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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 in vitro culture model of adult male Sprague-Dawley (SD) rats with trigeminal ganglion (TG) was used to study the effects of Gastrodia elata Blume on calcitonin gene-related peptide (TG) The level of expression and the associated intracellular signal transduction mechanisms. After the different concentrations of Gastrodin Saponins were incubated with TG, the number of CGRP-ir positive cells was compared by immunohistochemical staining. Compared with real-time RT-PCR, (P <0.05), while the expression of CGRP-mRNA under the action of flunarizine hydrochloride, etoposide and flunarizine hydrochloride showed a significant difference compared with PD98059 , U0126 phosphorylated ERK1 / 2 protein (phosphorylated ERK1 / 2, pERK1 / 2) levels change. The experimental results showed that the levels of CGRP-ir (+) cells in TG were remarkably reduced by the combination of gastrodin (5 and 10 mmol.L-1) and 1.2 mmol.L-1 sumatriptan succinate , 20 and 40 mmol.L-1) on CGRP-ir (+) cells expression and culture group no significant difference. Gastrodia saponin (5 and 10 mmol.L-1) significantly decreased the expression of CGRP-mRNA (P <0.01) with 1.2 mmol.L-1 sumatriptan succinate and 10 μmol.L-1 flunarizine hydrochloride, respectively. The results of Western blotting showed that the ability of Gastrodia saponin (5 and 10 mmol.L-1) to reduce pERK1 / 2 protein expression in TG was close to that of PD98059 and U0126 (10μmol.L-1). The results showed that Gastrodia saponin (5 and 10 mmol.L-1) could significantly inhibit the expression of CGRP-ir (+) cells in rat TG and the intensity of CGRP mRNA expression was comparable to that of sumatriptan succinate and fluocinonide The ability of pERK1 / 2 to decrease the expression of pERK1 / 2 in TG was close to that of ERK1 / 2 signal pathway-specific blocker, which indicated that it could up-regulate the expression of CGRP by ERK1 / 2 signal transduction pathway.