Department of Ophthalmology, Severance Hospital, Institute of Vision Research, Yonsei University College of Medicine, Seoul, Korea.
Department of Ophthalmology, Yongin Severance Hospital, Yonsei University College of Medicine, Yongin, Gyeonggi-do, Republic of Korea.
Invest Ophthalmol Vis Sci. 2021 Dec 1;62(15):25. doi: 10.1167/iovs.62.15.25.
This study investigated the effect of glycogen synthase kinase-3β (GSK-3β) inhibition on the fibrosis of human Tenon's fibroblasts (HTFs) induced by transforming growth factor-β (TGF-β).
Quantitative real-time PCR and Western blot analyses were performed to determine the expression levels of molecules associated with the fibrosis of HTFs by TGF-β (fibronectin, collagen Iα, and α-smooth muscle actin) and GSK-3β. The levels of phosphorylated Smad2 and Smad3 were also analyzed in the presence of the GSK-3β inhibitor CHIR 99021. The wound healing assay was performed to determine the effect of CHIR 99021 on the migration of HTFs. All experiments were conducted using primary cultured HTFs or human tenon tissues obtained from normal subjects and patients with glaucoma.
Treatment with TGF-β resulted in an increase in the levels of molecules associated with the fibrosis of HTFs. The expression levels of these molecules were higher in the tenon tissues obtained from patients with glaucoma than those from normal subjects. When the HTFs were treated with TGF-β, a significant increase in the active form of GSK-3β (Y216) was observed. A significant decrease in the active form of GSK-3β and molecules associated with fibrosis by TGF-β was noted in HTFs treated with CHIR 99021. CHIR 99021 treatment reduced the phosphorylated Smad2/Smad2 and phosphorylated Smad3/Smad3 ratios in HTFs and attenuated HTF migration.
Our results demonstrated the effect of GSK-3β inhibition on the regulation of TGF-β-mediated fibrosis of HTFs, suggesting GSK-3β to be a potential target for maintaining bleb function after glaucoma filtration surgery.
本研究旨在探讨糖原合成酶激酶-3β（GSK-3β）抑制对转化生长因子-β（TGF-β）诱导的人Tenon 氏成纤维细胞（HTFs）纤维化的影响。
通过 TGF-β（纤连蛋白、胶原 Iα 和 α-平滑肌肌动蛋白）和 GSK-3β，采用定量实时 PCR 和 Western blot 分析来确定与 HTFs 纤维化相关分子的表达水平。还分析了存在 GSK-3β 抑制剂 CHIR 99021 时磷酸化 Smad2 和 Smad3 的水平。进行划痕愈合实验以确定 CHIR 99021 对 HTFs 迁移的影响。所有实验均使用原代培养的 HTFs 或来自正常受试者和青光眼患者的人Tenon 组织进行。
TGF-β 处理导致与 HTFs 纤维化相关分子的水平增加。来自青光眼患者的 Tenon 组织中的这些分子表达水平高于来自正常受试者的组织。当 HTFs 用 TGF-β 处理时，观察到 GSK-3β 的活性形式（Y216）显著增加。用 CHIR 99021 处理的 HTFs 中，GSK-3β 和 TGF-β 相关纤维化分子的活性形式显著减少。CHIR 99021 处理降低了 HTFs 中磷酸化 Smad2/Smad2 和磷酸化 Smad3/Smad3 的比值，并减弱了 HTF 的迁移。
我们的结果表明 GSK-3β 抑制对 TGF-β 介导的 HTFs 纤维化的调节作用，提示 GSK-3β 可能成为青光眼滤过手术后维持 bleb 功能的潜在靶点。
