Effects of probucol on high glucose-induced specificity protein 1/Keap1/Nrf2/glutamate-cysteine ligase catalytic in the cultured human müller cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Li Chenxiang ¹ , Ai Shibei ² ,  Chen Zhongping ^1,3 , Zhou Xuxia ¹

1. Aier School of Ophthalmology, Central South University, Changsha 410015, China;
2. The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen 518000, China;
3. Changsha Aier Eye Hospital, Changsha 410015, China;

Corresponding author：

Chen Zhongping, Email: 13755047346@163.com

Keywords：

Probucol/therapeutic use; Sp1 transcription factor; NF-E2-related factor 2; Glutamate-cysteine ligase; Müller cell; Kelchlike ECH assoiated protein1

DOI：

10.3760/cma.j.issn.1005-1015.2019.02.015

Video：

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ObjectiveTo observe the expression of probucol on high glucose-induced specificity protein 1(SP1), kelchlike ECH associated protein1 (Keap1), NF-E2-related factor 2 (Nrf2) and glutamate-cysteine ligase catalytic (GCLC) in the cultured human müller cells and preliminary study the antioxidation of the probucol on müller cells.MethodsPrimary cultured human müller cells were randomly divided into four groups: normoglycaemia group (5.5 mmol/L glucose), normoglycaemia with probucol group (5.5 mmol/L glucose+100 μmol/L probucol), hyperglycemia group (25.0 mmol/L glucose), hyperglycemia with probucol group (25.0 mmol/L glucose + 100 μmol/L probucol). Immunofluorescence staining was used to assess distribution of SP1, Keap1, Nrf2, GCLC in human Müller cells. SP1, Keap1, Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR). Independent sample t test was used to compare the data between the two groups.ResultsAll müller cells expressed glutamine synthetase (＞95%), which confirmed the cultured cells in vitro were the purification of generations of müller cells. The expressions of SP1, Keap1, Nrf2, and GCLC protein were positive in human müller cells. qRT-PCR indicated that SP1 (t=28.30, P＜0.000), Keap1 (t=5.369, P=0.006), and Nrf2 (t=10.59, P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group; GCLC (t=4.633, P=0.010) mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group. However, SP1 (t=12.60, P=0.000) and Keap1 (t=4.076, P=0.015) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group; Nrf2 (t=12.90, P=0.000) and GCLC (t=15.96, P＜0.000) mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group.ConclusionProbucol plays an antioxidant role by inhibiting the expression of SP1, Keap1 and up-regulating the expression of Nrf2, GCLC in müller cells induced by high glucose.

Citation： Li Chenxiang, Ai Shibei, Chen Zhongping, Zhou Xuxia. Effects of probucol on high glucose-induced specificity protein 1/Keap1/Nrf2/glutamate-cysteine ligase catalytic in the cultured human müller cells. Chinese Journal of Ocular Fundus Diseases, 2019, 35(2): 187-191. doi: 10.3760/cma.j.issn.1005-1015.2019.02.015 Copy

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2.	Atkin M, Laight D, Cummings MH. The effects of garlic extract upon endothelial function, vascular inflammation, oxidative stress and insulin resistance in adults with type 2 diabetes at high cardiovascular risk. A pilot double blind randomized placebo controlled trial[J]. Diabetes Complications, 2016, 30(4): 723-727. DOI: 10.1016/j.jdiacomp.2016.01.003.
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4.	Vellanki S, Ferrigno A, Alanis Y, et al. High glucose and glucose deprivation modulate Müller cell viability and VEGF secretion[J]. Int J Ophthalmol Eye Sci, 2016, 4(2): 178-183.
5.	Dehdashtian E, Mehrzadi S, Yousefi B, et al. Diabetic retinopathy pathogenesis and the ameliorating effects of melatonin; involvement of autophagy, inflammation and oxidative stress[J]. Life Sci, 2018, 193: 20-33. DOI: 10.1016/j.lfs.2017.12.001.
6.	Hui Y, Yin Y. MicroRNA-145 attenuates high glucose-induced oxidative stress and inflammation in retinal endothelial cells through regulating TLR4/NF-κB signaling[J]. Life Sci, 2018, 207: 212-218. DOI: 10.1016/j.lfs.2018.06.005.
7.	Zhong Q, Mishra M, Kowluru RA. Transcription factor Nrf2-mediated antioxidant defense system in the development of diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2013, 54(6): 3941-3948. DOI: 10.1167/iovs.13-11598.
8.	Shelton P, Jaiswal AK. The transcription factor NF-E2-related factor 2(Nrf2): a protooncogene?[J]. FASEB J, 2013, 27(2): 414-423. DOI: 10.1096/fj.12-217257.
9.	胡崇宇, 李雅嘉, 王晨旭, 等. 普罗布考联合胰激肽原酶治疗老年糖尿病周围神经病变的临床效果[J]. 中国老年学杂志, 2018, 38(5): 1034-1036. DOI: 10.3969/j.issn.1005-9202.2018.05.004.Hu CY, Li WJ, Wang CX, et al. The clinical effect of probucol combined with pancreatic kininogenase in the treatment of senile diabetic peripheral neuropathy[J]. Chinese Journal of Gerontology, 2018, 38(5): 1034-1036. DOI: 10.3969/j.issn.1005-9202.2018.05.004.
10.	Duan SB, Liu GL, Wang YH, et al. Epithelial-to-mesenchymal transdifferentiation of renal tubular epithelial cell mediated by oxidative stress and intervention effect of probucol in diabetic nephropathy rats[J]. Ren Fail, 2012, 34(10): 1244-1251. DOI: 10.3109/0886022X.2012.718711.
11.	Müller D, Schopp P, Melchinger AE. Selection on expected maximum haploid breeding values can increase genetic gain in recurrent genomic selection[J]. G3(Bethesda), 2018, 8(4): 1173-1181. DOI: 10.1534/g3.118.200091.
12.	王心蕊, 王越晖, 辛颖, 等. Müller细胞在高血糖状态下的变化对维持视网膜结构与功能的影响[J]. 中国临床康复, 2003, 7(24): 3294-3295. DOI: 10.3321/j.issn:1673-8225.2003.24.002.Wang XR, Wang YH, Xin Y, et al. Effects of high glucose on ultrastructure of Müller cells and structure and function of retina[J]. Chinese Journal of Clinical Rehabilitation, 2003, 7(24): 3294-3295. DOI: 10.3321/j.issn:1673-8225.2003.24.002.
13.	Di Marco E, Jha JC, Sharma A, et al. Are reactive oxygen species still the basis for diabetic complications?[J]. Clin Sci (Lond), 2015, 129(2): 199-216. DOI: 10.1042/CS20150093.
14.	Batliwala S, Xavier C, Liu Y, et al. Involvement of Nrf2 in ocular diseases[J]. Oxid Med Cell Longev, 2017, 2017: 1703810. DOI: 10.1155/2017/1703810.
15.	Bellezza I, Giambanco I, Minelli A, et al. Nrf2-Keap1 signaling in oxidative and reductive stress[J]. Biochim Biophys Acta, 2018, 1865(5): 721-733. DOI: 10.1016/j.bbamcr.2018.02.010.
16.	Sanchez AP, Sharma K. Transcription factors in the pathogenesis of diabetic nephropathy[J]. Expert Rev Mol Med, 2009, 11: 13. DOI: 10.1017/S1462399409001057.
17.	Guo D, Wu B, Yan J, et al. A possible gene silencing mechanism: hypermethylation of the Keap1 promoter abrogates binding of the transcription factor Sp1 in lung cancer cells[J]. Biochem Biophys Res Commun, 2012, 428(1): 80-85. DOI: 10.1016/j.bbrc.2012.10.010.
18.	Mishra M, Zhong Q, Kowluru RA. Epigenetic modifications of Nrf2-mediated glutamate-cysteine ligase: implications for the development of diabetic retinopathy and the metabolic memory phenomenon associated with its continued progression[J]. Free Radic Biol Med, 2014, 75: 129-139. DOI: 10.1016/j.freeradbiomed.2014.07.001.
19.	Mishra M, Zhong Q, Kowluru RA. Epigenetic modifications of Keap1 regulate its interaction with the protective factor Nrf2 in the development of diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2014, 55(11): 7256-7265. DOI: 10.1167/iovs.14-15193.
20.	Kowluru RA. Effect of advanced glycation end products on accelerated apoptosis of retinal capillary cells under in vitro conditions[J]. Life Sci, 2005, 76(9): 1051-1060. DOI: 10.1016/j.lfs.2004.10.017.
21.	Kanwar M, Kowluru RA. Role of glyceraldehyde 3-phosphate dehydrogenase in the development and progression of diabetic retinopathy[J]. Diabetes, 2009, 58(1): 227-234. DOI: 10.2337/db08-1025.
22.	Kowluru RA, Mishra M. Epigenetic regulation of redox signaling in diabetic retinopathy: Role of Nrf2[J]. Free Radic Biol Med, 2017, 103: 155-164. DOI: 10.1016/j.freeradbiomed.2016.12.030.
23.	Fu H, Li G, Liu C, et al. Probucol prevents atrial remodeling by inhibiting oxidative stress and TNF-α/NF-κB/TGF-β signal transduction pathway in alloxan-induced diabetic rabbits[J]. J Cardiovasc Electrophysiol, 2015, 26(2): 211-222. DOI: 10.1111/jce.12540.
24.	Zhu H, Jin X, Zhao J, et al. Probucol protects against atherosclerosis through lipid-lowering and suppressing immune maturation of CD11c+ dendritic cells in STZ-induced diabetic LDLR-/-mice[J]. J Cardiovasc Pharmacol, 2015, 65(6): 620-627. DOI: 10.1097/FJC.0000000000000234.
25.	Ishitobi T, Hyogo H, Tokumo H, et al. Efficacy of probucol for the treatment of non-alcoholic steatohepatitis with dyslipidemia: An open-label pilot study[J]. Hepatol Res, 2014, 44(4): 429-435. DOI: 10.1111/hepr.12135.
26.	Agardh E, Hultberg B, Agardh C. Effects of inhibition of glycation and oxidative stress on the development of cataract and retinal vessel abnormalities in diabetic rats[J]. Curr Eye Res, 2000, 21(1): 543-549. DOI: 10.1076/0271-3683(200007)2111-ZFT543.
27.	陈忠平, 张入铭. 普罗布考治疗高血脂非增生型糖尿病视网膜病变疗效观察[J]. 中华眼底病杂志, 2012, 28(5): 477-481. DOI: 10.3760/cma.j.issn.1005-1015.2012.05.011.Chen ZP, Zhang RM. Probucol for non-proliferative diabetic retinopathy with hyperlipidemia[J]. Chin J Ocul Fundus Dis, 2012, 28(5): 477-481. DOI: 10.3760/cma.j.issn.1005-1015.2012.05.011.
28.	陈忠平, 唐仕波, 王启常, 等. 普罗布考辅助治疗高血脂糖尿病黄斑水肿的临床观察[J]. 中华眼底病杂志, 2013, 29(5): 490-494. DOI: 10.3760/cma.j.issn.1005-1015.2013.05.011.Chen ZP, Tang SB, Wang QC. Clinical study of probucol in the treatment of hyperlipidemia diabetic macular edema[J]. Chin J Ocul Fundus Dis, 2013, 29(5): 490-494. DOI: 10.3760/cma.j.issn.1005-1015.2013.05.011.

1. Song P, Yu J, Chan KY, et al. Prevalence, risk factors and burden of diabetic retinopathy in China: a systematic review and meta-analysis[J/OL]. J Glob Health, 2018, 8(1): 010803[2018-06-09]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997368/. DOI: 10.7189/jogh.08.010803.
2. Atkin M, Laight D, Cummings MH. The effects of garlic extract upon endothelial function, vascular inflammation, oxidative stress and insulin resistance in adults with type 2 diabetes at high cardiovascular risk. A pilot double blind randomized placebo controlled trial[J]. Diabetes Complications, 2016, 30(4): 723-727. DOI: 10.1016/j.jdiacomp.2016.01.003.
3. Vecino E, Rodriguez FD, Ruzafa N, et al. Glia-neuron interactions in the mammalian retina[J]. Prog Retin Eye Res, 2016, 51: 1-40. DOI: 10.1016/j.preteyeres.2015.06.003.
4. Vellanki S, Ferrigno A, Alanis Y, et al. High glucose and glucose deprivation modulate Müller cell viability and VEGF secretion[J]. Int J Ophthalmol Eye Sci, 2016, 4(2): 178-183.
5. Dehdashtian E, Mehrzadi S, Yousefi B, et al. Diabetic retinopathy pathogenesis and the ameliorating effects of melatonin; involvement of autophagy, inflammation and oxidative stress[J]. Life Sci, 2018, 193: 20-33. DOI: 10.1016/j.lfs.2017.12.001.
6. Hui Y, Yin Y. MicroRNA-145 attenuates high glucose-induced oxidative stress and inflammation in retinal endothelial cells through regulating TLR4/NF-κB signaling[J]. Life Sci, 2018, 207: 212-218. DOI: 10.1016/j.lfs.2018.06.005.
7. Zhong Q, Mishra M, Kowluru RA. Transcription factor Nrf2-mediated antioxidant defense system in the development of diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2013, 54(6): 3941-3948. DOI: 10.1167/iovs.13-11598.
8. Shelton P, Jaiswal AK. The transcription factor NF-E2-related factor 2(Nrf2): a protooncogene?[J]. FASEB J, 2013, 27(2): 414-423. DOI: 10.1096/fj.12-217257.
9. 胡崇宇, 李雅嘉, 王晨旭, 等. 普罗布考联合胰激肽原酶治疗老年糖尿病周围神经病变的临床效果[J]. 中国老年学杂志, 2018, 38(5): 1034-1036. DOI: 10.3969/j.issn.1005-9202.2018.05.004.Hu CY, Li WJ, Wang CX, et al. The clinical effect of probucol combined with pancreatic kininogenase in the treatment of senile diabetic peripheral neuropathy[J]. Chinese Journal of Gerontology, 2018, 38(5): 1034-1036. DOI: 10.3969/j.issn.1005-9202.2018.05.004.
10. Duan SB, Liu GL, Wang YH, et al. Epithelial-to-mesenchymal transdifferentiation of renal tubular epithelial cell mediated by oxidative stress and intervention effect of probucol in diabetic nephropathy rats[J]. Ren Fail, 2012, 34(10): 1244-1251. DOI: 10.3109/0886022X.2012.718711.
11. Müller D, Schopp P, Melchinger AE. Selection on expected maximum haploid breeding values can increase genetic gain in recurrent genomic selection[J]. G3(Bethesda), 2018, 8(4): 1173-1181. DOI: 10.1534/g3.118.200091.
12. 王心蕊, 王越晖, 辛颖, 等. Müller细胞在高血糖状态下的变化对维持视网膜结构与功能的影响[J]. 中国临床康复, 2003, 7(24): 3294-3295. DOI: 10.3321/j.issn:1673-8225.2003.24.002.Wang XR, Wang YH, Xin Y, et al. Effects of high glucose on ultrastructure of Müller cells and structure and function of retina[J]. Chinese Journal of Clinical Rehabilitation, 2003, 7(24): 3294-3295. DOI: 10.3321/j.issn:1673-8225.2003.24.002.
13. Di Marco E, Jha JC, Sharma A, et al. Are reactive oxygen species still the basis for diabetic complications?[J]. Clin Sci (Lond), 2015, 129(2): 199-216. DOI: 10.1042/CS20150093.
14. Batliwala S, Xavier C, Liu Y, et al. Involvement of Nrf2 in ocular diseases[J]. Oxid Med Cell Longev, 2017, 2017: 1703810. DOI: 10.1155/2017/1703810.
15. Bellezza I, Giambanco I, Minelli A, et al. Nrf2-Keap1 signaling in oxidative and reductive stress[J]. Biochim Biophys Acta, 2018, 1865(5): 721-733. DOI: 10.1016/j.bbamcr.2018.02.010.
16. Sanchez AP, Sharma K. Transcription factors in the pathogenesis of diabetic nephropathy[J]. Expert Rev Mol Med, 2009, 11: 13. DOI: 10.1017/S1462399409001057.
17. Guo D, Wu B, Yan J, et al. A possible gene silencing mechanism: hypermethylation of the Keap1 promoter abrogates binding of the transcription factor Sp1 in lung cancer cells[J]. Biochem Biophys Res Commun, 2012, 428(1): 80-85. DOI: 10.1016/j.bbrc.2012.10.010.
18. Mishra M, Zhong Q, Kowluru RA. Epigenetic modifications of Nrf2-mediated glutamate-cysteine ligase: implications for the development of diabetic retinopathy and the metabolic memory phenomenon associated with its continued progression[J]. Free Radic Biol Med, 2014, 75: 129-139. DOI: 10.1016/j.freeradbiomed.2014.07.001.
19. Mishra M, Zhong Q, Kowluru RA. Epigenetic modifications of Keap1 regulate its interaction with the protective factor Nrf2 in the development of diabetic retinopathy[J]. Invest Ophthalmol Vis Sci, 2014, 55(11): 7256-7265. DOI: 10.1167/iovs.14-15193.
20. Kowluru RA. Effect of advanced glycation end products on accelerated apoptosis of retinal capillary cells under in vitro conditions[J]. Life Sci, 2005, 76(9): 1051-1060. DOI: 10.1016/j.lfs.2004.10.017.
21. Kanwar M, Kowluru RA. Role of glyceraldehyde 3-phosphate dehydrogenase in the development and progression of diabetic retinopathy[J]. Diabetes, 2009, 58(1): 227-234. DOI: 10.2337/db08-1025.
22. Kowluru RA, Mishra M. Epigenetic regulation of redox signaling in diabetic retinopathy: Role of Nrf2[J]. Free Radic Biol Med, 2017, 103: 155-164. DOI: 10.1016/j.freeradbiomed.2016.12.030.
23. Fu H, Li G, Liu C, et al. Probucol prevents atrial remodeling by inhibiting oxidative stress and TNF-α/NF-κB/TGF-β signal transduction pathway in alloxan-induced diabetic rabbits[J]. J Cardiovasc Electrophysiol, 2015, 26(2): 211-222. DOI: 10.1111/jce.12540.
24. Zhu H, Jin X, Zhao J, et al. Probucol protects against atherosclerosis through lipid-lowering and suppressing immune maturation of CD11c+ dendritic cells in STZ-induced diabetic LDLR-/-mice[J]. J Cardiovasc Pharmacol, 2015, 65(6): 620-627. DOI: 10.1097/FJC.0000000000000234.
25. Ishitobi T, Hyogo H, Tokumo H, et al. Efficacy of probucol for the treatment of non-alcoholic steatohepatitis with dyslipidemia: An open-label pilot study[J]. Hepatol Res, 2014, 44(4): 429-435. DOI: 10.1111/hepr.12135.
26. Agardh E, Hultberg B, Agardh C. Effects of inhibition of glycation and oxidative stress on the development of cataract and retinal vessel abnormalities in diabetic rats[J]. Curr Eye Res, 2000, 21(1): 543-549. DOI: 10.1076/0271-3683(200007)2111-ZFT543.
27. 陈忠平, 张入铭. 普罗布考治疗高血脂非增生型糖尿病视网膜病变疗效观察[J]. 中华眼底病杂志, 2012, 28(5): 477-481. DOI: 10.3760/cma.j.issn.1005-1015.2012.05.011.Chen ZP, Zhang RM. Probucol for non-proliferative diabetic retinopathy with hyperlipidemia[J]. Chin J Ocul Fundus Dis, 2012, 28(5): 477-481. DOI: 10.3760/cma.j.issn.1005-1015.2012.05.011.
28. 陈忠平, 唐仕波, 王启常, 等. 普罗布考辅助治疗高血脂糖尿病黄斑水肿的临床观察[J]. 中华眼底病杂志, 2013, 29(5): 490-494. DOI: 10.3760/cma.j.issn.1005-1015.2013.05.011.Chen ZP, Tang SB, Wang QC. Clinical study of probucol in the treatment of hyperlipidemia diabetic macular edema[J]. Chin J Ocul Fundus Dis, 2013, 29(5): 490-494. DOI: 10.3760/cma.j.issn.1005-1015.2013.05.011.

Chinese Journal of Ocular Fundus Diseases

Effects of probucol on high glucose-induced specificity protein 1/Keap1/Nrf2/glutamate-cysteine ligase catalytic in the cultured human müller cells

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