Effect of high expression of polypyrimidine tract-binding protein-associated splicing factor on retinal microvascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Liang Jingli , Kou Zhenyu , Cao Jingjing , Li Hui , Teng He , Liu Aihua , Zhang Chuanli ,  Dong Lijie

Tianjin Medical University Eye Hospital, School of Optometry, Eye Institute, Tianjin International Joint Research Center of Ophthalmology and Visual Science, Tianjin 300384, China;

Corresponding?author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Human retinal vascular endothelial cells; Polypyrimidine tract-binding protein-associated splicing factor; 4-hydroxynonenal; Reactive oxygen species; Heme oxygenase-1; Threonine protein kinase; Cell experiment

DOI：

10.3760/cma.j.cn511434-20210222-00090

Video：

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Objective To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism. Methods The HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. Results The number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups (t=12.311, 15.643, 17.346; P＜0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups (t=16.311, 14.833, 18.442; P＜0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences (t=17.342, 16.813, 18.794; P＜0.001). Conclusion PSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.

Citation： Liang Jingli, Kou Zhenyu, Cao Jingjing, Li Hui, Teng He, Liu Aihua, Zhang Chuanli, Dong Lijie. Effect of high expression of polypyrimidine tract-binding protein-associated splicing factor on retinal microvascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2023, 39(4): 324-329. doi: 10.3760/cma.j.cn511434-20210222-00090 Copy

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1. Mittler R. ROS are good[J]. Trends Plant Sci, 2017, 22(1): 11-19. DOI: 10.1016/j.tplants.2016.08.002.
2. Morozumi Y, Ino R, Takaku M, et al. Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing[J]. Nucleic Acids Res, 2012, 40(7): 3031-3041. DOI: 10.1093/nar/gkr1229.
3. 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.
4. Xu T, Liu S, Ma T, et al. Aldehyde dehydrogenase 2 protects against oxidative stress associated with pulmonary arterial hypertension[J]. Redox Biol, 2017, 11: 286-296. DOI: 10.1016/j.redox.2016.12.019.
5. Iwami H, Pruessner J, Shiraki K, et al. Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress[J]. Exp Eye Res, 2014, 124: 37-47. DOI: 10.1016/j.exer.2014.04.014.
6. Teng H, Hong Y, Cao J, et al. Senescence marker protein30 protects lens epithelial cells against oxidative damage by restoring mitochondrial function[J]. Bioengineered, 2022, 13(5): 12955-12971. DOI: 10.1080/21655979.2022.2079270.
7. 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.
8. Kim YM, Kim SJ, Tatsunami R, et al. ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis[J]. Am J Physiol Cell Physiol, 2017, 312(6): C749-C764. DOI: 10.1152/ajpcell.00346.2016.
9. 徐嫚鴻, 王林妮, 林婷婷, 等. 多聚嘧啶序列結合蛋白相關剪接因子對缺氧誘導人視網膜微血管內皮細胞功能的影響[J]. 中華眼底病雜志, 2020, 36(2): 135-142. DOI: 10.3760/cma.j.issn.1005-1015.2020.02.010.Xu MH, Wang LN, Lin TT, et al. Effect of polypyrimidine sequence-binding protein-associated splicing factors on hypoxia-induced function of human retinal microvascular endothelial cells[J]. Chin J Fundus Dis, 2020, 36(2): 135-142. DOI: 10.3760/cma.j.issn.1005-1015.2020.02.010.
10. Bellezza I, Giambanco I, Minelli A, et al. Nrf2-Keap1 signaling in oxidative and reductive stress[J]. Biochim Biophys Acta Mol Cell Res, 2018, 1865(5): 721-733. DOI: 10.1016/j.bbamcr.2018.02.010.
11. Chen J, Wang L, Chen Y, et al. Phosphatidylinositol 3 kinase pathway and 4-hydroxy-2-nonenal-induced oxidative injury in the RPE[J]. Invest Ophthalmol Vis Sci, 2009, 50(2): 936-942. DOI: 10.1167/iovs.08-2439.
12. Zakaria A, Rady M, Mahran L, et al. Pioglitazone attenuates lipopolysaccharide-induced oxidative stress, dopaminergic neuronal loss and neurobehavioral impairment by activating Nrf2/ARE/HO-1[J]. Neurochem Res, 2019, 44: 2856-2868. DOI: 10.1007/s11064-019-02907-0.
13. Sun X, Chen L, He Z. PI3K/Akt-Nrf2 and anti-inflammation effect of macrolides in chronic obstructive pulmonary disease[J]. Curr Drug Metab, 2019, 20(4): 301-304. DOI: 10.2174/1389200220666190227224748.
14. Wen Z, Hou W, Wu W, et al. 6'-O-galloylpaeoniflorin attenuates cerebral ischemia reperfusion-induced neuroinflammation and oxidative stress via PI3K/Akt/Nrf2 activation[J/OL]. Oxid Med Cell Longev, 2018, 2018: 8678267[2018-03-25]. https://europepmc.org/article/MED/29765506. DOI:10.1155/2018/8678267.
15. Wang L, Chen Y, Sternberg P, et al. Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE[J]. Invest Ophthalmol Vis Sci, 2008, 49(4): 1671-1678. DOI 10.1167/iovs.07-1099.

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Chinese Journal of Ocular Fundus Diseases

Effect of high expression of polypyrimidine tract-binding protein-associated splicing factor on retinal microvascular endothelial cells

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