Luo Guo-Gang, Fan Wen-Jing, Yuan Xing-Yun, Yuan Bo-Bo, Lü She-Min, Cao Yong-Xiao, Xu Cang-Bao
Department of Neurology, First Affiliated Hospital, Key Laboratory of Environment and Genes Related Disease, Ministry of Education, Medical School of Xi'an Jiaotong University, Xi'an 710061, China.
Yao Xue Xue Bao. 2011 Dec;46(12):1451-6.
The Chinese herbal medicine Tianma (Gastrodia elata) has been used for treating and preventing primary headache over thousands of years, but the exact pharmacological mechanism of the main bioactive ingredient gastrodin remains unclear. In present study, the effects of gastrodin on calcitonin gene-related peptide (CGRP) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) expression were observed in rat trigeminal ganglion (TG) after in vitro organ culture to explore the underlying intracellular mechanism of gastrodin on primary vascular-associated headache. CGRP-immunoreactivity (CGRP-ir) positive neurons count, positive area, mean optical density and integrated optical density by means of immunohistochemistry stain were compared at different concentrations of gastrodin, which was separately co-incubated with DMEM in SD rat TG for 24 hours. Only at 5 or 10 mmol L(-1) concentration, gastrodin demonstrated significantly concentration-dependent reduction of CGRP-ir (+) expression and its action closed to 1.2 mmol L(-1) sumatriptan succinate. While at 2.5, 20, and 40 mmol L(-1) concentration, gastrodin did not show remarkable effects on CGRP-ir (+) expression. The optimal concentration of gastrodin (5 and 10 mmol L(-1)) similarly inhibited CGRP-mRNA expression level separately compared with 1.2 mmol L(-1) sumatriptan succinate and 10 micromol L(-1) flunarizine hydrochloride, which was quantitatively analyzed by real-time PCR (RT-PCR). pERK1/2 level was examined by Western blotting after co-cultured with optimal concentration of gastrodin and effective specific ERK1/2 pathway inhibitors PD98059, U0126. The result indicated that gastrodin significantly reduced pERK1/2 protein actions similarly to ERK1/2 pathway specific blockade. It suggests ERK1/2 signaling transduction pathway may be involved in gastrodin intracellular mechanism. This study indicates gastrodin (5 and 10 mmol L(-1)) can remarkably reduce CGRP-ir (+) neuron, CGRP-mRNA and pERK1/2 expression level in cultured rat TG, with its actions similar to the effective concentration of sumatriptan succinate, flunarizine hydrochloride and specific ERK1/2 pathway blocker. The intracellular signaling transduction ERK1/2 pathway may be involved in the gastrodin reducing CGRP up-regulation in rat TG after organ culture.
中药天麻（天麻）用于治疗和预防原发性头痛已有数千年历史，但其主要生物活性成分天麻素的确切药理机制仍不清楚。在本研究中，通过体外器官培养观察天麻素对大鼠三叉神经节（TG）中降钙素基因相关肽（CGRP）和磷酸化细胞外信号调节激酶1/2（pERK1/2）表达的影响，以探讨天麻素对原发性血管相关性头痛的潜在细胞内机制。在不同浓度的天麻素下，通过免疫组织化学染色比较CGRP免疫反应性（CGRP-ir）阳性神经元计数、阳性面积、平均光密度和积分光密度，天麻素分别与SD大鼠TG中的DMEM共孵育24小时。仅在5或10 mmol L（-1）浓度下，天麻素显示出CGRP-ir（+）表达的显著浓度依赖性降低，其作用接近1.2 mmol L（-1）舒马曲坦琥珀酸盐。而在2.5、20和40 mmol L（-1）浓度下，天麻素对CGRP-ir（+）表达未显示出显著影响。与1.2 mmol L（-1）舒马曲坦琥珀酸盐和10 μmol L（-1）盐酸氟桂利嗪相比，天麻素的最佳浓度（5和10 mmol L（-1））同样分别抑制了CGRP-mRNA表达水平，通过实时PCR（RT-PCR）进行定量分析。在与最佳浓度的天麻素和有效的特异性ERK1/2途径抑制剂PD98059、U0126共同培养后，通过蛋白质印迹法检测pERK1/2水平。结果表明，天麻素显著降低了pERK1/2蛋白作用，类似于ERK1/2途径特异性阻断。这表明ERK1/2信号转导途径可能参与天麻素的细胞内机制。本研究表明，天麻素（5和10 mmol L（-1））可显著降低培养的大鼠TG中CGRP-ir（+）神经元、CGRP-mRNA和pERK1/2的表达水平，其作用类似于舒马曲坦琥珀酸盐、盐酸氟桂利嗪和特异性ERK1/2途径阻滞剂的有效浓度。细胞内信号转导ERK1/2途径可能参与天麻素降低器官培养后大鼠TG中CGRP上调的过程。
