Department of Clinical Chemistry and Biochemistry, Medical University of Lodz, 90-419 Lodz, Poland.
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA.
Int J Mol Sci. 2021 Apr 26;22(9):4494. doi: 10.3390/ijms22094494.
Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG.
原发性开角型青光眼 (POAG) 是最常见的青光眼类型。新出现的证据表明，内质网 (ER) 应激和蛋白激酶 RNA 样内质网激酶 (PERK) 介导的未折叠蛋白反应 (UPR) 信号通路在 POAG 发病机制中起关键作用。因此，本研究的主要目的是评估 PERK 抑制剂 LDN-0060609 在原发性人眼小梁细胞 (HTM) 细胞青光眼细胞模型中的疗效。为了评估 ER 应激标志物蛋白的水平，使用 Western blot 和 TaqMan 基因表达分析进行了检测。通过 XTT、LDH 测定和吉姆萨染色测定细胞毒性，通过彗星试验测定遗传毒性。通过相差显微镜评估细胞形态变化。通过 caspase-3 测定和流式细胞术 (FC) 分析细胞凋亡，通过 FC 分析细胞周期进程。结果表明，LDN-0060609 在诱导 ER 应激条件下的 HTM 细胞中触发 ER 应激标志物蛋白的显著下降。此外，LDN-0060609 有效地增加了 HTM 细胞的活力，减少了 DNA 损伤，增加了增殖，恢复了正常形态，减少了凋亡，并恢复了诱导 ER 应激条件下的 HTM 细胞的正常细胞周期分布。因此，PERK 抑制剂，如 LDN-0060609，可能为 POAG 提供一种创新的、突破性的治疗策略。
