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Carvacrol protects the ARPE19 retinal pigment epithelial cells against high glucose-induced oxidative stress, apoptosis, and inflammation by suppressing the TRPM2 channel signaling pathways

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Abstract

Purpose

The concentration of plasma high glucose (HGu) in diabetes mellitus (DM) induces the retinal pigment epithelial cell (ARPE19) death via the increase of inflammation, cytosolic (cytROS), and mitochondrial (mitROS) free oxygen radical generations. Transient potential melastatin 2 (TRPM2) cation channel is stimulated by cytROS and mitROS. Hence, the cytROS and mitROS-mediated excessive Ca2+ influxes via the stimulation of TRPM2 channel cause to the induction of DM-mediated retina oxidative cytotoxicity. Because of the antioxidant role of carvacrol (CRV), it may modulate oxidative cytotoxicity via the attenuation of TRPM2 in the ARPE19. We aimed to investigate the modulator action of CRV treatment on the HGu-mediated TRPM2 stimulation, oxidative stress, and apoptosis in the ARPE19 cell model.

Material and methods

The ARPE19 cells were divided into four groups as normal glucose (NGu), NGu + Carv, HGu, and HGu + CRV.

Results

The levels of cell death (propidium iodide/Hoechst rate) and apoptosis markers (caspases 3, 8, and 9), cytokine generations (IL-1β and TNF-α), ROS productions (cytROS, mitROS, and lipid peroxidation), TRPM2 currents, and intracellular free Ca2+ (Fluo/3) were increased in the HGu group after the stimulations of hydrogen peroxide and ADP-ribose, although their levels were diminished via upregulation of glutathione and glutathione peroxidase by the treatments of CRV and TRPM2 blockers.

Conclusion

Current results confirmed that the HGu-induced overload Ca2+ influx and oxidative retinal toxicity in the ARPE19 cells were induced by the stimulation of TRPM2, although they were modulated via the inhibition of TRPM2 by CRV. CRV may be noted as a potential therapeutic antioxidant to the TRPM2 activation-mediated retinal oxidative injury.

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Data availability

The data analyses in the current study were performed in the BSN Health, Analyses, Innov., Consul., Org., Agricul. and Industry Ltd (Isparta, Turkey) https://www.bsnsaglik.com.tr/. The details of current analyses are available from Prof. Dr. M. Nazıroğlu on reasonable request.

Abbreviations

2APB:

2-Aminoethoxydiphenyl borate

ACA:

N-(p-amylcinnamoyl)anthranilic acid

ADPR:

ADP-ribose

ARPE19:

Adult retinal pigment epithelial 19

BF:

Bright field

Ca2+ :

Calcium ion

CASP/3:

Caspase 3

CASP/8:

Caspase 8

CASP/9:

Caspase 9

CLSM:

Confocal laser scanning microscope

CRV:

Carvacrol

cytCa2+ :

Cytosolic free calcium ion

cytROS:

Cytosolic free reactive oxygen radicals

cytZn2+ :

Cytosolic free Zn2+

DM:

Diabetes mellitus

DRG:

Dorsal root ganglion

ROS:

Free reactive oxygen radicals

GPx:

Glutathione peroxidase

HGu:

High glucose

LPO:

Lipid peroxidation

mitROS:

Mitochondrial free reactive oxygen radicals

mitMP:

Mitochondrial membrane potential

NGu:

Normal glucose

PARP1:

Poly [ADP-ribose] polymerase 1

SERCA:

Sarco(endo)plasmic reticulum Ca2+-ATPase

TRP:

Transient receptor potential

TRPM2 :

Transient receptor potential melastatin 2

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Acknowledgements

The authors would like to thank Dr. Xinhua Shu for his valuable contributions to preparation of ARPE19 cells.

Funding

The study was performed with a financial support of the company (BSN Health) (Project No: 2021–03. Project owner: Dr. Hatice Daldal).

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Authors and Affiliations

  1. Department of Ophthalmology, Faculty of Medicine, Usak University, TR-64100, Usak, Turkey

    Hatice Daldal

  2. Departments of Biophysics and Neuroscience, Neuroscience Research Center, School of Medicine, Suleyman Demirel University, TR-32260, Isparta, Turkey

    Mustafa Nazıroğlu

  3. Drug Discovery Unit, BSN Health, Analyses, Innov., Consult., Org., Agricul., Ltd., TR-32260, Isparta, Turkey

    Mustafa Nazıroğlu

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  1. Hatice Daldal
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Contributions

H Daldal: The design, data analyses, manuscript preparation, conception, and critical revision of present study. M. Nazıroğlu: The analyses and preparation of figures in the manuscript. The manuscript submission was approved by both authors.

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Correspondence to Mustafa Nazıroğlu.

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Daldal, H., Nazıroğlu, M. Carvacrol protects the ARPE19 retinal pigment epithelial cells against high glucose-induced oxidative stress, apoptosis, and inflammation by suppressing the TRPM2 channel signaling pathways. Graefes Arch Clin Exp Ophthalmol 260, 2567–2583 (2022). https://doi.org/10.1007/s00417-022-05731-5

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