芍药苷减轻 TGF-β2 介导的人眼小梁细胞细胞外基质重塑和氧化应激。
Paeoniflorin alleviates TGF-β2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.
机构信息
Department of Ophthalmology, The First People's Hospital of Jiangxia District, Wuhan, No.1 Wenhua Avenue, Zhifang Street, Jiangxia District, Wuhan, 430200, China.
出版信息
Int Ophthalmol. 2024 May 25;44(1):229. doi: 10.1007/s10792-024-02917-0.
BACKGROUND
The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-β2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment.
METHODS
Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-β2 (5 ng/mL). PAE (300 μM) was added to serum-starved confluent cultures of HTM cells for 2 h, followed by incubation with TGF-β2 for 22 h. SB-431542, a TGF-β receptor inhibitor (10 μM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-β2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells.
RESULTS
PAE attenuated TGF-β2-induced oxidative stress and suppressed TGF-β2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-β2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-β2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells.
CONCLUSION
PAE alleviates TGF-β2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.
背景
多功能促纤维化细胞因子转化生长因子-β2（TGF-β2）与原发性开角型青光眼的病理生理学有关。芍药苷（PAE）是一种具有多种药理作用的单萜糖苷，如抗氧化、抗纤维化和抗炎特性。研究表明，芍药苷可保护人角膜上皮细胞、视网膜色素上皮细胞和视网膜小胶质细胞免受损伤。在这里，研究了 PAE 在 TGF-β2 依赖性小梁网（TM）微环境细胞外基质（ECM）重塑中的生物学作用。
方法
在无血清培养基中培养原代或转化（GTM3）人 TM（HTM）细胞，并用 TGF-β2（5ng/mL）孵育。PAE（300μM）添加到血清饥饿的 HTM 细胞汇合培养物中 2 小时，然后用 TGF-β2 孵育 22 小时。SB-431542，一种 TGF-β 受体抑制剂（10μM），用作阳性对照。通过 CellROX 绿色染料评估细胞内 ROS 水平。Western blot 用于测量 TGF-β2/Smad2/3 信号相关分子的水平。通过 RT-qPCR 评估胶原 1α1、胶原 4α1 和结缔组织生长因子（CTGF）的表达。通过免疫荧光测定法测量 HTM 细胞中胶原 I/IV 的表达。鬼笔环肽染色测定法用于评估细胞中的 F-肌动蛋白应力纤维形成。
结果
PAE 减弱了 TGF-β2 诱导的氧化应激，并抑制了原代或转化 HTM 细胞中 TGF-β2 诱导的 Smad2/3 信号。此外，PAE 抑制了 TGF-β2 诱导的胶原 1α1、胶原 4α1 和 CTGF 表达的上调，并减少了 TGF-β2 介导的胶原 I/IV 表达和 F-肌动蛋白应力纤维形成。
结论
PAE 通过失活 Smad2/3 信号减轻了 TGF-β2 诱导的 HTM 细胞中 ECM 沉积和氧化应激。
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