A novel NADPH oxidase inhibitor targeting Nox4 in TGFβ-induced lens epithelial to mesenchymal transition
A novel NADPH oxidase inhibitor targeting Nox4 in TGFβ-induced lens epithelial to mesenchymal transition
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DOI：
10.1016/j.exer.2019.107692
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发表时间：
2019-08-01
影响因子：
3.4
通讯作者：
Lovicu, Frank J.
中科院分区：
文献类型：
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作者：
Das, Shannon;Wikstrom, Per;Lovicu, Frank J.
许多基于小分子的NADPH氧化酶活性抑制剂在很大程度上不足以证实广泛的说法，通常表现出缺乏nox -异构体特异性，有时仅作为活性氧（ROS）的清除剂。在本研究中，我们使用一种新开发的高选择性Nox4抑制剂GLX7013114来调节TGF β诱导的晶状体上皮向间充质转化（EMT）。用0.3 μ M GLX7013114预处理大鼠晶状体上皮外植体，再用200 pg/ml tgf - β 2诱导晶状体EMT。使用超氧化物荧光探针二氢乙二铵（DHE）在显微镜下观察ROS的产生。使用相衬显微镜记录EMT过程，并对分子EMT标记物进行免疫标记。qPCR观察emt相关基因的变化。TGF β诱导的ROS在培养8 h时表现明显，GLX7013114诱导的ROS强度显著降低，与未处理外植体的ROS水平相当。在抑制剂存在的情况下，通过相衬显微镜观察TGF β诱导的EMT 5天，外植体晶状体上皮细胞在第2天变成肌成纤维细胞，并在第5天发生进行性凋亡，形成裸晶状体囊。TGF β和GLX7013114处理的外植体细胞存活率有所提高；然而，这些差异并不显著。首次发现GLX7013114抑制Nox4可降低TGF β诱导的α -平滑肌肌动蛋白（α SMA）、胶原蛋白和纤维连接蛋白的基因表达。鉴于GLX7013114似乎可以阻断TGF β诱导的EMT的某些方面，包括ROS的产生，因此它可能是一种新的有用的nox4选择性抑制剂，值得进一步研究。
Many of the small molecule-based inhibitors of NADPH oxidase activity are largely inadequate to substantiate broad claims, often exhibiting a lack of Nox-isoform-specificity, and sometimes only acting as scavengers of reactive oxygen species (ROS). In the present study, we use a newly developed highly selective Nox4 inhibitor, GLX7013114, to modulate TGF beta-induced lens epithelial to mesenchymal transition (EMT). Rat lens epithelial explants were pre-treated with 0.3 mu M of GLX7013114, and then treated with 200 pg/ml of TGF-beta 2 to induce lens EMT. ROS production was visualized microscopically using the superoxide fluorogenic probe, dihydroethidium (DHE). The EMT process was documented using phase-contrast microscopy, and molecular EMT markers were immunolabeled. qPCR was also performed to observe changes in EMT-associated genes. TGF beta-induced ROS was evident at 8 h of culture and its intensity was found to be significantly reduced when GLX7013114 was applied, comparable to ROS levels measured in untreated explants. Using phase-contrast microscopy to follow TGF beta-induced EMT over 5 days in the presence of the inhibitor, lens epithelial cells in explants became myofibroblastic by day 2 and underwent progressive apoptosis to reveal a bare lens capsule by day 5. Explants treated with TGF beta and GLX7013114 had some increased cell survival; however, these differences were not significant. For the first time, Nox4 inhibition by GLX7013114 was shown to reduce the TGF beta-induced gene expression of alpha-smooth muscle actin (alpha SMA), collagen la and fibronectin. GLX7013114, given that it appears to block aspects of TGF beta-induced EMT, including ROS production, may be a new useful Nox4-selective inhibitor for further studies.