Jiang Yingle, Wen Xuewei, Jian Xiaoyu, Chen Qianbo, Li Yan
Department of Ophthalmology, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China.
Int J Mol Med. 2025 Mar;55(3). doi: 10.3892/ijmm.2025.5486. Epub 2025 Jan 10.
Retinal pigment epithelial (RPE) cells undergoing epithelial‑mesenchymal transition (EMT) are a key factor in promoting the progression of subretinal fibrosis. The klotho protein and gene exert anti‑fibrotic effects in multiple fibrotic diseases. However, the mechanisms involved in the role of klotho are unclear in subretinal fibrosis. The aim of the present study was to explore the effects of klotho on subretinal fibrosis induced by laser photocoagulation in mice and EMT induced by TGF‑β1 in RPE cells and the underlying molecular mechanisms. , klotho overexpression or knockdown was performed in ARPE‑19 cells (adult retinal Pigment Epithelial‑19), then TGF‑β1 treatment was applied. Using western blotting, expression of epithelial markers (zonula occludens‑1), mesenchymal signs (α‑smooth muscle actin, α‑SMA, N‑cadherin, N‑cad and collagen I), and the ERK1/2 and Wnt/β‑catenin signaling pathways were assessed. The proliferative ability of ARPE‑19 cells was examined by CCK‑8 and EdU test, and the migratory ability was examined by wound healing and Transwell assays. Furthermore, to explore the underlying molecular pathway of klotho overexpression, RNA‑sequencing (seq) was performed. , photocoagulation was used to induce subretinal fibrosis in mice, which occurs as a result of choroidal neovascularization (CNV), then recombinant mouse klotho protein was administered intravitreally. Upregulation of epithelial and downregulation of mesenchymal markers demonstrated that klotho overexpression prevented TGF‑β1‑induced EMT; klotho knockdown resulted in the opposite effects. Additionally, klotho overexpression suppressed cell proliferation and migration and attenuated ERK1/2 and Wnt/β‑catenin signaling activated by TGF‑β1. RNA‑seq results demonstrated that several signaling pathways, including cellular senescence and the TNF signaling pathway, were associated with anti‑fibrotic effects of klotho overexpression. , subretinal fibrotic areas were attenuated following klotho treatment in laser‑induced CNV lesions, as illustrated by immunofluorescence and Masson staining of the mouse eyes. Western blotting results that the protein levels of mesenchymal markers were significantly downregulated and those of epithelial markers were upregulated. In summary, the present study suggested that klotho may have therapeutic value in management of fibrotic vitreoretinal disorders such as subretinal fibrosis.
经历上皮-间质转化（EMT）的视网膜色素上皮（RPE）细胞是促进视网膜下纤维化进展的关键因素。klotho蛋白和基因在多种纤维化疾病中发挥抗纤维化作用。然而，在视网膜下纤维化中，klotho发挥作用的机制尚不清楚。本研究的目的是探讨klotho对小鼠激光光凝诱导的视网膜下纤维化以及对RPE细胞中TGF-β1诱导的EMT的影响及其潜在分子机制。在ARPE-19细胞（成人视网膜色素上皮-19）中进行klotho过表达或敲低，然后施加TGF-β1处理。使用蛋白质印迹法评估上皮标志物（紧密连接蛋白-1）、间质标志物（α-平滑肌肌动蛋白、α-SMA、N-钙黏蛋白、N-cad和I型胶原）的表达以及ERK1/2和Wnt/β-连环蛋白信号通路。通过CCK-8和EdU试验检测ARPE-19细胞的增殖能力，通过伤口愈合和Transwell试验检测其迁移能力。此外，为了探索klotho过表达的潜在分子途径，进行了RNA测序（seq）。使用光凝诱导小鼠视网膜下纤维化，其由脉络膜新生血管（CNV）引起，然后玻璃体内注射重组小鼠klotho蛋白。上皮标志物上调和间质标志物下调表明klotho过表达可预防TGF-β1诱导的EMT；klotho敲低则产生相反的效果。此外，klotho过表达抑制细胞增殖和迁移，并减弱TGF-β1激活的ERK1/2和Wnt/β-连环蛋白信号。RNA测序结果表明，包括细胞衰老和TNF信号通路在内的几种信号通路与klotho过表达的抗纤维化作用相关。在激光诱导的CNV病变中，klotho处理后视网膜下纤维化区域减小，小鼠眼睛的免疫荧光和Masson染色表明了这一点。蛋白质印迹结果显示间质标志物的蛋白水平显著下调，上皮标志物的蛋白水平上调。总之，本研究表明klotho在治疗诸如视网膜下纤维化等纤维化玻璃体视网膜疾病中可能具有治疗价值。
