Abstract：
BACKGROUND: Dysfunction of dopamine homeostasis (DAH), which is regulated by vesicular monoamine transporter 2 (VMAT2), is a vital cause of dopamine (DA) neurotoxicity and motor deficits in Parkinson\'s disease (PD). Gastrodin (4-hydroxybenzyl alcohol 4-O-β-D-glucoside; GTD), a natural active compound derived from Gastrodia elata Blume, can be used to treat multiple neurological disorders, including PD. However, whether GTD regulates VMAT2-mediated DAH dysfunction in PD models remains unclear.
OBJECTIVE: To explore whether GTD confers dopaminergic neuroprotection by facilitating DA vesicle storage and maintaining DAH in PD models.
METHODS: Mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and PC12 cells with 1-methyl-4-phenyl-pyridinium (MPP+) to induce PD characteristics. Multiple behavioural tests were performed to evaluate the motor functions of the mice. HPLC was used to measure DA and 3,4-dihydroxyphenylacetic acid (DOPAC) levels. Transmission electron microscopy was used to observe synaptic vesicles. Molecular docking and molecular dynamics were used to determine the binding affinity of GTD to the target protein. Reserpine (Res, a VMAT2 inhibitor) and PD0325901 (901, a MEK inhibitor) were employed to investigate the mechanism of GTD. Western blotting and immunohistochemistry were used to assess the expression of the target proteins.
RESULTS: GTD attenuated motor deficits and dopaminergic neuronal injury, reversed the imbalance of DAH, and increased VMAT2 levels and vesicle volume in MPTP-induced mice. GTD ameliorated cell damage, ROS release, and dysfunction of DAH in MPP+-induced PC12 cells. Moreover, the neuroprotective effects of GTD were reversed by Res in vitro and in vivo. Furthermore, GTD can activate the MEK/ERK/CREB pathway to upregulate VMAT2 in vitro and in vivo. Interestingly, 901 reversed the effects of GTD on VMAT2 and dopaminergic neuronal impairment.
CONCLUSIONS: GTD relieved PD-related motor deficits and dopaminergic neuronal impairment by facilitating MEK-depended VMAT2 to regulate DAH, which offers new insights into its therapeutic potential.
OBJECTIVE: To explore whether GTD confers dopaminergic neuroprotection by facilitating DA vesicle storage and maintaining DAH in PD models.
METHODS: Mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and PC12 cells with 1-methyl-4-phenyl-pyridinium (MPP+) to induce PD characteristics. Multiple behavioural tests were performed to evaluate the motor functions of the mice. HPLC was used to measure DA and 3,4-dihydroxyphenylacetic acid (DOPAC) levels. Transmission electron microscopy was used to observe synaptic vesicles. Molecular docking and molecular dynamics were used to determine the binding affinity of GTD to the target protein. Reserpine (Res, a VMAT2 inhibitor) and PD0325901 (901, a MEK inhibitor) were employed to investigate the mechanism of GTD. Western blotting and immunohistochemistry were used to assess the expression of the target proteins.
RESULTS: GTD attenuated motor deficits and dopaminergic neuronal injury, reversed the imbalance of DAH, and increased VMAT2 levels and vesicle volume in MPTP-induced mice. GTD ameliorated cell damage, ROS release, and dysfunction of DAH in MPP+-induced PC12 cells. Moreover, the neuroprotective effects of GTD were reversed by Res in vitro and in vivo. Furthermore, GTD can activate the MEK/ERK/CREB pathway to upregulate VMAT2 in vitro and in vivo. Interestingly, 901 reversed the effects of GTD on VMAT2 and dopaminergic neuronal impairment.
CONCLUSIONS: GTD relieved PD-related motor deficits and dopaminergic neuronal impairment by facilitating MEK-depended VMAT2 to regulate DAH, which offers new insights into its therapeutic potential.
摘要：
背景：多巴胺稳态（DAH）功能障碍，它由囊泡单胺转运蛋白2（VMAT2）调节，是帕金森病（PD）多巴胺（DA）神经毒性和运动障碍的重要原因。天麻素（4-羟基苯甲醇4-O-β-D-葡萄糖苷；GTD），一种来自天麻的天然活性化合物，可用于治疗多种神经系统疾病，包括PD.然而,在PD模型中,GTD是否调节VMAT2介导的DAH功能障碍尚不清楚.
目的：在PD模型中，探讨GTD是否通过促进DA囊泡储存和维持DAH而赋予多巴胺能神经保护作用。
方法:小鼠用1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)和PC12细胞用1-甲基-4-苯基-吡啶(MPP+)处理以诱导PD特征。进行多种行为测试以评估小鼠的运动功能。HPLC用于测量DA和3,4-二羟基苯基乙酸(DOPAC)水平。透射电镜观察突触囊泡。使用分子对接和分子动力学来确定GTD与靶蛋白的结合亲和力。利血平（Res，VMAT2抑制剂）和PD0325901（901，MEK抑制剂）用于研究GTD的机制。使用蛋白质印迹和免疫组织化学来评估靶蛋白的表达。
结果：GTD减轻了运动缺陷和多巴胺能神经元损伤，扭转了DAH的不平衡，MPTP诱导小鼠的VMAT2水平和囊泡体积增加。GTD改善了细胞损伤，ROS释放，MPP+诱导的PC12细胞中DAH的功能障碍。此外，GTD的神经保护作用在体外和体内被Res逆转。此外，GTD可以激活MEK/ERK/CREB途径，在体外和体内上调VMAT2。有趣的是,901逆转了GTD对VMAT2和多巴胺能神经元损伤的影响。
结论：GTD通过促进MEK依赖性VMAT2调节DAH，缓解PD相关的运动障碍和多巴胺能神经元损伤，这为其治疗潜力提供了新的见解。
目的：在PD模型中，探讨GTD是否通过促进DA囊泡储存和维持DAH而赋予多巴胺能神经保护作用。
方法:小鼠用1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)和PC12细胞用1-甲基-4-苯基-吡啶(MPP+)处理以诱导PD特征。进行多种行为测试以评估小鼠的运动功能。HPLC用于测量DA和3,4-二羟基苯基乙酸(DOPAC)水平。透射电镜观察突触囊泡。使用分子对接和分子动力学来确定GTD与靶蛋白的结合亲和力。利血平（Res，VMAT2抑制剂）和PD0325901（901，MEK抑制剂）用于研究GTD的机制。使用蛋白质印迹和免疫组织化学来评估靶蛋白的表达。
结果：GTD减轻了运动缺陷和多巴胺能神经元损伤，扭转了DAH的不平衡，MPTP诱导小鼠的VMAT2水平和囊泡体积增加。GTD改善了细胞损伤，ROS释放，MPP+诱导的PC12细胞中DAH的功能障碍。此外，GTD的神经保护作用在体外和体内被Res逆转。此外，GTD可以激活MEK/ERK/CREB途径，在体外和体内上调VMAT2。有趣的是,901逆转了GTD对VMAT2和多巴胺能神经元损伤的影响。
结论：GTD通过促进MEK依赖性VMAT2调节DAH，缓解PD相关的运动障碍和多巴胺能神经元损伤，这为其治疗潜力提供了新的见解。
