title: IMM-H004, a novel courmarin derivative, protects against oxygen-and glucose-deprivation/restoration-induced apoptosis in PC12 cells.
abstract: 7-Hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin (IMM-H004) is a novel coumarin derivative synthesized in our laboratory. The purpose of the current study was to determine the neuroprotective effects of IMM-H004 on PC12 cells and its potential mechanism of action. PC12 cells were subject to oxygen and glucose deprivation (OGD) followed by the restoration of oxygen and glucose (R), which mimics ischemia and reperfusion in vivo. Cell viability was determined by MTT assay. DNA fragmentation was analyzed by DNA ladder. ROS and mitochondrial membrane potential were measured by fluorescent microscope and quantified by Image-Pro Express 6.0 software. ATP was measured by luciferin-luciferase assay. The activation of signal-regulated molecules was assessed by the Western blot analysis. OH formation was determined using the Electron Spin Resonance (ESR) trapping technique in combination with 5, 5-dimethyl-1-pyrroline-N-oxide. OGD/R reduced cell viability and induced cell apoptosis, which were both dose-dependently attenuated by IMM-H004. The accumulation of intracellular reactive oxygen species (ROS) and reduced mitochondrial membrane potential observed in PC12 cells treated with OGD/R, which switch on the mitochondrion-dependent apoptotic pathway, were reversed by IMM-H004. ATP production in OGD/R-treated PC12 cells was elevated by IMM-H004, which suggests that it restored the functions of the mitochondria. OGD/R-induced cytochrome c release from the mitochondria reduced the ratio of apoptotic proteins, Bcl-2/Bax, and induced caspase-3 activation and DNA fragmentation. These changes were significantly inhibited by IMM-H004. IMM-H004 also significantly inhibited OH formation, determined by electron spin resonance, which indicates that it is a potent free-radical scavenger. This study has demonstrated that IMM-H004 protects PC12 cells against OGD/R-induced apoptosis, at least in part, by scavenging excessive ROS and inhibiting the mitochondrion-dependent apoptotic pathway.
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China.
authors.forename: Hai-jie
authors.initials: HJ
authors.lastname: Ji
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China.
authors.forename: Dong-mei
authors.initials: DM
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China.
authors.forename: Jin-feng
authors.initials: JF
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China.
authors.forename: Ming-na
authors.initials: MN
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China.
authors.forename: Zhi-peng
authors.initials: ZP
authors.lastname: Li
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China.
authors.forename: Dong-hui
authors.initials: DH
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China; Tsinghua-Peking Center for Life Sciences, School of Medicine, Tsinghua University, Haidian District, Beijing 100084, PR China; Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Haidian District, Beijing 100084, PR China. Electronic address: gangliu27@tsinghua.edu.cn.
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nanwei Road, Xicheng District, Beijing 100050, PR China. Electronic address: chennh@imm.ac.cn.
authors.forename: Nai-hong
authors.initials: NH
authors.lastname: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D003374:Coumarins / Q000494:pharmacology*; D053938:DNA Fragmentation; D005947:Glucose / Q000172:deficiency*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018696:Neuroprotective Agents / Q000494:pharmacology*; D016716:PC12 Cells; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C494828:Bax protein, rat; D003374:Coumarins; C588372:IMM-H004; D018696:Neuroprotective Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D000255:Adenosine Triphosphate; D005947:Glucose
keywords: Apoptosis; Coumarin derivative; IMM-H004; Oxygen- and glucose-deprivation/restoration; PC12 cells; Stroke