天麻皂苷对三叉神经节离体培养后降钙素基因相关肽表达影响的机制研究

The pharmacological mechanism of gastrodin on calcitonin gene-related peptide of cultured rat trigeminal ganglion

利用成年雄性Sprague-Dawley(SD)大鼠三叉神经节(trigeminal ganglion,TG)离体培养模型,深入研究天麻活性成分天麻皂苷对TG内降钙素基因相关肽(calcitonin gene-related peptide,CGRP)表达水平的影响以及相关的细胞内信号转导机制。将不同质量浓度的天麻皂苷与TG孵育后,免疫组织化学染色比较CGRP免疫反应(CGRP-immunoreactivity,CGRP-ir)阳性细胞数;实时定量PCR(real-time RT-PCR)对比与琥珀酸舒马普坦和盐酸氟桂利嗪作用下CGRP-mRNA表达变化;Western blotting检测进一步对比与细胞外信号调节激酶1/2(extracellular signal-regulated kinase1/2,ERK1/2)通路特异性阻滞剂PD98059、U0126作用下,磷酸化ERK1/2蛋白(phosphorylated ERK1/2,pERK1/2)水平变化。实验结果显示,天麻皂苷(5和10 mmol.L-1)与1.2 mmol.L-1琥珀酸舒马普坦程度相当地显著降低TG内CGRP-ir(+)细胞表达,而天麻皂苷(2.5、20和40 mmol.L-1)对CGRP-ir(+)细胞表达与培养组比较无显著差别。天麻皂苷(5和10 mmol.L-1)与1.2 mmol.L-1琥珀酸舒马普坦和10μmol.L-1盐酸氟桂利嗪分别显著降低CGRP-mRNA表达水平(P<0.01)。Western blotting检测结果显示,天麻皂苷(5和10 mmol.L-1)降低TG内pERK1/2蛋白表达水平的能力接近于PD98059和U0126(10μmol.L-1)。研究结果表明,天麻皂苷(5和10 mmol.L-1)能显著抑制大鼠TG内CGRP-ir(+)细胞表达,抑制CGRP mRNA表达的强度相当于琥珀酸舒马普坦和盐酸氟桂利嗪;降低TG内pERK1/2蛋白表达的能力接近于ERK1/2信号通路特异性阻滞剂的作用,提示天麻皂苷可能通过细胞内ERK1/2信号转导通路抑制CGRP上调表达。

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 μmol-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.