TSPO-神经甾体-GABA(A)受体通路介导钩吻的中枢神经毒性作用

Gelsemium elegans is a highly toxic plant, which of toxic mechanism and detoxification has been rarely understood. Our previous study suggested that the neuropharmacolgocial mechanism of Gelsemium elegans potentially connected to TSPO-neurosteroids-GABA(A) receptor pathway, and diazepam significantly inhibited the neurotoxicity of Gelsemium elegans. In the present project, Gelsemium elegans, collected from Guangxi province, will be systematically isolated and purified for its toxic principles, which then will be examined by toxicity tests. In vivo and in vitro, the isotope-labelling method will be used to examine the affinity of TSPO with Gelsemium toxic principles (GTP); HPLC-MS, ELISA, qRT-PCR and western blot will be used to measure the levels of neurosteroids by GTP; the whole-cell patch clamp techniques will be used to test the activity of GABA(A) receptor with GTP; The tool medicines with the ability of blocking or inhibiting TSPO-neurosteroids-GABA(A) pathway enzymes/receptors will be used to confirm the key target of GTP to mediate neurosteroids by TSPO to GABA(A) receptor. Finally, according to the mechanism, the effective antagonists against Gelsemium neurotoxicity will be screened to obtain an optimal therapy for Gelsemium intoxication. After that, the expectation of this project is to elucidate the mechanism of Gelsemium neurotoxicity and to supply a remedy for Gelsemium intoxication, which might be benefit to the clinic treatment against Gelsemium poisoning.

钩吻为剧毒植物，其毒性机制和解毒方法亟待研究。本课题组前期研究提示，钩吻的神经作用机制可能与脑内TSPO-神经甾体-GABA(A)受体通路有关；地西泮能有效抑制钩吻生物碱的神经毒性。本项目拟采集桂产钩吻，通过系统地提取、分离和毒性试验，明确主要的钩吻毒性组分和购物毒素。在组织、细胞和整体动物水平上，通过放射受体分析法检测钩吻与脑内TSPO的亲和力；采用HPLC-MS、ELISA、qRT-PCR和western blot法探测钩吻对脑内神经甾体水平的影响；膜片钳全细胞纪录技术明确钩吻与GABA(A)受体的功能；并通过相关工具药的拮抗作用，确认钩吻的关键作用靶点，以期探明钩吻神经毒性作用与脑内TSPO-神经甾体-GABA(A)受体通路的关联机制。并在此基础上，筛选钩吻中毒的特效解药，探索钩吻中毒的最佳解救方案。本研究将有助于钩吻毒性机制的理解，对钩吻中毒治疗具有推动作用。

钩吻为剧毒植物，但其毒性机制和防治并不明确，其神经毒性机制可能与脑内TSPO-神经甾体-GABA(A)受体通路的关系亟待研究。本项目通过液质联用检测桂产钩吻的11种生物碱的含量，对含量较高的钩吻生物碱单体进行急性毒性研究，确认以毒性最显著的钩吻素己作为钩吻毒素进行中枢毒性机制的研究。通过液质联用和ELISA法测定钩吻素己中毒大鼠海马、前额叶皮质、延髓、小脑和外周血中神经甾体的含量，其中钩吻素己能增加海马区以别孕烯醇酮为代表的神经甾体含量，而外周血的别孕烯醇酮则下降；RT-PCR和蛋白印迹检测结果提示，在钩吻素己中毒大鼠海马区中，与别孕烯醇酮合成有关的TSPO、P450 SCC酶和5-α-还原酶的mRNA和蛋白表达量均上调；蛋白质组学、蛋白印迹、免疫组化和液质联用的结果提示，钩吻素己能上调海马区GAD1蛋白表达，增加GABA的含量，下调GABARAPL2蛋白表达，导致GABA(A)受体表达下调，引起海马区GABA能神经元功能紊乱而产生毒性症状；GABA(A)受体激动剂地西泮和咪达唑仑能有效拮抗钩吻素己的神经毒性，而GABA(A)受体拮抗剂氟马西尼则会增加钩吻素己的毒性。由此可见，钩吻素己作为钩吻毒素的代表，其中枢神经毒性与海马区TSPO-神经甾体- GABA(A)受体通路密切相关，其中苯二氮䓬类药物可能是防治钩吻素己急性中毒的特效药物。本研究阐明了钩吻的神经毒性机制，并提出潜在的特效药，并有助于深化理解TSPO-神经甾体- GABA(A)受体通路的中枢神经毒性的生物学机制。