Life sciences · 2025
In vitro anti-neovascularization and anti-angiogenic activities of Platycladus orientalis (L.) Franco in human retinal endothelial cells via Raf/MEK 1/2 and AKT1/mTOR signaling.
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Lim Jing Xian, Yong Phaik Har, Lim Siew Huah, Ng Zhi Xiang
Аннотация
AIMS: This study investigated the anti-neovascularization and anti-angiogenic potential of Platycladus orientalis ethyl acetate fraction (POEA) in human retinal endothelial cells (HRECs) under varying glucose conditions.
METHODS: The phytochemicals in POEA were identified with liquid chromatography-mass spectrometry while POEA in vitro cytotoxicity was evaluated. Both gene and protein expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), serine/threonine kinase 1 (AKT1), mammalian target of rapamycin (mTOR), rapidly accelerated fibrosarcoma-1 (Raf-1), mitogen-activated extracellular signal-regulated kinase 1 and 2 (MEK1/2) in POEA-treated group were compared to the control. Cell migration and tube formation assays were conducted using VEGF and the MEK1/2 inhibitor, U0126 as reference controls.
KEY FINDINGS: Phytochemical analysis revealed monoterpene esters, diterpenoids, and phenolic glycosides in POEA. POEA with an IC₅₀ of 0.413 mg/ml preserved the HRECs viability above 80 % under high glucose stress. POEA treatment significantly attenuated high glucose-induced upregulation of VEGF, VEGFR2, mTOR, Raf-1, and MEK1/2 at both gene and protein levels while enhancing AKT1 activation, suggesting restoration of cell survival pathways. Functional assays demonstrated dose-dependent inhibition of glucose- and VEGF-induced HRECs migration and tube formation by POEA, comparable to the MEK1/2 inhibitor U0126. Mechanistically, POEA suppressed phosphorylation of VEGFR2, Raf-1, and MEK1/2, and downregulated mTOR signaling, thereby disrupting angiogenic signaling cascades critical for endothelial proliferation and migration. Combined POEA and U0126 treatment showed additive inhibitory effects, supporting a synergistic mechanism through dual blockade of VEGF-driven pathways.
SIGNIFICANCE: POEA could mitigate diabetic retinopathy-related neovascularization and angiogenesis through modulation of VEGF/VEGFR2, AKT1/mTOR, and Raf-1/MEK1/2 signaling in HRECs.
METHODS: The phytochemicals in POEA were identified with liquid chromatography-mass spectrometry while POEA in vitro cytotoxicity was evaluated. Both gene and protein expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), serine/threonine kinase 1 (AKT1), mammalian target of rapamycin (mTOR), rapidly accelerated fibrosarcoma-1 (Raf-1), mitogen-activated extracellular signal-regulated kinase 1 and 2 (MEK1/2) in POEA-treated group were compared to the control. Cell migration and tube formation assays were conducted using VEGF and the MEK1/2 inhibitor, U0126 as reference controls.
KEY FINDINGS: Phytochemical analysis revealed monoterpene esters, diterpenoids, and phenolic glycosides in POEA. POEA with an IC₅₀ of 0.413 mg/ml preserved the HRECs viability above 80 % under high glucose stress. POEA treatment significantly attenuated high glucose-induced upregulation of VEGF, VEGFR2, mTOR, Raf-1, and MEK1/2 at both gene and protein levels while enhancing AKT1 activation, suggesting restoration of cell survival pathways. Functional assays demonstrated dose-dependent inhibition of glucose- and VEGF-induced HRECs migration and tube formation by POEA, comparable to the MEK1/2 inhibitor U0126. Mechanistically, POEA suppressed phosphorylation of VEGFR2, Raf-1, and MEK1/2, and downregulated mTOR signaling, thereby disrupting angiogenic signaling cascades critical for endothelial proliferation and migration. Combined POEA and U0126 treatment showed additive inhibitory effects, supporting a synergistic mechanism through dual blockade of VEGF-driven pathways.
SIGNIFICANCE: POEA could mitigate diabetic retinopathy-related neovascularization and angiogenesis through modulation of VEGF/VEGFR2, AKT1/mTOR, and Raf-1/MEK1/2 signaling in HRECs.
Предметные рубрики MeSH
HumansTOR Serine-Threonine KinasesEndothelial CellsProto-Oncogene Proteins c-aktPlant ExtractsAngiogenesis InhibitorsCell MovementSignal TransductionRetinaMAP Kinase Kinase 1Vascular Endothelial Growth Factor Receptor-2Proto-Oncogene Proteins c-rafMAP Kinase Kinase 2Cells, CulturedMAP Kinase Signaling System