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. PURPOSE 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. STUDY DESIGN 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. CONCLUSION 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.

中文翻译：

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Clivorine是来自Ligularia物种的一种Ocinecine吡咯嗪核生物碱，通过培养的PC12细胞中NGF诱导的信号传导途径损害神经元分化。

背景技术吡咯并立烷生物碱（PAs）通常在许多植物中发现，包括那些用于药物治疗的植物。PAs的肝毒性已在体内和体外得到证实。但是，很少提及PA的神经毒性。目的在这项研究中，我们旨在研究在体外培养的PC12细胞中，在各种Ligularia物种中发现的PA之一，盐酸克利夫林的体外神经毒性。使用Study DESIGN PC12细胞系首先阐明了盐酸氯氟碱的神经毒性和潜在机制，包括细胞活力和形态变化，神经元分化标志物和信号传导途径。方法用浓度系列的食草碱和/或神经生长因子（NGF）攻击PC12细胞。收集细胞裂解物用于MTT分析，台盼蓝染色，免疫细胞荧光染色，qRT-PCR和蛋白质印迹。结果山楂碱可抑制细胞增殖和神经元分化，MTT分析证实了这一点，并且剂量依赖性地减少了神经突的长出。此外，食黄素降低了编码神经元分化标记（例如神经丝和TrkA（NGF受体））的mRNA的水平。此外，山楂碱在培养的PC12细胞中减少了NGF诱导的TrkA，蛋白激酶B和cAMP反应元件结合蛋白的磷酸化。结论综上所述，我们的结果表明，食黄素可能通过下调NGF / TrkA / Akt信号通路在PC12细胞中具有神经毒性。PA不仅会损害肝脏，而且还具有神经毒性，这可能会导致脑部疾病，例如抑郁。结果山楂碱可抑制细胞增殖和神经元分化，MTT分析证实了这一点，并且剂量依赖性地减少了神经突的长出。此外，食黄素降低了编码神经元分化标记（例如神经丝和TrkA（NGF受体））的mRNA的水平。此外，山楂碱在培养的PC12细胞中减少了NGF诱导的TrkA，蛋白激酶B和cAMP反应元件结合蛋白的磷酸化。结论综上所述，我们的结果表明，食黄素可能通过下调NGF / TrkA / Akt信号通路在PC12细胞中具有神经毒性。PA不仅会损害肝脏，而且还具有神经毒性，这可能会导致脑部疾病，例如抑郁。结果山楂碱可抑制细胞增殖和神经元分化，MTT分析证实了这一点，并且剂量依赖性地减少了神经突的长出。此外，食黄素降低了编码神经元分化标记（例如神经丝和TrkA（NGF受体））的mRNA的水平。此外，山楂碱在培养的PC12细胞中减少了NGF诱导的TrkA，蛋白激酶B和cAMP反应元件结合蛋白的磷酸化。结论综上所述，我们的结果表明，食黄素可能通过下调NGF / TrkA / Akt信号通路在PC12细胞中具有神经毒性。PA不仅会损害肝脏，而且还具有神经毒性，这可能会导致脑部疾病，例如抑郁症。