Table1_Dopamine Homeostasis Imbalance and Dopamine Receptors-Mediated AC/cAMP/PKA Pathway Activation are Involved in Aconitine-Induced Neurological Impairment in Zebrafish and SH-SY5Y Cells.DOCX

Aconitine is one of the main bioactive and toxic ingredients of Aconitum species. Increasingly, aconitine has been reported to induce neurotoxicity. However, whether aconitine has effects on the dopaminergic nervous system remains unclear. In this study, zebrafish embryos at 6-days postfertilization were exposed to aconitine at doses of 0.5, 1, and 2 μM for 24 h, and SH-SY5Y cells were treated with 50, 100, and 200 μM of aconitine for 24 h. Results demonstrated that aconitine treatment induced deformities and enhanced the swimming behavior of zebrafish larvaes. Aconitine exposure suppressed cell proliferation and increased the number of reactive oxygen species and apoptosis in zebrafish larvaes and SH-SY5Y cells. Aconitine altered the levels of dopamine and its metabolites by regulating the expression of genes and proteins related to dopamine synthesis, storage, degradation, and reuptake in vivo and in vitro. Moreover, aconitine activated the AC/cAMP/PKA pathway by activating the dopamine D1 receptor (D1R) and inhibiting the dopamine D2 receptor (D2R) to disturb intracellular calcium homeostasis, eventually leading to the damage of nerve cells. Furthermore, the D1R antagonist SCH23390 and D2R agonist sumanirole pretreatment effectively attenuated the excitatory state of larvaes. Sumanirole and PKA antagonist H-89 pretreatment effectively decreased intracellular Ca2+ accumulation induced by aconitine in vivo. SCH23390 and sumanirole also reduced aconitine-induced cytotoxicity by inhibiting the AC/cAMP/PKA pathway in vitro. These results suggested that dopamine homeostasis imbalance and dopamine receptors (DRs)-mediated AC/cAMP/PKA pathway activation might be vital mechanisms underlying aconitine-induced neurological injury.

乌头碱（aconitine）是乌头属（Aconitum）植物主要的生物活性与毒性成分之一。现有研究越来越多地报道乌头碱可诱发神经毒性。然而，乌头碱对多巴胺能神经系统是否具有影响，目前仍不明确。本研究以受精后6天的斑马鱼胚胎为对象，分别以0.5、1、2 μM的乌头碱染毒24小时；同时以50、100、200 μM的乌头碱处理SH-SY5Y细胞24小时。结果显示，乌头碱处理可诱导斑马鱼幼体出现畸形，并增强其游泳行为。乌头碱暴露可抑制斑马鱼幼体与SH-SY5Y细胞的细胞增殖，并增加活性氧（reactive oxygen species, ROS）水平与细胞凋亡数量。在体内与体外模型中，乌头碱可通过调控多巴胺合成、储存、降解及再摄取相关基因与蛋白的表达，改变多巴胺及其代谢产物的水平。此外，乌头碱可通过激活多巴胺D1受体（dopamine D1 receptor, D1R）并抑制多巴胺D2受体（dopamine D2 receptor, D2R），激活腺苷酸环化酶（AC）/环磷酸腺苷（cAMP）/蛋白激酶A（PKA）通路，进而扰乱细胞内钙稳态，最终造成神经细胞损伤。进一步实验显示，预先使用D1R拮抗剂SCH23390与D2R激动剂舒马尼洛（sumanirole）处理，可有效缓解斑马鱼幼体的兴奋状态。预先使用舒马尼洛与PKA拮抗剂H-89处理，可在体内有效减少乌头碱诱导的细胞内钙蓄积。在体外实验中，SCH23390与舒马尼洛也可通过抑制AC/cAMP/PKA通路，减轻乌头碱诱导的细胞毒性。上述结果表明，多巴胺稳态失衡以及多巴胺受体（dopamine receptors, DRs）介导的AC/cAMP/PKA通路激活，可能是乌头碱诱发神经损伤的关键机制。