title: Clivorine, an otonecine pyrrolizidine alkaloid from Ligularia species, impairs neuronal differentiation via NGF-induced signaling pathway in cultured PC12 cells.
abstract: BACKGROUND
  Pyrrolizidine alkaloids (PAs) are commonly found in many plants including those used in medical therapeutics. The hepatotoxicities of PAs have been demonstrated both in vivo and in vitro; however, the neurotoxicities of PAs are rarely mentioned.
  
  
  OBJECTIVE
  In this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PC12 cells.
  
  
  METHODS
  PC12 cell line was employed to first elucidate the neurotoxicity and the underlying mechanism of clivorine, including cell viability and morphology change, neuronal differentiation marker and signaling pathway.
  
  
  METHODS
  PC12 cells were challenged with series concentrations of clivorine and/or nerve growth factor (NGF). The cell lysates were collected for MTT assay, trypan blue staining, immunocytofluorescent staining, qRT-PCR and western blotting.
  
  
  RESULTS
  Clivorine inhibited cell proliferation and neuronal differentiation evidenced by MTT assay and dose-dependently reducing neurite outgrowth, respectively. In addition, clivorine decreased the level of mRNAs encoding for neuronal differentiation markers, e.g. neurofilaments and TrkA (NGF receptor). Furthermore, clivorine reduced the NGF-induced the phosphorylations of TrkA, protein kinase B and cAMP response element-binding protein in cultured PC12 cells.
  
  
  CONCLUSIONS
  Taken together, our results suggest that clivorine might possess neurotoxicities in PC12 cells via down-regulating the NGF/TrkA/Akt signaling pathway. PAs not only damage the liver, but also possess neurotoxicities, which could possibly result in brain disorders, such as depression.
authors.affiliation: The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China; Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Aizhen
authors.initials: A
authors.lastname: Xiong
authors.affiliation: Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Artemis Lu
authors.initials: AL
authors.lastname: Yan
authors.affiliation: Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Cathy W C
authors.initials: CW
authors.lastname: Bi
authors.affiliation: Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Kelly Y C
authors.initials: KY
authors.lastname: Lam
authors.affiliation: Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Gallant K L
authors.initials: GK
authors.lastname: Chan
authors.affiliation: Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Kitty K M
authors.initials: KK
authors.lastname: Lau
authors.affiliation: Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Tina T X
authors.initials: TT
authors.lastname: Dong
authors.affiliation: Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Huangquan
authors.initials: H
authors.lastname: Lin
authors.affiliation: The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China.
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Division of Life Science, Center for Chinese Medicine and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China. Electronic address: botsim@ust.hk.
authors.forename: Karl W K
authors.initials: KW
authors.lastname: Tsim
mesh_terms: D000818:Animals; D019659:Asteraceae / Q000737:chemistry*; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects*; D051058:Oncogene Protein v-akt / Q000187:drug effects; D016716:PC12 Cells; D010766:Phosphorylation; D011763:Pyrrolizidine Alkaloids / Q000494:pharmacology*; D051381:Rats; D020917:Receptor, trkA / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: C502240:NGF protein, human; D011763:Pyrrolizidine Alkaloids; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D051058:Oncogene Protein v-akt; C028677:clivorine
keywords: Clivorine; Ligularia species; Neuronal differentiation; Neurotoxicity; Protein kinase B; Pyrrolizidine alkaloids