Luteolin inhibits subretinal fibrosis and epithelial-mesenchymal transition in laser-induced mouse model via suppression of Smad2/3 and YAP signaling,Phytomedicine

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Luteolin inhibits subretinal fibrosis and epithelial-mesenchymal transition in laser-induced mouse model via suppression of Smad2/3 and YAP signaling
Phytomedicine ( IF 6.7 ) Pub Date : 2023-05-09 , DOI: 10.1016/j.phymed.2023.154865
Chaoyang Zhang ₁ , Yao Zhang ₂ , Xin Hu ₃ , Zhenzhen Zhao ₄ , Ziang Chen ₂ , Xi Wang ₄ , Zhihua Zhang ₁ , Haiying Jin ₂ , Jingfa Zhang ₁

Affiliation

Background

Subretinal fibrosis (SF) accounts for vision loss in patients with neovascular age-related macular degeneration (nAMD) even treated with adequate intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) drugs. Currently, there is no treatment available to prevent or treat SF caused by nAMD.

Purpose

This study aims to investigate the potential effects of luteolin on SF and epithelial-mesenchymal transition (EMT) as well as the underlying molecular pathways both in vivo and in vitro.

Methods

Seven-week-old male C57BL/6J mice were employed to establish laser-induced choroidal neovascularization (CNV) and SF. One day after the laser induction, luteolin was administered intravitreally. SF and CNV were assessed with the immunolabeling of collagen type I (collagen I) and isolectin B4 (IB4), respectively. RPE65 and α-SMA colocalization in the lesions was used to evaluate the extent of EMT in retinal pigment epithelial (RPE) cells by using immunofluorescence. In vitro, luteolin was administered to TGFβ1-treated primary human RPE (phRPE) cells. RT-qPCR, Western blot and immunofluorescence were employed to evaluate the change of EMT-related molecules, epithelial markers, and relevant signaling pathways. The functional changes associated with EMT were investigated using the scratch assay, Transwell migration assay, and collagen gel contraction assay. CCK-8 was used to determine the cell viability of phRPE cells.

Results

On day 7 and 14 after laser induction in mice, intravitreal injection of luteolin dramatically decreased the immunolabeled sizes of both collagen I and IB4, as well as the amount of colocalized double immunostaining of α-SMA and RPE65 in laser-induced SF lesions. In vitro, TGFβ1-treated phRPE cells demonstrated increased cell migration and contraction capacity, accompanied with considerable overexpression of fibronectin, α-SMA, N-cadherin and vimentin, as well as downregulation of E-cadherin and ZO-1. The above changes were largely inhibited by luteolin co-incubation. Mechanistically, luteolin could evidently decrease the phosphorylation of Smad2/3, whereas increase the phosphorylation of YAP in TGFβ1-treated phRPE cells.

Conclusion

This study demonstrates that luteolin exhibits the anti-fibrotic effect in a laser-induced mouse model by inhibiting EMT of RPE cells via deactivating Smad2/3 and YAP signaling, which provides a potential natural compound for the prevention and treatment of SF and fibrosis-related diseases.

中文翻译：

木犀草素通过抑制 Smad2/3 和 YAP 信号抑制激光诱导小鼠模型中的视网膜下纤维化和上皮-间质转化

背景

视网膜下纤维化 (SF) 是新生血管性年龄相关性黄斑变性 (nAMD) 患者视力丧失的原因，即使在玻璃体内注射足够的抗血管内皮生长因子 (抗 VEGF) 药物后也是如此。目前，没有可用于预防或治疗由 nAMD 引起的 SF 的治疗方法。

目的

本研究旨在研究木犀草素对 SF 和上皮-间质转化 (EMT) 的潜在影响以及体内和体外的潜在分子通路。

方法

七周大的雄性 C57BL/6J 小鼠被用来建立激光诱导的脉络膜新生血管 (CNV) 和 SF。激光诱导一天后，木犀草素被玻璃体内给药。SF 和 CNV 分别用 I 型胶原蛋白（胶原蛋白 I）和异凝集素 B4（IB4）的免疫标记进行评估。RPE65 和 α-SMA 在病变中的共定位用于通过免疫荧光评估视网膜色素上皮 (RPE) 细胞中 EMT 的程度。体外，将木犀草素施用于 TGFβ1 处理的原代人 RPE (phRPE) 细胞。采用 RT-qPCR、Western blot 和免疫荧光评估 EMT 相关分子、上皮标志物和相关信号通路的变化。使用划痕试验、Transwell 迁移试验和胶原凝胶收缩试验研究了与 EMT 相关的功能变化。CCK-8 用于确定 phRPE 细胞的细胞活力。

结果

在小鼠激光诱导后第 7 天和第 14 天，玻璃体内注射木犀草素显着降低了 I 型胶原蛋白和 IB4 的免疫标记大小，以及激光诱导的 SF 损伤中 α-SMA 和 RPE65 的共定位双重免疫染色量。在体外，TGFβ1 处理的 phRPE 细胞表现出增加的细胞迁移和收缩能力，伴随着纤连蛋白、α-SMA、N-钙粘蛋白和波形蛋白的大量过度表达，以及 E-钙粘蛋白和 ZO-1 的下调。木犀草素共孵育很大程度上抑制了上述变化。从机制上讲，木犀草素可以明显降低 Smad2/3 的磷酸化，同时增加 TGFβ1 处理的 phRPE 细胞中 YAP 的磷酸化。