ABSTRACT
PURPOSE: The main characteristic of proliferative vitreoretinopathy (PVR) is migration, adhesion, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE). Eupatilin is a naturally occurring flavone that has the potential to inhibit cell proliferation and EMT. However, its efficacy on the PVR model induced by transforming growth factor-2 (TGF-ß2) is unknown. In this study, the potential effect of eupatilin on proliferation and EMT in the treatment of RPE was investigated. METHODS: Serum starved human RPE cells (ARPE-19) were treated with 10 ng/ml TGF-ß2 alone or co-treated with 25 µM eupatilin for 48 h. Quantitative real-time PCR and Western blot analysis were used to assess targets at the mRNA and protein expression level, respectively. Apoptosis and cell cycle progression was assessed by image-based cytometry. The effect of treatment on cell migration was evaluated by wound healing assay. RESULTS: Eupatilin inhibited TGF-ß2-induced RPE cell proliferation via regulating the cell cycle and inducing apoptosis. TGF-ß2 upregulated mRNA expression of mesenchymal markers fibronectin and vimentin was significantly downregulated by the treatment, while the epithelial markers E-cadherin and occludin expression was upregulated. The therapy significantly suppressed TGF-ß2 encouraged cell migration through downregulating the expression of transcription factors Twist, Snail, and ZEB1 induced by TGF-ß2. Furthermore, eupatilin significantly inhibited the expression of MMP-1, -7, and -9, and suppressed NF-κB signalling. CONCLUSION: These results suggest that eupatilin could inhibit the proliferation and transformation into fibroblast-like cells of RPE cells; thus the agent may be a potential therapeutic value in treating PVR.
Subject(s)
Epithelial Cells/drug effects , Epithelial-Mesenchymal Transition/drug effects , Flavonoids/pharmacology , Retinal Pigment Epithelium/cytology , Antigens, CD/genetics , Cadherins/genetics , Cell Line , Cell Physiological Phenomena/drug effects , Epithelial Cells/metabolism , Fibronectins/genetics , Humans , Matrix Metalloproteinases/genetics , Nuclear Proteins/genetics , Occludin/genetics , Snail Family Transcription Factors/genetics , Transforming Growth Factor beta2 , Twist-Related Protein 1/genetics , Vimentin/genetics , Vimentin/metabolism , Vitreoretinopathy, Proliferative/drug therapy , Vitreoretinopathy, Proliferative/genetics , Vitreoretinopathy, Proliferative/metabolism , Zinc Finger E-box-Binding Homeobox 1/geneticsABSTRACT
PURPOSE: Age-related macular degeneration (AMD) is the most common cause of visual loss. The dry AMD is characterized by retinal pigment epithelium (RPE) death and changes in AMD lead to severe loss of vision. Coumarin-derived esculetin has a number of therapeutic and pharmacological effects such as anti-inflammatory and antioxidant with various mechanisms. The purpose of this study was to investigate the effects of esculetin treatment on lipopolysaccharide (LPS)-induced inflammation, oxidative stress, and cell survival. MATERIAL AND METHODS: Human RPE cells (ARPE-19) were incubated for 24-72 h with 5 µg/ml LPS to induce inflammation and oxidative stress. Esculetin (5 µM) was used to protect the cells from LPS-induced damage. The cell viability was evaluated by quantitative 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Interleukin 6 (IL-6), IL-12, and vascular endothelial growth factor (VEGF) levels were determined by enzyme-linked immunosorbent assay (ELISA). IL-1ß, tumor necrosis factor receptor (TNFR), TNF-related apoptosis-inducing ligand (TRAIL), catalase, glutathione peroxidase (GPx), superoxide dismutase 1 (CuZnSOD) and SOD2 (MnSOD) mRNA expressions were analyzed by RT-quantitative polymerase chain reaction. Apoptosis was monitored by cell-based cytometer. NF-kappa B (NF-κB) p65/RelA levels were determined by ELISA, and NF-κB protein expression and extracellular signal-regulated kinase (ERK1/2) phosphorylation were evaluated by Western blot analysis. RESULTS: Esculetin treatment significantly suppressed LPS-induced cell death mediated by apoptosis and necrosis in a concentration-dependent manner. While LPS caused significant inflammation with cytokine increase in cells, esculetin reduced the expression of LPS-induced cytokines, VEGF, TNFR, and TRAIL. Furthermore, exposure to LPS increased the expression of GPx and mitochondrial MnSOD, leading to oxidative stress in the cells. Esculetin treatment attenuated phosphorylation of ERK1/2 and NF-κB expression mediated by LPS. CONCLUSIONS: These results suggest that esculetin may be an alternative treatment option for endotoxin-induced inflammation and oxidative stress, which therefore may inhibit the development of LPS-mediated AMD.
