Res Commun, 1997, 236(3): 712-715. DOI: 10.1006/bbrc.1997.7036.
13.	Zone H, Ward M, Madden A, et al. Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycationend-products (RAGE)[J]. Diabetologia, 2010, 53(12): 2656-2666. DOI: 10.1007/s00125-010-1900-z.
14.	Ai J, Liu Y, Sun JH. Advanced glycation end-products stimulate basic fibroblast growth factor expression in cultured Müller cells[J]. Mol Med Rep, 2013, 7(1): 16-20. DOI: 10.3892/mmr.2012.1152.
15.	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. Inhibition of oxygen induced retinal neovascularization by recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor intraocular injection in mice[J]. Chin J Ocul Fundus Dis, 2014, 30(5): 504-508. DOI: 10.3760/cma.j.issn.1005-1015.2014.05.019.
16.	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. Inhibition of PTB-associated splicing factor on IGF-1/VEGF signaling 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.
17.	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. The regulation of PTB-associated splicing factor on phosphatidylinositol 3 kinase/Akt signaling pathway in 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.
18.	Li XY, Zhang MN, Zhou HF. The morphological features and mitochondrial oxidative stress mechanism of the retinal neurons apoptosis in early diabetic rats[J/OL]. Diabetes Res, 2014, 2014: 678123[2014-01-02]. https://dx.doi.org/10.1155/2014/678123. DOI: 10.1155/2014/678123.
19.	Tu BP, Weissman JS. Oxidative protein folding in eukaryotes: mechanisms and consequences[J]. Cell Biol, 2004, 164(3): 341-346. DOI: 10.1083/jcb.200311055.
20.	Curtis TM, Hamilton R, Yong OH, et al. Müller glial dysfunction during diabetic retinopathy in rats is linked to accumulation of advanced glycation end-products and advanced lipoxidation end-products[J]. Diabetologia, 2011, 54(3): 690-698. DOI: 10.1007/s00125-010-1971-x.
21.	董传芳, 刘雪平, 王美霞, 等. 糖基化终末产物对SH-SY5Y细胞氧化应激及凋亡的影响[J]. 山东大学学报(医学版), 2012, 50(1): 9-14. DOI: 10.6040/j.issn.1671-7554.2012.01.003.Dong CF, Liu XP, Wang MX, et al. Effect of advanced glycation end products on oxidative stress and apoptosis of SH-SY5Y cells[J]. Journal of Shandong University (Health Science), 2012, 50(1): 9-14. DOI: 10.6040/j.issn.1671-7554.2012.01.003.
22.	汪峻岭, 刘瑶, 郑杰, 等. 糖基化终产物对体外培养兔视网膜Müller细胞的影响[J]. 现代医学, 2006, 34(4): 229-233. DOI: 10.3969/j.issn.1671-7562.2006.04.004.Wang JL, Liu Y, Zheng J, et al. Effects of advanced glycation end products on the cultured rabbit retinal Müller cells[J]. Modern Medical Journal, 2006, 34(4): 229-233. DOI: 10.3969/j.issn.1671-7562.2006.04.004.
23.	张敏, 王强, 王康, 等. 糖基化终末产物对体外培养牛眼小梁细胞氧化应激及凋亡的影响[J]. 眼科新进展, 2014, 34(7): 640-642. DOI: 10.13389/j.cnki.rao.2014.0175.Zhang M, Wang Q, Wang K, et al. Effects of advanced glycation end products on oxidative stress and apoptosis of bovine trabecular meshwork cells cultured in vitro[J]. Rec Adv Ophthalmol, 2014, 34(7): 640-642. DOI: 10.13389/j.cnki.rao.2014.0175.

1. Paaon JG, Mayer SA, Tempst P, et al. Characterization and molecularcloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing[J]. Genes Dev, 1991, 5(7): 1237-1251. DOI: 10.1101/gad.5.7.1237.
2. Patton JG, Porto EB, Galceran J, et al. Cloning and characterization of PSF, a novel pre-mRNA splicing factor[J]. Genes Dev, 1993, 7(3): 393-406. DOI: 10.1101/gad.7.3.393.
3. Dye BT, Patton JG. An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles[J]. Exp Cell Res, 2001, 1263(1): 131-144. DOI: 10.1006/excr.2000.5097.
4. 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.
5. Lowery LA, Rubin J, Sive H. Whitesnake/sfpq is required for cell survival and neuronal development in the zebrafish[J]. Dev Dyn, 2007, 236(5): 1347-1357. DOI: 10.1002/dvdy.21132.
6. Elsaeidi F, Bemben MA, Zhao XF, et al. Jak/Stat signaling stimulates zebra fish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq[J]. Neurosci, 2014, 34(7): 2632-2644. DOI: 10.1523/JNEUROSCI.3898-13.2014.
7. Smith PD, Sun F, Park KK, et al. SOCS3 deletion promotes optic nerve regeneration in vivo[J]. Neuron, 2009, 64(5): 617-623. DOI: 10.1016/j.neuron.2009.11.021.
8. Kowluru RA. Mitochondria damage in the pathogenesis of diabetic retinopathy and in the metabolic memory associated with its continued progression[J]. Curr Med Chem, 2013, 20(26): 3226-3233. DOI: 10.2174/09298673113209990029.
9. Tam J, Dhamdhere KP, Tiruveedhula P, et al. Subclinical capillary changes in non-proliferative diabetic retinopathy[J]. Optom Vis Sci, 2012, 89(5): 692-703. DOI: 10.1097/OPX.0b013e3182548b07.
10. Berweck S, Thieme H, Lepple-Wienhues A, et al. Insulin-induced hyperpolarization in retinal capillary pericytes[J]. Invest Ophthalmol Vis Sci, 1993, 34(12): 3402-3407.
11. Lecleire-Collet A, Tessier LH, Massin P. et al Advanced glycation end products can induce glial reaction and neuronal degeneration in retinal explants[J]. Br J Ophthalmol, 2005, 89(12): 1631-1633. DOI: 10.1136/bjo.2005.079491.
12. Hirata C, Nakano K, Nakamura N, et al. Advanced glycation end products induce expression of vascular endothelial growth factor by retinal Müller cells[J]. Biochem Biophys Res Commun, 1997, 236(3): 712-715. DOI: 10.1006/bbrc.1997.7036.
13. Zone H, Ward M, Madden A, et al. Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycationend-products (RAGE)[J]. Diabetologia, 2010, 53(12): 2656-2666. DOI: 10.1007/s00125-010-1900-z.
14. Ai J, Liu Y, Sun JH. Advanced glycation end-products stimulate basic fibroblast growth factor expression in cultured Müller cells[J]. Mol Med Rep, 2013, 7(1): 16-20. DOI: 10.3892/mmr.2012.1152.
15. 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. Inhibition of oxygen induced retinal neovascularization by recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor intraocular injection in mice[J]. Chin J Ocul Fundus Dis, 2014, 30(5): 504-508. DOI: 10.3760/cma.j.issn.1005-1015.2014.05.019.
16. 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. Inhibition of PTB-associated splicing factor on IGF-1/VEGF signaling 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.
17. 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. The regulation of PTB-associated splicing factor on phosphatidylinositol 3 kinase/Akt signaling pathway in 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.
18. Li XY, Zhang MN, Zhou HF. The morphological features and mitochondrial oxidative stress mechanism of the retinal neurons apoptosis in early diabetic rats[J/OL]. Diabetes Res, 2014, 2014: 678123[2014-01-02]. https://dx.doi.org/10.1155/2014/678123. DOI: 10.1155/2014/678123.
19. Tu BP, Weissman JS. Oxidative protein folding in eukaryotes: mechanisms and consequences[J]. Cell Biol, 2004, 164(3): 341-346. DOI: 10.1083/jcb.200311055.
20. Curtis TM, Hamilton R, Yong OH, et al. Müller glial dysfunction during diabetic retinopathy in rats is linked to accumulation of advanced glycation end-products and advanced lipoxidation end-products[J]. Diabetologia, 2011, 54(3): 690-698. DOI: 10.1007/s00125-010-1971-x.
21. 董传芳, 刘雪平, 王美霞, 等. 糖基化终末产物对SH-SY5Y细胞氧化应激及凋亡的影响[J]. 山东大学学报(医学版), 2012, 50(1): 9-14. DOI: 10.6040/j.issn.1671-7554.2012.01.003.Dong CF, Liu XP, Wang MX, et al. Effect of advanced glycation end products on oxidative stress and apoptosis of SH-SY5Y cells[J]. Journal of Shandong University (Health Science), 2012, 50(1): 9-14. DOI: 10.6040/j.issn.1671-7554.2012.01.003.
22. 汪峻岭, 刘瑶, 郑杰, 等. 糖基化终产物对体外培养兔视网膜Müller细胞的影响[J]. 现代医学, 2006, 34(4): 229-233. DOI: 10.3969/j.issn.1671-7562.2006.04.004.Wang JL, Liu Y, Zheng J, et al. Effects of advanced glycation end products on the cultured rabbit retinal Müller cells[J]. Modern Medical Journal, 2006, 34(4): 229-233. DOI: 10.3969/j.issn.1671-7562.2006.04.004.
23. 张敏, 王强, 王康, 等. 糖基化终末产物对体外培养牛眼小梁细胞氧化应激及凋亡的影响[J]. 眼科新进展, 2014, 34(7): 640-642. DOI: 10.13389/j.cnki.rao.2014.0175.Zhang M, Wang Q, Wang K, et al. Effects of advanced glycation end products on oxidative stress and apoptosis of bovine trabecular meshwork cells cultured in vitro[J]. Rec Adv Ophthalmol, 2014, 34(7): 640-642. DOI: 10.13389/j.cnki.rao.2014.0175.

Chinese Journal of Ocular Fundus Diseases

Effects of polypyramidine tract binding protein-associated splicing factor overexpression on apoptosis of human Müller cells under advanced glycation end products treatment

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