State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
Life Sci. 2021 Jan 15;265:118741. doi: 10.1016/j.lfs.2020.118741. Epub 2020 Nov 10.
Autophagy has been reported to play an essential role in fibrotic disorders. Known as fibrotic cataract, posterior capsular opacification (PCO) result from pathological epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). This study aims to identify the role and potential mechanism of autophagy in TGF-β2-induced EMT in LECs.
Primary rabbit LECs were treated with TGF-β2 to induce EMT as a model of fibrotic cataract in vitro. 3-methyladenine, chloroquine, bafilomycin A1, and gene silencing of autophagy-related protein 7 (ATG7) were treated in LECs for autophagy inhibition, while rapamycin was utilized for autophagy activation. The expression levels of EMT/autophagy-associated markers were analyzed by qRT-PCR, western blotting, immunofluorescence and transmission electron microscopy. We additionally examined cell migration ability with transwell migration assay and wound healing assay.
TGF-β2 promoted autophagy flux during EMT progression of LECs in a time-dependent manner. Autophagy activation by rapamycin enhanced TGF-β2-triggered fibrogenic responses and cell migration in LECs, whereas pharmacological inhibition of autophagy alleviated TGF-β2-induced increases of EMT markers and cell migration of LECs. In addition, the phosphorylation of Smad2/3 induced by TGF-β2 was suppressed through autophagy inhibition, while it was promoted upon autophagy activation, indicating that TGF-β2/Smad signaling was involved in the modulation of autophagy on EMT in LECs. Furthermore, ATG7-silenced LECs exerted anti-fibrosis effect induced by TGF-β2 through downregulation of autophagy.
Intervention/inhibition of autophagy could attenuate TGF-β2-induced EMT in LECs, which provides autophagy-related insights on preventing and treating the fibrotic cataract or other fibrotic diseases.
自噬在纤维性疾病中发挥着重要作用，这已得到报道。纤维性白内障又被称为后发性白内障，是由晶状体上皮细胞（LEC）的病理性上皮-间充质转化（EMT）引起的。本研究旨在确定自噬在 TGF-β2 诱导的 LECs 中 EMT 中的作用和潜在机制。
体外采用 TGF-β2 处理原代兔 LECs 以诱导 EMT，作为纤维性白内障模型。用 3-甲基腺嘌呤、氯喹、巴弗洛霉素 A1 和自噬相关蛋白 7（ATG7）的基因沉默处理 LECs 以抑制自噬，而用雷帕霉素激活自噬。通过 qRT-PCR、western blot、免疫荧光和透射电镜分析 EMT/自噬相关标志物的表达水平。我们还通过 Transwell 迁移实验和划痕愈合实验检测了细胞迁移能力。
TGF-β2 在 LECs 的 EMT 进展过程中呈时间依赖性促进自噬流。雷帕霉素激活自噬增强了 TGF-β2 触发的 LECs 纤维化反应和细胞迁移，而自噬抑制减轻了 TGF-β2 诱导的 EMT 标志物增加和 LECs 的细胞迁移。此外，TGF-β2 诱导的 Smad2/3 磷酸化通过自噬抑制得到抑制，而在自噬激活时则得到促进，表明 TGF-β2/Smad 信号参与了 LECs 中自噬对 EMT 的调节。此外，沉默 ATG7 的 LECs 通过下调自噬发挥 TGF-β2 诱导的抗纤维化作用。
自噬的干预/抑制可以减轻 TGF-β2 诱导的 LECs 中的 EMT，这为预防和治疗纤维性白内障或其他纤维性疾病提供了自噬相关的见解。
