• The Korean Journal of Physiology and Pharmacology
• /
• 제27권5호
• /
• pp.449-456
• /
• 2023

N-methyl-D-aspartate (NMDA) receptors are ionic glutamine receptors involved in brain development and functions such as learning and memory formation. NMDA receptor inhibition is associated with autophagy activation. In this study, we investigated whether the NMDA receptor antagonists, memantine and ifenprodil, induce autophagy in human retinal pigment epithelial cells (ARPE-19) to remove N-retinylidene-N-retinylethanolamine (A2E), an intracellular lipofuscin component. Fluorometric analysis using labeled A2E (A2E-BDP) and confocal microscopic examination revealed that low concentrations of NMDA receptor antagonists, which did not induce cytotoxicity, significantly reduced A2E accumulation in ARPE-19 cells. In addition, memantine and ifenprodil activated autophagy in ARPE-19 cells as measured by microtubule-associated protein 1A/1B-light chain3-II formation and phosphorylated p62 protein levels. Further, to understand the correlation between memantine- and ifenprodil-mediated A2E degradation and autophagy, autophagy-related 5 (ATG5) was depleted using RNA interference. Memantine and ifenprodil failed to degrade A2E in ARPE-19 cells lacking ATG5. Taken together, our study indicates that the NMDA receptor antagonists, memantine and ifenprodil, can remove A2E accumulated in cells via autophagy activation in ARPE-19 cells.

• Molecules and Cells
• /
• 제43권7호
• /
• pp.632-644
• /
• 2020

The molecular mechanism underlying autophagy impairment in the retinal pigment epithelium (RPE) in dry age-related macular degeneration (AMD) is not yet clear. Based on the causative role of poly(ADP-ribose) polymerase 1 (PARP1) in RPE necrosis, this study examined whether PARP1 is involved in the autophagy impairment observed during dry AMD pathogenesis. We found that autophagy was downregulated following H₂O₂-induced PARP1 activation in ARPE-19 cells and olaparib, PARP1 inhibitor, preserved the autophagy process upon H₂O₂ exposure in ARPE-19 cells. These findings imply that PARP1 participates in the autophagy impairment upon oxidative stress in ARPE-19 cells. Furthermore, PARP1 inhibited autolysosome formation but did not affect autophagosome formation in H₂O₂-exposed ARPE-19 cells, demonstrating that PARP1 is responsible for impairment of late-stage autophagy in particular. Because PARP1 consumes NAD⁺ while exerting its catalytic activity, we investigated whether PARP1 impedes autophagy mediated by sirtuin1 (SIRT1), which uses NAD⁺ as its cofactor. A NAD⁺ precursor restored autophagy and protected mitochondria in ARPE-19 cells by preserving SIRT1 activity upon H₂O₂. Moreover, olaparib failed to restore autophagy in SIRT1-depleted ARPE-19 cells, indicating that PARP1 inhibits autophagy through SIRT1 inhibition. Next, we further examined whether PARP1-induced autophagy impairment occurs in the retinas of dry AMD model mice. Histological analyses revealed that olaparib treatment protected mouse retinas against sodium iodate (SI) insult, but not in retinas cotreated with SI and wortmannin, an autophagy inhibitor. Collectively, our data demonstrate that PARP1-dependent inhibition of SIRT1 activity impedes autophagic survival of RPE cells, leading to retinal degeneration during dry AMD pathogenesis.

• Journal of Ginseng Research
• /
• 제47권2호
• /
• pp.311-318
• /
• 2023

Background: The beneficial effects of compound K (CK) on different chronic diseases have been shown to be at least related to antioxidant action. Nevertheless, since its antioxidant activity in human retinal pigment epithelial (RPE) cells is still unknown, here we investigated whether CK alleviates oxidative stress-stimulated damage in RPE ARPE-19 cells. Methods: The cytoprotective consequence of CK in hydrogen peroxide (H₂O₂)-treated cells was evaluated by cell viability, DNA damage, and apoptosis assays. Fluorescence analysis and immunoblotting were performed to investigate the inhibitory action of CK on reactive oxygen species (ROS) production and mitochondrial dysfunction. Results: H₂O₂-promoted cytotoxicity, oxidative stress, DNA damage, mitochondrial impairment, and apoptosis were significantly attenuated by CK in ARPE-19 cells. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation level and its shuttling to the nucleus were increased, which was correlated with upregulated activation of heme oxygenase-1 (HO-1). However, zinc protoporphyrin, a blocker of HO-1, significantly abrogated the preventive action of CK in H₂O₂-treated ARPE-19 cells. Conclusion: This study indicates that activation of Nrf2/HO-1 signaling by CK plays an important role in rescuing ARPE-19 cells from oxidative cellular damage.

• BMB Reports
• /
• 제44권6호
• /
• pp.405-409
• /
• 2011

The present study demonstrated that valproic acid (VPA) transcriptionally regulates human GM3 synthase (hST3Gal V), which catalyzes ganglioside GM3 biosynthesis in ARPE-19 human retinal pigment epithelial cells. For this, we characterized the promoter region of the hST3Gal V gene. Functional analysis of the 5'-flanking region of the hST3Gal V gene revealed that the -177 to -83 region functions as the VPA-inducible promoter and that the CREB/ATF binding site at -143 is crucial for VPA-induced expression of hST3Gal V in ARPE-19 cells. In addition, the transcriptional activity of hST3Gal V induced by VPA in ARPE-19 cells was inhibited by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. In summary, our results identified the core promoter region in the hST3Gal V promoter and for the first time demonstrated that ATF2 binding to the CREB/ATF binding site at -143 is essential for transcriptional activation of hST3Gal V in VPA-induced ARPE-19 cells.

• Journal of Ginseng Research
• /
• 제47권1호
• /
• pp.65-73
• /
• 2023

Background: Age-related macular degeneration (AMD) is a significant visual disease that induces impaired vision and irreversible blindness in the elderly. However, the effects of ginseng berry extract (GBE) on the retina have not been studied. Therefore, this study aimed to investigate the protective effects of GBE on blue light (BL)-induced retinal damage and elucidate its underlying mechanisms in human retinal pigment epithelial cells (ARPE-19 cells) and Balb/c retina. Methods: To investigate the effects and underlying mechanisms of GBE on retinal damage in vitro, we performed cell viability assay, pre-and post-treatment of sample, reactive oxygen species (ROS) assay, quantitative real-time PCR (qRT-PCR), and western immunoblotting using A2E-laden ARPE-19 cells with BL exposure. In addition, Balb/c mice were irradiated with BL to induce retinal degeneration and orally administrated with GBE (50, 100, 200 mg/kg). Using the harvested retina, we performed histological analysis (thickness of retinal layers), qRT-PCR, and western immunoblotting to elucidate the effects and mechanisms of GBE against retinal damage in vivo. Results: GBE significantly inhibited BL-induced cell damage in ARPE-19 cells by activating the SIRT1/PGC-1α pathway, regulating NF-kB translocation, caspase 3 activation, PARP cleavage, expressions of apoptosis-related factors (BAX/BCL-2, LC3-II, and p62), and ROS production. Furthermore, GBE prevented BL-induced retinal degeneration by restoring the thickness of retinal layers and suppressed inflammation and apoptosis via regulation of NF-kB and SIRT1/PGC-1α pathway, cleavage of caspase 3 and PARP, and expressions of apoptosis-related factors in vivo. Conclusions: GBE could be a potential agent to prevent dry AMD and progression to wet AMD.

• 생명과학회지
• /
• 제17권9호통권89호
• /
• pp.1204-1210
• /
• 2007

Totopoisomerase II 저해제인 etoposide는 핵안에 DNA double strand breaks를 일으키므로써 세포의 DNA에 손상을 초래한다. 본 연구에서는 인간 망막색소상피세포인 ARPE-19 세포에서의 세포성장 및 아폽토시스에서 etoposide의 역할을 살펴보았다. Etoposide는 세포의 성장을 크게 감소시켰으며 TUNEL에서 아폽토시스를 나타내는 DNA fragmentation의 증가를 유도하였다. 게다가, etoposide는 산화적 손상에 대해 세포나 조직을 보호하는 역할을 하는 것으로 알려진 세포내 항산화효소인 heme oxygenase-1 (HO-1)의 발현을 크게 증가시켰다. Etoposide에 의한 HO-1 발현증가는 항산화물질 NAC에 의해 억제되었는데, 이는 etoposide에 의한 세포내 ROS의 증가가 HO-1 발현에 중요한 역할을 한다는 것을 의미한다. 또한 HO-1 발현을 억제하기 위하여 HO-1 siRNA 방법을 사용하였다. 흥미롭게도, HO-1 유전자의 knock-down은 etoposide에 의해 유도되는 DNA fragmentation의 정도를 증가시켰다. 이들 결과를 종합해볼 때, etoposide에 의해 자극되어진 ARPE-19 세포에서 발현증가된 HO-1은 etoposide에 의한 아폽토시스 유발과정에서 세포를 보호하는 항아폽토시스의 기능을 한다는 것을 시사한다.

• IMMUNE NETWORK
• /
• 제20권3호
• /
• pp.26.1-26.9
• /
• 2020

Cereblon (CRBN), a negative modulator of AMP-activated protein kinase (AMPK), is highly expressed in the retina. We confirmed the expression of CRBN in ARPE-19 human retinal cells by Western blotting. We also demonstrated that CRBN knock-down (KD) could effectively downregulate IL-6 and MCP-1 protein and gene expression in LPS-stimulated ARPE-19 cells. Additionally, CRBN KD increased the phosphorylation of AMPK/acetylcoenzyme A carboxylase (ACC) and the expression of heme oxygenase-1 (HO-1) in ARPE-19 cells. Furthermore, CRBN KD significantly reduced LPS-induced nuclear translocation of NF-κB p65 and activation of NF-κB promoter activity. However, these processes could be inactivated by compound C (inhibitor of AMPK) and zinc protoporphyrin-1 (ZnPP-1; inhibitor of HO-1). In conclusion, compound C and ZnPP-1 can rescue LPS-induced levels of proinflammatory cytokines (IL-6 and MCP-1) in CRBN KD ARPE-19 cells. Our data demonstrate that CRBN deficiency negatively regulates proinflammatory cytokines via the activation of AMPK/HO-1 in the retina.

• 생명과학회지
• /
• 제32권11호
• /
• pp.865-871
• /
• 2022

황반 변성은 현대 사회에서 발병율이 급격히 증가한 질환 중 하나이다. 황반 변성의 대표적인 원인은 노화로 인한 산화 스트레스로 널리 알려져 있고 최근 노령화로 인해 발병 연령은 40대부터 급격하게 증가하고 있다. 따라서 본 연구에서는 TMH를 이용해 산화적 스트레스에 대한 항산화 효능 및 세포 사멸 억제연구를 통해 눈 건강 개선 소재를 개발하였다. 먼저 TMH의 인간 망막색소상피세포 사멸 억제 효능 평가를 위해 MTS assay로 세포 생존율을 측정하였다. 망막색소상피세포에서 H₂O₂에 의한 산화적 스트레스로 인해 약 60%의 세포 생존율을 확인 할 수 있었고 TMH에 의해 농도 의존적으로 세포 사멸을 억제됨을 확인할 수 있었다. 또한 LDH release assay를 통해 세포막 보호 효능을 확인 하였으며 세포 내 ROS 측정을 통해 세포 내 활성산소종 조절을 통한 항산화 효능을 확인 할 수 있었다. 마지막으로 세포 생존과 사멸에 관여하는 세포 내 신호 단백질인 MAPKs의 인산화 조절, 항산화 효소인 HO-1 발현 조절, 세포 사멸 관여 단백질인 Bax/Bcl-2의 발현 그리고 cleaved caspase-3 단백질 분석을 통해 TMH의 산화 스트레스에 대한 항산화 그리고 세포 사멸 억제 경로를 확인 하였다. 위와 같은 결과 도출을 통해 TMH의 눈 건강 기능성 소재로서의 가능성을 확인하였다.

• 한국축산식품학회지
• /
• 제44권3호
• /
• pp.699-709
• /
• 2024

Oxya chinensis sinuosa (OC) is a well-known edible insect. Several researches on the health benefits of OC consumption have been performed to date; however, their effect on eye health remains largely unknown. This study aimed to assess the protective effects of OC extracts on the oxidative stress on the retinal pigment epithelium (RPE) cells. Oxidative damage has been identified as one of the key regulatory factors in agerelated macular degeneration. H₂O₂-induced reactive oxygen species (ROS) production, a well-known oxidative stress factor, can cause cell death in retinal pigment epithelia cells. In this study, we found that three OC extracts effectively prevented H₂O₂-induced ROS production and subsequent death of ARPE-19 cells in a dose-dependent manner. In addition, the OC extracts inhibited the phosphorylation of mitogen-activated protein kinases including p38, JNK, and ERK. The OC extracts restored IκBα degradation induced by H₂O₂, indicating that OC extracts suppressed the activation of nuclear factorκB. Furthermore, the three OC extracts were shown to have antioxidant effects by upregulating the intracellular expression of key antioxidant proteins such as SOD, NQO, and HO-1. Here we demonstrated the antioxidant and anti-apoptotic effects of the OC extracts on ARPE-19, indicating their potential role in improving eye health. These results suggest that three OC extracts plays a critical role in oxidative stress-induced cell death protects in ARPE-19 cells.

• Parasites, Hosts and Diseases
• /
• 제57권2호
• /
• pp.83-92
• /
• 2019

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and $H_2O_2$ levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.