title: Madecassic Acid protects against hypoxia-induced oxidative stress in retinal microvascular endothelial cells via ROS-mediated endoplasmic reticulum stress.
abstract: Madecassic acid (MA) is an abundant triterpenoid in Centella asiatica (L.) Urban. (Apiaceae) that has been used as a wound-healing, anti-inflammatory and anti-cancer agent. Up to now, the effects of MA against oxidative stress remain unclear. In this study, we investigated the effect of MA and its mechanisms on hypoxia-induced human Retinal Microvascular Endothelial Cells (hRMECs). hRMECs were pre-treated with different concentrations of MA (0-50μM) for 30min before being incubated under hypoxia condition (37°C, 5% CO2and 95% N2). Cell apoptosis was evaluated with MTT assay and TUNEL staining, and the expression of apoptosis- and endoplasmic reticulum (ER) stress-related molecules was assessed with western blotting and RT-PCR analysis. Intracellular ROS level was evaluated using DCFH-DA. Intracellular malondialdehyde (MDA), dehydrogenase (LDH), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) were evaluated using related Kits. Activating transcription factor 4 (ATF4) nuclear translocation was assessed with western blotting analysis and immunofluorescence staining. MA significantly reduced oxidative stress in hypoxia-induced hRMECs, as shown by increased cell viability, SOD and GSH-PX leakage, decreased TUNEL- and ROS-positive cell ratio, LDH and MDA leakage, caspase-3 and -9 activity, and Bax/Bcl-2 ratio. In addition, MA also attenuated hypoxia-induced ER stress in hRMECs, as shown by reduced mRNA levels of glucose-regulated protein 78 (GRP78), C/EBP homologous transcription factor (CHOP), protein levels of cleaved activating transcription factor 6 (ATF6) and inositol-requiring kinase/endonuclease 1 alpha (IRE1α), phosphorylation of pancreatic ER stress kinase (PERK) and eukaryotic initiation factor 2 alpha (eIF2α), cleaved caspase-12 and ATF4 translocation to nucleus. The current study indicated that the regulation of oxidative stress and ER stress by MA would be a promising therapy to reverse the process and development of hypoxia-induced hRMECs dysfunction.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510000, Guangdong Province, People's Republic of China.
authors.forename: Boyu
authors.initials: B
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510000, Guangdong Province, People's Republic of China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510000, Guangdong Province, People's Republic of China.
authors.forename: Yaguang
authors.initials: Y
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510000, Guangdong Province, People's Republic of China.
authors.forename: Yiwen
authors.initials: Y
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510000, Guangdong Province, People's Republic of China.
authors.forename: Xi
authors.initials: X
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510000, Guangdong Province, People's Republic of China.
authors.forename: Ching Kit
authors.initials: CK
authors.lastname: Tsui
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510000, Guangdong Province, People's Republic of China. Electronic address: lulin888@126.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510000, Guangdong Province, People's Republic of China. Electronic address: liangxlsums@qq.com.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Liang
mesh_terms: D051701:Activating Transcription Factor 4 / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008315:Malondialdehyde / Q000378:metabolism; D055806:Microvessels / Q000473:pathology*; D018384:Oxidative Stress / Q000187:drug effects*; D020011:Protective Agents / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012160:Retina / Q000473:pathology; D013482:Superoxide Dismutase / Q000378:metabolism; D014315:Triterpenes / Q000737:chemistry / Q000494:pharmacology*; D056811:Unfolded Protein Response / Q000187:drug effects
chemical_list: C497109:ATF4 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D020011:Protective Agents; D017382:Reactive Oxygen Species; D014315:Triterpenes; D051701:Activating Transcription Factor 4; D008315:Malondialdehyde; D007770:L-Lactate Dehydrogenase; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; C001669:madecassic acid
keywords: ATF4; Apoptosis; Endoplasmic reticulum stress; Human retinal microvascular endothelial cells; Madecassic acid; Oxidative stress; ROS