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Redox Biol. 2017 Apr;11:524-534. doi: 10.1016/j.redox.2016.12.036. Epub 2017 Jan 5.

Tert-butyl hydroperoxide (t-BHP) induced apoptosis and necroptosis in endothelial cells: Roles of NOX4 and mitochondrion.

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State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.
Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, PR China.
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China. Electronic address:


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-BHPL and t-BHPH induced endothelial cell death was determined. T-BHPL induced 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-BHPH induced 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-BHPH induced 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-BHPL and 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-BHPL and t-BHPH-induced apoptosis and necroptosis, respectively.


Apoptosis; Endothelial cells; Mitochondria; NOX4; Necroptosis; Reactive oxygen species

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