배양된 사람 각막내피세포에서 Cyclosporine A로 유도된 노화의 효과 = Effect of Cyclosporine A-induced Senescence on Cultured Human Corneal Endothelial Cells

다국어 초록 (Multilingual Abstract)

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.

국문 초록 (Abstract)

목적: 생체 외에서 배양된 사람 각막내피세포에서 cyclosporine A (CsA)로 인한 노화의 영향에 대해 알아보고자 하였다.
대상과 방법: 사람 각막내피세포를 배양하고 0-100 μM CsA와 incubation을 시행하였다. 노화 연관 베타 갈락토시다제(senescenceassociated β-Galactosidase, SA-β-gal) 염색을 시행하였다. WST-8 분석 키트를 사용하여 미토콘드리아 탈수소효소의 활성을 평가하고, JC-1 dye를 이용하여 미토콘드리아막 전위(ΔΨm)를 측정하였다. 세포 내와 미토콘드라아에서 활성 산소 형성을 측정하였다. 세포 내와 미토콘드리아의 칼슘 수치를 각각 Fluo-4와 Rhod-2를 통해 측정하였다. 단백질 발현은 웨스턴 블롯(western blot)에 의해 평가되었다.
결과: 농도 의존적으로 CsA는 SA-β-gal 양성 세포의 비율을 증가시켰고(p=0.003) 미토콘드리아 탈수소효소의 활성과 ΔΨm을 감소시켰다(p=0.029, p=0.004). 세포 내 및 미토콘드리아의 활성 산소는 CsA와 배양하는 동안 증가하였다(p=0.005). 100 μM의 CsA에서 미토콘드리아 칼슘 수치가 높아진 반면(p=0.001), 세포 내 칼슘 수치는 낮아졌다(p=0.029). CsA는 GSK3β와 ERK1/2를 활성화시키고 ZO-1 발현을 감소시켰다.
결론: 사람 각막내피세포에서 CsA는 산화 스트레스의 유도와 미토콘드리아, GSK3β 및 ERK1/2 경로를 통해 노화를 유도한다. 따라서 고농도의 CsA 사용시 주의하여 사용하여야 한다.

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