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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
  • Sowmya Shree Gopal
  • Ganesan Ponesakki
Original Article
  • 29 Downloads

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

Glucose ARPE-19 Oxidative stress Redox transcription factors Antioxidant enzymes 

Abbreviations

ARE

Antioxidant response element

ARPE-19

Adult retinal pigment epithelial cell line-19

ATCC

American type culture collection

CCCP

Carbonyl cyanide m-chlorophenyl hydrazine

CRALBP

Cellular retinaldehyde-binding protein

DCF

Dichlorofluorescein

DCFH-DA

2′,7′-Dichlorofluorescein diacetate

DMEM/F12

Dulbecco’s modified eagle medium nutrient mixture F-12

ER

Endoplasmic reticulum

FBS

Fetal bovine serum

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HO-1

Heme oxygenase 1

H2O2

Hydrogen peroxide

MMP

Mitochondrial membrane potential

NFκB

Nuclear factor kappa B

Nrf2

Nuclear factor erythroid-2 related factor 2

POS

Photoreceptor outer segment

ROS

Reactive oxygen species

RPE

Retinal pigmental epithelium

RPE-65

Retinal pigment epithelium-specific 65 kDa protein

SOD-2/MnSOD

Superoxide dismutase 2/manganese-dependent superoxide dismutase

WST-1

Water-soluble tetrazolium salt-1

XBP-1

X-box binding protein

ZO-1

Zonula occludens-1

Notes

Acknowledgements

The author, Arpitha H S acknowledges the University Grants Commission (UGC), New Delhi, India for granting Research Fellowship. The authors thank the Academy of Scientific & Innovative Research (AcSIR) and the Director, CSIR-CFTRI for the constant support to carry out this work.

Funding

This study was funded by the 12th Five Year Plan Project (BSC-0404) of CSIR, New Delhi.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Molecular NutritionCSIR-Central Food Technological Research Institute (CFTRI)MysoreIndia

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