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. 2019 Feb;46(1):1263-1274.
doi: 10.1007/s11033-019-04597-x. Epub 2019 Jan 9.

An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells

Arpitha Haranahalli Shivarudrappa  1 , Sowmya Shree Gopal  1 , Ganesan Ponesakki  2
Affiliations

An in vitro protocol to study the effect of hyperglycemia on intracellular redox signaling in human retinal pigment epithelial (ARPE-19) cells

Arpitha Haranahalli Shivarudrappa et al. Mol Biol Rep. 2019 Feb.

Abstract

DMEM/F12 nutrient mixture, a recommended media for ARPE-19 culture, contains glucose concentration of 17.5 mM. But, several recent studies employed normal glucose media (5.5 mM) that was shown to affect the growth and function of ARPE-19 cells. Here, we set a protocol to study the effect of hyperglycemia on intracellular oxidative stress and redox status in ARPE-19 using DMEM/F12 as control. The WST-1 assay was performed to analyze the viability of ARPE-19 upon glucose treatment. The intracellular oxidative stress was measured by a dichlorofluorescein assay. The mitochondrial membrane potential (MMP) was monitored by using a JC-10 MMP assay kit. The expression of antioxidant marker proteins was analyzed by western blotting. Exogenous addition of glucose (7.5 and 12.5 mM) for 24 and 48 h did not change the viability and morphology of ARPE-19 cells. Hyperglycemia increased intracellular ROS level and decreased MMP in a dose-dependent manner. High-glucose treatment for 24 h down-regulated the protein expression of redox-specific transcription factors Nrf-2, XBP-1 and NF-κB, and subsequently decreased the expression of HO-1, catalase, and SOD-2. This study offers baseline information for the subsequent use of DMEM/F12 nutrient mixture to study glucose-mediated changes in intracellular oxidative stress and redox status of ARPE-19 without affecting its basic functions.

Keywords: ARPE-19; Antioxidant enzymes; Glucose; Oxidative stress; Redox transcription factors.

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References

    1. Hamann S (2002) Molecular mechanisms of water transport in the eye. Int Rev Cytol 215:395–431 - DOI - PubMed
    1. Simo R, Villarroel M, Corraliza L, Hernandez C, Garcia-Ramirez M (2010) The retinal pigment epithelium: Something more than a constituent of the blood-retinal barrier-implications for the pathogenesis of diabetic retinopathy. J Biomed Biotechnol 2010:190724 - DOI - PubMed - PMC
    1. Davidson AE, Millar ID, Urquhart JE, Burgess-Mullan R, Shweikh Y, Parry N, O’Sullivan J, Maher GJ, McKibbin M, Downes SM, Lotery AJ (2009) Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa. Am J Hum Genet 85:581–592 - DOI - PubMed - PMC
    1. Bianchi E, Scarinci F, Ripandelli G, Feher J, Pacella E, Magliulo G, Gabrieli CB, Plateroti R, Plateroti P, Mignini F, Artico M (2013) Retinal pigment epithelium, age-related macular degeneration and neurotrophic keratouveitis. Int J Mol Med 31:232–242 - DOI - PubMed
    1. Dunn KC, Aotaki-keen AE, Putkey FR, Hjelmeland LM (1996) ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62:155–170 - DOI - PubMed

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