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KMID : 0360220200610090999
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Journal of the Korean Ophthalmological Society
2020 Volume.61 No. 9 p.999 ~ p.1009
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Effect of Cyclosporine A-induced Senescence on Cultured Human Corneal Endothelial Cells
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Jang Yoon-Kyung
Chung Tae-Young
Shin Young-Joo
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Abstract
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Purpose: To investigate the in vitro effect of cyclosporine A (CsA)-induced senescence on human corneal endothelial cells (HCECs).
Methods: HCECs were cultured and incubated with 0-100 μM CsA. Senescence-associated β?galactosidase (SA-β-gal) staining was performed. Mitochondrial dehydrogenase activity was assessed using a WST-8 assay kit and mitochondrial membrane potential (ΔΨm) was measured using JC-1 dye. Intracellular and mitochondrial formation of reactive oxygen species (ROS) was measured with 2’,7’-dichlorodihydrofluorescein diacetate and MitoSOX probes. Intracellular and mitochondrial calcium levels were measured using Fluo-4 and Rhod-2, respectively. Protein expression was evaluated by Western blotting.
Results: CsA increased the percentage of SA-β-gal-positive cells (p = 0.003) and decreased mitochondrial dehydrogenase activity and ΔΨm in a dose-dependent manner (p = 0.029, p = 0.004). Intracellular and mitochondrial ROS levels increased during incubation with CsA (p = 0.005). CsA at 100 μM increased mitochondrial calcium levels (p = 0.001), whereas intracellular calcium levels decreased at 100 μM CsA (p = 0.029). CsA activated GSK3β and ERK1/2 and reduced ZO-1 expression.
Conclusions: CsA induces senescence in HCECs through oxidative stress and via mitochondria-, GSK3β-, and ERK1/2-dependent pathways. Thus, concentrations of CsA should be monitored.
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KEYWORD
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Cyclosporine A, Human corneal endothelial cells, Mitochondrial membrane potential, Mitochondrial oxidative stress, Senescence
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