SB431542 inhibits the effects of high glucose environment on RF/6A cells by mediating the PTEN-induced putative kinase 1/Parkin pathway to regulate mitochondrial autophagy_Chinese Journal of Ocular Fundus Diseases

Authors：

Zhuang Tongtong , Wang Qing , Wang Yong , Dong Lijie ,  Liang Jingli

Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;

Corresponding author：

Liang Jingli, Email: youyou_leung@hotmail.com

Keywords：

Monkey choroidal-retinal endothelial cells; High glucose; Diabetic retinopathy; Transforming growth factor-β inhibitor; Mitochondria autophagy

DOI：

10.3760/cma.j.cn511434-20250421-00180

Video：

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Objective To explore the effect of SB431542 on monkey choroidal-retinal endothelial (RF/6A) cells in high glucose state and its mechanism of regulating mitochondrial autophagy by mediating the PINK1/Parkin pathway. Methods Cell experiments. The minimum effective drug concentration of SB431542 was determined by using the Cell Counting Kit-8 (CCK-8). RF/6A cells cultured in vitro were divided into normal group (NC group), mannitol group, high glucose group (HG group), high glucose with dimethyl sulfoxide group (HG + DMSO group), and high glucose + SB431542 group (HG + SB431542 group). CCK-8 and cell scratch assay were used to detect the proliferation and migration of RF/6A cells induced by high glucose. The expression of autophagosomes was detected by autophagy staining kit; the expression level of reactive oxygen species was detected by reactive oxygen species kit; the expression level of mitochondrial superoxide in cells was detected by MitoSOX fluorescent probe; the mitochondrial membrane potential level in cells was detected by JC-10 staining; the morphology of mitochondria was observed by MitoTracker staining, and the total area of mitochondria, average shape factor and branch length were quantitatively analyzed.Cellular immunofluorescence (IF) staining was used to detect the fluorescence expression of EndMT markers vimentin and VE-cadherin; Western blotting (WB) was used to detect the protein expression of vimentin, VE-cadherin, and mitochondrial autophagy-related proteins TOMM20, LC3, P62, PINK1, and Parkin; one-way analysis of variance was used for comparisons among multiple groups.Results The minimum effective drug concentration of SB431542 was 5 μmol/L. SB431542 significantly inhibited the proliferation and migration of RF/6A cells induced by high glucose (F = 81.92、87.84, P＜0.000 1). SB431542 suppressed the expression of reactive oxygen species and mitochondrial superoxide induced by high glucose (F = 429.50, 450.20; P＜0.000 1), restored the mitochondrial membrane potential level (F = 315.3, P＜0.000 1), and restored the mitochondrial morphology (F = 209.50, P＜0.000 1). IF and WB confirmed that SB431542 inhibited the expression of Vimentin induced by high glucose (F = 117.30、51.11; P＜0.000 1) and upregulated the expression of VE-cadherin (F = 136.80、27.54; P＜0.000 1). WB further confirmed that SB431542 upregulated the protein expression of LC3, PINK1, and Parkin (F = 16.64, 37.72, 32.63; P＜0.05) and inhibited the protein expression of TOMM20 and P62 (F = 33.87, 67.77; P＜0.01). Conclusion SB431542 upregulates mitochondrial autophagy expression through activation of the PINK1/Parkin pathway, effectively restores mitochondria-related functions to maintain homeostasis, and inhibits high glucose-induced RF/6A cell proliferation,migration,and EndMT formation.

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2.	Korhonen A, Gucciardo E, Lehti K, et al. Proliferative diabetic retinopathy transcriptomes reveal angiogenesis, anti-angiogenic therapy escape mechanisms, fibrosis and lymphatic involvement[J/OL]. Sci Rep, 2021, 11(1): 18810[2021-09-22]. https://pubmed.ncbi.nlm.nih.gov/34552123/. DOI: 10.1038/s41598-021-97970-5.
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4.	Jiménez-Loygorri JI, Benítez-Fernández R, Viedma-Poyatos Á, et al. Mitophagy in the retina: viewing mitochondrial homeostasis through a new lens[J/OL]. Prog Retin Eye Res, 2023, 96: 101205[2023-07-15]. https://pubmed.ncbi.nlm.nih.gov/37454969/. DOI: 10.1016/j.preteyeres.2023.101205.
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7.	Guo L, Wen X, Hou Y, et al. Dihydroartemisinin inhibits endothelial cell migration via the TGF-β1/ALK5/SMAD2 signaling pathway[J/OL]. Exp Ther Med, 2021, 22(1): 709[2021-05-03]. https://pubmed.ncbi.nlm.nih.gov/34007318/. DOI: 10.3892/etm.2021.10141.
8.	Zhang J, Li R, Liu Q, et al. SB431542-loaded liposomes alleviate liver fibrosis by suppressing TGF-β signaling[J]. Mol Pharm, 2020, 17(11): 4152-4162. DOI: 10.1021/acs.molpharmaceut.0c00633.
9.	Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
10.	Chaudhry A, Shi R, Luciani DS. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells[J/OL]. Am J Physiol Endocrinol Metab, 2020, 318(2): E87-101[2020-02-01]. https://pubmed.ncbi.nlm.nih.gov/31846372/. DOI: 10.1152/ajpendo.00457.2019.
11.	Valente AJ, Maddalena LA, Robb EL, et al. A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture[J]. Acta Histochem, 2017, 119(3): 315-326. DOI: 10.1016/j.acthis.2017.03.001.
12.	何珍, 寇振宇, 东莉洁, 等. 组织蛋白酶L抑制剂经线粒体途径抑制氧化应激诱导的视网膜色素上皮细胞凋亡[J]. 中华眼底病杂志, 2024, 40(5): 379-386. DOI: 10.3760/cma.j.cn511434-20231207-00474.He Z, Kou ZY, Dong LJ, et al. Cathepsin L inhibitor suppresses oxidative stress-induced apoptosis of retinal pigment epithelial cells by targeting mitochondria[J]. Chin J Ocul Fundus Dis, 2024, 40(5): 379-386. DOI: 10.3760/cma.j.cn511434-20231207-00474.
13.	Callan A, Jha S, Valdez L, et al. TGF-β signaling pathways in the development of diabetic retinopathy[J/OL]. Int J Mol Sci, 2024, 25(5): 3052[2024-04-06]. https://pubmed.ncbi.nlm.nih.gov/38474297/. DOI: 10.3390/ijms25053052.
14.	Wang M, Sheng KJ, Fang JC, et al. Redox signaling in diabetic retinopathy and opportunity for therapeutic intervention through natural products[J/OL]. Eur J Med Chem, 2022, 244: 114829[2022-12-15]. https://pubmed.ncbi.nlm.nih.gov/36209631/. DOI: 10.1016/j.ejmech.2022.114829.
15.	Rodriguez R, Lowe K, Keniry M, et al. Involvement of TGFβ signaling pathway in oxidative stress and diabetic retinopathy[J]. Arch Clin Exp Ophthalmol, 2021, 3(2): 23-28.
16.	Wang H, Ramshekar A, Kunz E, et al. 7-ketocholesterol induces endothelial-mesenchymal transition and promotes fibrosis: implications in neovascular age-related macular degeneration and treatment[J]. Angiogenesis, 2021, 24(3): 583-595. DOI: 10.1007/s10456-021-09770-0.
17.	曹靖靖, 韩菲菲, 寇振宇, 等. SB431542对高糖诱导的RPE细胞自噬及上皮-间充质转化影响的实验研究[J]. 中华眼科杂志, 2025, 61(3): 202-210. DOI: 10.3760/cma.j.cn112142-20240311-00109.Cao JJ, Han FF, Kou ZY, et al. Effect of SB431542 on autophagy and epithelial mesenchymal transition in retinal pigment epithelial cells induced by high glucose[J]. Chin J Ocul Fundus Dis, 2025, 61(3): 202-210. DOI: 10.3760/cma.j.cn112142-20240311-00109.
18.	Nijim W, Moustafa M, Humble J, et al. Endothelial to mesenchymal cell transition in diabetic retinopathy: targets and therapeutics[J/OL]. Front Ophthalmol (Lausanne), 2023, 3: 1230581[2023-09-07]. https://pubmed.ncbi.nlm.nih.gov/38983088/. DOI: 10.3389/fopht.2023.1230581.
19.	Rossmann MP, Dubois SM, Agarwal S, et al. Mitochondrial function in development and disease[J/OL]. Dis Model Mech, 2021, 14(6): dmm048912[2021-06-01]. https://pubmed.ncbi.nlm.nih.gov/34114603/. DOI: 10.1242/dmm.048912.
20.	Wu Y, Zou H. Research progress on mitochondrial dysfunction in diabetic retinopathy[J/OL]. Antioxidants (Basel), 2022, 11(11): 2250[2022-11-15]. https://pubmed.ncbi.nlm.nih.gov/36421435/. DOI: 10.3390/antiox11112250.
21.	Kowluru RA. Mitochondrial stability in diabetic retinopathy: lessons learned from epigenetics[J]. Diabetes, 2019, 68(2): 241-247. DOI: 10.2337/dbi18-0016.
22.	Tanaka K. The PINK1-Parkin axis: an overview[J]. Neurosci Res, 2020, 159: 9-15. DOI: 10.1016/j.neures.2020.01.006.
23.	Lenka DR, Dahe SV, Antico O, et al. Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans[J/OL]. Elife, 2024, 13: RP96699[2024-09-02]. https://pubmed.ncbi.nlm.nih.gov/39221915/. DOI: 10.7554/eLife.96699.
24.	Narendra DP, Youle RJ. The role of PINK1-Parkin in mitochondrial quality control[J]. Nat Cell Biol, 2024, 26(10): 1639-1651. DOI: 10.1038/s41556-024-01513-9.
25.	Sauvé V, Sung G, MacDougall EJ, et al. Structural basis for feedforward control in the PINK1/Parkin pathway[J/OL]. EMBO J, 2022, 41(12): e109460[2022-06-14]. https://pubmed.ncbi.nlm.nih.gov/35491809/. DOI: 10.15252/embj.2021109460.
26.	Fakih R, Sauvé V, Gehring K. Feedforward activation of PRKN/parkin[J]. Autophagy, 2023, 19(2): 729-730. DOI: 10.1080/15548627.2022.2100615.
27.	Qiu Y, Wang J, Li H, et al. Emerging views of OPTN (optineurin) function in the autophagic process associated with disease[J]. Autophagy, 2022, 18(1): 73-85. DOI: 10.1080/15548627.2021.1908722.
28.	Zhang MY, Zhu L, Bao X, et al. Inhibition of Drp1 ameliorates diabetic retinopathy by regulating mitochondrial homeostasis[J/OL]. Exp Eye Res, 2022, 220: 109095[2022-04-28]. https://pubmed.ncbi.nlm.nih.gov/35490835/. DOI: 10.1016/j.exer.2022.109095.
29.	Zhang Y, Xi X, Mei Y, et al. High-glucose induces retinal pigment epithelium mitochondrial pathways of apoptosis and inhibits mitophagy by regulating ROS/PINK1/Parkin signal pathway[J]. Biomed Pharmacother, 2019, 111: 1315-1325. DOI: 10.1016/j.biopha.2019.01.034.
30.	Huang L, Yao T, Chen J, et al. Effect of Sirt3 on retinal pigment epithelial cells in high glucose through Foxo3a/ PINK1-Parkin pathway mediated mitophagy[J/OL]. Exp Eye Res, 2022, 218: 109015[2022-02-28]. https://pubmed.ncbi.nlm.nih.gov/35240195/. DOI: 10.1016/j.exer.2022.109015.
31.	Li A, Gao M, Liu B, et al. Mitochondrial autophagy: molecular mechanisms and implications for cardiovascular disease[J/OL]. Cell Death Dis, 2022, 13(5): 444[2022-05-09]. https://pubmed.ncbi.nlm.nih.gov/35534453/. DOI: 10.1038/s41419-022-04906-6.

1. Kour V, Swain J, Singh J, et al. A review on diabetic retinopathy[J/OL]. Curr Diabetes Rev. 2024, 20(6): e201023222418[2024-04-12]. https://pubmed.ncbi.nlm.nih.gov/37867267/. DOI: 10.2174/0115733998253672231011161400.
2. Korhonen A, Gucciardo E, Lehti K, et al. Proliferative diabetic retinopathy transcriptomes reveal angiogenesis, anti-angiogenic therapy escape mechanisms, fibrosis and lymphatic involvement[J/OL]. Sci Rep, 2021, 11(1): 18810[2021-09-22]. https://pubmed.ncbi.nlm.nih.gov/34552123/. DOI: 10.1038/s41598-021-97970-5.
3. Ferrington DA, Fisher CR, Kowluru RA. Mitochondrial defects drive degenerative retinal diseases[J]. Trends Mol Med, 2020, 26(1): 105-118. DOI: 10.1016/j.molmed.2019.10.008.
4. Jiménez-Loygorri JI, Benítez-Fernández R, Viedma-Poyatos Á, et al. Mitophagy in the retina: viewing mitochondrial homeostasis through a new lens[J/OL]. Prog Retin Eye Res, 2023, 96: 101205[2023-07-15]. https://pubmed.ncbi.nlm.nih.gov/37454969/. DOI: 10.1016/j.preteyeres.2023.101205.
5. Hombrebueno JR, Cairns L, Dutton LR, et al. Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy[J/OL]. JCI Insight, 2019, 4(23): e129760[2019-12-05]. https://pubmed.ncbi.nlm.nih.gov/31661466/. DOI: 10.1172/jci.insight.129760.
6. D'Amico AG, Maugeri G, Magrì B, et al. Targeting the PINK1/Parkin pathway: a new perspective in the prevention and therapy of diabetic retinopathy[J/OL]. Exp Eye Res, 2024, 247: 110024[2024-08-06]. https://pubmed.ncbi.nlm.nih.gov/39117133/. DOI: 10.1016/j.exer.2024.110024.
7. Guo L, Wen X, Hou Y, et al. Dihydroartemisinin inhibits endothelial cell migration via the TGF-β1/ALK5/SMAD2 signaling pathway[J/OL]. Exp Ther Med, 2021, 22(1): 709[2021-05-03]. https://pubmed.ncbi.nlm.nih.gov/34007318/. DOI: 10.3892/etm.2021.10141.
8. Zhang J, Li R, Liu Q, et al. SB431542-loaded liposomes alleviate liver fibrosis by suppressing TGF-β signaling[J]. Mol Pharm, 2020, 17(11): 4152-4162. DOI: 10.1021/acs.molpharmaceut.0c00633.
9. Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
10. Chaudhry A, Shi R, Luciani DS. A pipeline for multidimensional confocal analysis of mitochondrial morphology, function, and dynamics in pancreatic β-cells[J/OL]. Am J Physiol Endocrinol Metab, 2020, 318(2): E87-101[2020-02-01]. https://pubmed.ncbi.nlm.nih.gov/31846372/. DOI: 10.1152/ajpendo.00457.2019.
11. Valente AJ, Maddalena LA, Robb EL, et al. A simple ImageJ macro tool for analyzing mitochondrial network morphology in mammalian cell culture[J]. Acta Histochem, 2017, 119(3): 315-326. DOI: 10.1016/j.acthis.2017.03.001.
12. 何珍, 寇振宇, 东莉洁, 等. 组织蛋白酶L抑制剂经线粒体途径抑制氧化应激诱导的视网膜色素上皮细胞凋亡[J]. 中华眼底病杂志, 2024, 40(5): 379-386. DOI: 10.3760/cma.j.cn511434-20231207-00474.He Z, Kou ZY, Dong LJ, et al. Cathepsin L inhibitor suppresses oxidative stress-induced apoptosis of retinal pigment epithelial cells by targeting mitochondria[J]. Chin J Ocul Fundus Dis, 2024, 40(5): 379-386. DOI: 10.3760/cma.j.cn511434-20231207-00474.
13. Callan A, Jha S, Valdez L, et al. TGF-β signaling pathways in the development of diabetic retinopathy[J/OL]. Int J Mol Sci, 2024, 25(5): 3052[2024-04-06]. https://pubmed.ncbi.nlm.nih.gov/38474297/. DOI: 10.3390/ijms25053052.
14. Wang M, Sheng KJ, Fang JC, et al. Redox signaling in diabetic retinopathy and opportunity for therapeutic intervention through natural products[J/OL]. Eur J Med Chem, 2022, 244: 114829[2022-12-15]. https://pubmed.ncbi.nlm.nih.gov/36209631/. DOI: 10.1016/j.ejmech.2022.114829.
15. Rodriguez R, Lowe K, Keniry M, et al. Involvement of TGFβ signaling pathway in oxidative stress and diabetic retinopathy[J]. Arch Clin Exp Ophthalmol, 2021, 3(2): 23-28.
16. Wang H, Ramshekar A, Kunz E, et al. 7-ketocholesterol induces endothelial-mesenchymal transition and promotes fibrosis: implications in neovascular age-related macular degeneration and treatment[J]. Angiogenesis, 2021, 24(3): 583-595. DOI: 10.1007/s10456-021-09770-0.
17. 曹靖靖, 韩菲菲, 寇振宇, 等. SB431542对高糖诱导的RPE细胞自噬及上皮-间充质转化影响的实验研究[J]. 中华眼科杂志, 2025, 61(3): 202-210. DOI: 10.3760/cma.j.cn112142-20240311-00109.Cao JJ, Han FF, Kou ZY, et al. Effect of SB431542 on autophagy and epithelial mesenchymal transition in retinal pigment epithelial cells induced by high glucose[J]. Chin J Ocul Fundus Dis, 2025, 61(3): 202-210. DOI: 10.3760/cma.j.cn112142-20240311-00109.
18. Nijim W, Moustafa M, Humble J, et al. Endothelial to mesenchymal cell transition in diabetic retinopathy: targets and therapeutics[J/OL]. Front Ophthalmol (Lausanne), 2023, 3: 1230581[2023-09-07]. https://pubmed.ncbi.nlm.nih.gov/38983088/. DOI: 10.3389/fopht.2023.1230581.
19. Rossmann MP, Dubois SM, Agarwal S, et al. Mitochondrial function in development and disease[J/OL]. Dis Model Mech, 2021, 14(6): dmm048912[2021-06-01]. https://pubmed.ncbi.nlm.nih.gov/34114603/. DOI: 10.1242/dmm.048912.
20. Wu Y, Zou H. Research progress on mitochondrial dysfunction in diabetic retinopathy[J/OL]. Antioxidants (Basel), 2022, 11(11): 2250[2022-11-15]. https://pubmed.ncbi.nlm.nih.gov/36421435/. DOI: 10.3390/antiox11112250.
21. Kowluru RA. Mitochondrial stability in diabetic retinopathy: lessons learned from epigenetics[J]. Diabetes, 2019, 68(2): 241-247. DOI: 10.2337/dbi18-0016.
22. Tanaka K. The PINK1-Parkin axis: an overview[J]. Neurosci Res, 2020, 159: 9-15. DOI: 10.1016/j.neures.2020.01.006.
23. Lenka DR, Dahe SV, Antico O, et al. Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans[J/OL]. Elife, 2024, 13: RP96699[2024-09-02]. https://pubmed.ncbi.nlm.nih.gov/39221915/. DOI: 10.7554/eLife.96699.
24. Narendra DP, Youle RJ. The role of PINK1-Parkin in mitochondrial quality control[J]. Nat Cell Biol, 2024, 26(10): 1639-1651. DOI: 10.1038/s41556-024-01513-9.
25. Sauvé V, Sung G, MacDougall EJ, et al. Structural basis for feedforward control in the PINK1/Parkin pathway[J/OL]. EMBO J, 2022, 41(12): e109460[2022-06-14]. https://pubmed.ncbi.nlm.nih.gov/35491809/. DOI: 10.15252/embj.2021109460.
26. Fakih R, Sauvé V, Gehring K. Feedforward activation of PRKN/parkin[J]. Autophagy, 2023, 19(2): 729-730. DOI: 10.1080/15548627.2022.2100615.
27. Qiu Y, Wang J, Li H, et al. Emerging views of OPTN (optineurin) function in the autophagic process associated with disease[J]. Autophagy, 2022, 18(1): 73-85. DOI: 10.1080/15548627.2021.1908722.
28. Zhang MY, Zhu L, Bao X, et al. Inhibition of Drp1 ameliorates diabetic retinopathy by regulating mitochondrial homeostasis[J/OL]. Exp Eye Res, 2022, 220: 109095[2022-04-28]. https://pubmed.ncbi.nlm.nih.gov/35490835/. DOI: 10.1016/j.exer.2022.109095.
29. Zhang Y, Xi X, Mei Y, et al. High-glucose induces retinal pigment epithelium mitochondrial pathways of apoptosis and inhibits mitophagy by regulating ROS/PINK1/Parkin signal pathway[J]. Biomed Pharmacother, 2019, 111: 1315-1325. DOI: 10.1016/j.biopha.2019.01.034.
30. Huang L, Yao T, Chen J, et al. Effect of Sirt3 on retinal pigment epithelial cells in high glucose through Foxo3a/ PINK1-Parkin pathway mediated mitophagy[J/OL]. Exp Eye Res, 2022, 218: 109015[2022-02-28]. https://pubmed.ncbi.nlm.nih.gov/35240195/. DOI: 10.1016/j.exer.2022.109015.
31. Li A, Gao M, Liu B, et al. Mitochondrial autophagy: molecular mechanisms and implications for cardiovascular disease[J/OL]. Cell Death Dis, 2022, 13(5): 444[2022-05-09]. https://pubmed.ncbi.nlm.nih.gov/35534453/. DOI: 10.1038/s41419-022-04906-6.

Chinese Journal of Ocular Fundus Diseases

Latest ArticlesSB431542 inhibits the effects of high glucose environment on RF/6A cells by mediating the PTEN-induced putative kinase 1/Parkin pathway to regulate mitochondrial autophagy

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