title: Tert-butyl hydroperoxide (t-BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and mitochondrion.
abstract: Oxidative stress causes endothelial death while underlying mechanisms remain elusive. Herein, the pro-death effect of tert-butyl hydroperoxide (t-BHP) was investigated with low concentration (50μM) of t-BHP (t-BHPL) and high concentration (500μM) of t-BHP (t-BHPH). Both t-BHPLand t-BHPHinduced endothelial cell death was determined. T-BHPLinduced caspase-dependent apoptosis and reactive oxygen species (ROS) generation, which was inhibited by N-acetyl-L-cysteine (NAC). Furthermore, NADPH oxidase inhibitor diphenyleneiodonium (DPI), NOX4 siRNA, and NOX4 inhibitor GKT137831 reduced t-BHPL-induced ROS generation while mitochondrial respiratory chain inhibitors rotenone (Rot), 2-thenoyltrifluoroacetone (TTFA), and antimycin A (AA) failed to do so. NOX4 overexpression resulted in increased ROS generation and Akt expression but decreased sensitivity to t-BHPL. In contrast, T-BHPHinduced LDH release, PI uptake, and cell translucent cytoplasm. RIP1 inhibitor necrostatin-1 (Nec-1), MLKL inhibitor necrosulfonamide (NSA) and silencing RIP1, RIP3, and MLKL inhibited t-BHPH-induced cell death while pan-caspase inhibitor Z-VAD-FMK showed no effect. T-BHPH-induced ROS production was inhibited by TTFA, AA and Rot while DPI showed no effect. T-BHPHinduced RIP1/RIP3 interaction, which was decreased by Rot, TTFA, and AA. Silence RIP1 and RIP3 but not MLKL inhibited t-BHPH-induced mitochondrial membrane potential (MMP) decrease and ROS production. Moreover, P38MAPK inhibitor SB203580 reversed both t-BHPLand t-BHPH-induced cell death while inhibitors for ERKs and JNKs showed no obvious effect. These data suggested that t-BHP induced both apoptosis and necroptosis in endothelial cells which was mediated by ROS and p38MAPK. ROS derived from NADPH oxidase and mitochondria contributed to t-BHPLand t-BHPH-induced apoptosis and necroptosis, respectively.
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Wenwen
authors.initials: W
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Haitao
authors.initials: H
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Sun
authors.affiliation: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, PR China.
authors.forename: Kang
authors.initials: K
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
authors.forename: Jin-Jian
authors.initials: JJ
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China. Electronic address: xpchen@umac.mo.
authors.forename: Xiuping
authors.initials: X
authors.lastname: Chen
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001343:Autophagy / Q000187:drug effects / Q000235:genetics*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D000074663:NADPH Oxidase 4 / Q000037:antagonists & inhibitors / Q000235:genetics*; D018384:Oxidative Stress / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D047069:Pyrazolones; D011725:Pyridines / Q000494:pharmacology; D011728:Pyridones; D017382:Reactive Oxygen Species / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: D007093:Imidazoles; D011720:Pyrazoles; D047069:Pyrazolones; D011725:Pyridines; D011728:Pyridones; D017382:Reactive Oxygen Species; C576694:setanaxib; D020122:tert-Butylhydroperoxide; D000074663:NADPH Oxidase 4; C498910:NOX4 protein, human; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; D000111:Acetylcysteine
keywords: Apoptosis; Endothelial cells; Mitochondria; NOX4; Necroptosis; Reactive oxygen species