Graduate Institute of Medical Sciences and Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, 250 Wu-Hsing St. Taipei 110, Taiwan.
School of Pharmacy, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing St. Taipei 110, Taiwan.
Phytomedicine. 2019 Mar 15;56:207-214. doi: 10.1016/j.phymed.2018.11.002. Epub 2018 Nov 7.
Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that has been found to induce matrix metalloproteinase-9 (MMP-9) activation and result in eventual retinal dysfunction. Proinflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-1β (IL-1β) were also found to be involved in disease progression by mediating MMP-9 production. We previously reported that fungal derivative theissenolactone C (LC53) could exert ocular protective effects by suppressing neuroinflammation in experimental uveitis.
The aim of this study was to investigate the retinoprotective effects of natural compound LC53 on the high IOP-induced ischemia/reperfusion (I/R)-injury model of glaucoma and its cellular mechanisms.
A high IOP-induced I/R-injury model was manipulated by normal saline injection into the anterior chamber of the rat eye. MCP-1-stimulated monocytes and IL-1β-activated primary astrocytes were used to investigate the cellular mechanisms of LC53. Retinal function was evaluated with the scotopic threshold response (STR) and combined rod-cone response by electroretinography (ERG). As a positive control, rats were treated with memantine. MMP-9 gelatinolysis, mRNA expression and protein expression were analyzed by gelatin zymography, RT-PCR, and Western Blot, respectively. The phosphorylation levels of MAPKs and NF-κB p65 were tested by Western Blot. Additionally, the levels of inflammatory MCP-1 and IL-1β were determined by ELISA.
The present study revealed that LC53 preserved the retina functional deficiency assessed by scotopic threshold response (STR) and combined rod-cone response of ERG after high IOP-induced I/R injury. These retinal protective effects of LC53 were positively correlated with inhibitory activities in I/R injury-elicited ocular MMP-9 activation and expression. The increased level of MCP-1 was not affected, and the enhanced IL-1β production was partially reduced by LC53 in the retina after I/R injury. According to cellular studies, LC53 significantly and concentration-dependently abrogated MMP-9 activation and expression in MCP-1-stimulated THP-1 monocytes. We found the inhibitory activities of LC53 were through the ERK- and NF-κB-dependent pathways. In addition, LC53 dramatically suppressed IL-1β-induced MMP-9 activation and expression in primary astrocytes. The phosphorylation of 65-kD protein (p65) of NF-κB was substantially blocked by LC53 in IL-1β-stimulated primary astrocytes.
LC53 exerted a retinal protective effect through NF-κB inhibition and was highly potent against MMP-9 activities after high IOP-induced I/R injury, suggesting that LC53 would be a promising drug lead for glaucoma or related medical conditions attributed to retinal ischemia.
眼内压（IOP）升高是青光眼的一个主要危险因素，已发现其可诱导基质金属蛋白酶-9（MMP-9）的激活，并导致最终的视网膜功能障碍。研究发现，促炎细胞因子如单核细胞趋化蛋白-1（MCP-1）和白细胞介素-1β（IL-1β）也通过介导 MMP-9 的产生而参与疾病的进展。我们之前的研究报告称，真菌衍生物噻嗪内酯 C（LC53）可通过抑制实验性葡萄膜炎中的神经炎症来发挥眼部保护作用。
本研究旨在探讨天然化合物 LC53 对高眼压诱导的缺血/再灌注（I/R）损伤型青光眼模型的视网膜保护作用及其细胞机制。
通过向大鼠眼前房注射生理盐水来操纵高眼压诱导的 I/R 损伤模型。用 MCP-1 刺激的单核细胞和 IL-1β 激活的原代星形胶质细胞来研究 LC53 的细胞机制。通过视网膜电图（ERG）检测暗适应阈值反应（STR）和混合棒锥反应来评估视网膜功能。作为阳性对照，用美金刚胺治疗大鼠。通过明胶酶谱、RT-PCR 和 Western Blot 分别分析 MMP-9 凝胶溶解、mRNA 表达和蛋白表达。通过 Western Blot 检测 MAPKs 和 NF-κB p65 的磷酸化水平。此外，通过 ELISA 测定炎症性 MCP-1 和 IL-1β 的水平。
本研究表明，LC53 可在高眼压诱导的 I/R 损伤后保留由 STR 和 ERG 检测到的视网膜功能缺陷。LC53 的这些视网膜保护作用与抑制 I/R 损伤诱导的眼部 MMP-9 激活和表达呈正相关。LC53 对 I/R 损伤后视网膜中 MCP-1 水平的增加没有影响，对 IL-1β 产生的增强部分有抑制作用。根据细胞研究，LC53 可显著且浓度依赖性地抑制 MCP-1 刺激的 THP-1 单核细胞中的 MMP-9 激活和表达。我们发现 LC53 的抑制活性是通过 ERK 和 NF-κB 依赖性途径实现的。此外，LC53 可显著抑制原代星形胶质细胞中 IL-1β 诱导的 MMP-9 激活和表达。LC53 可显著阻断 IL-1β 刺激的原代星形胶质细胞中 p65 的磷酸化。
LC53 通过抑制 NF-κB 发挥视网膜保护作用，并对高眼压诱导的 I/R 损伤后 MMP-9 的活性具有高度活性，提示 LC53 可能成为青光眼或与视网膜缺血相关的相关疾病的有前景的药物先导。
