Clivorine, an otonecine pyrrolizidine alkaloid from Ligularia species, impairs neuronal differentiation via NGF-induced signaling pathway in cultured PC12 cells

Abstract

BackgroundPyrrolizidine 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. PurposeIn this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PC12 cells. Study designPC12 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. MethodsPC12 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. ResultsClivorine 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. ConclusionTaken 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.