芎麻汤药效部位对偏头痛的治疗机制研究

发布时间：2018-06-16 00:30

本文选题：芎麻汤药效部位 + 偏头痛　；参考：《湖南中医药大学》2015年博士论文

【摘要】：研究目的建立硝酸甘油(NTG)型偏头痛大鼠模型,观察芎麻汤药效部位(乙酸乙酯及正丁醇提取部位)对偏头痛发作时外周血及三叉神经颈髓复合体(TCC)内CGRP、5-HT、NF-κB的影响,确定各指标与芎麻汤药效部位的相关性；及对TCC内CGRP-CRLR/RAMP1信号转导通路的干预作用,以期阐明芎麻汤药效部位治疗偏头痛的作用原理。实验方法采用不同极性有机溶剂(石油醚、乙酸乙酯、正丁醇)对芎麻汤进行梯度提取,得芎麻汤药效部位(乙酸乙酯、正丁醇提取部位混合物)。健康SD雄性大鼠96只,按体质量随机分为Ⅰ、Ⅱ两大组(Ⅰ组60只,Ⅱ组36只。各大组内包括空白对照组(KB组),模型空白组(M组),芎麻汤药效部位低、中、高剂量组(D、Z、G组),琥珀酸舒马普坦片阳性对照组(Y组)),除KB组,M组外,其余组均对应药物预处理7d后,皮下注射NTG复制偏头痛模型,造模后的第30、120min时,灌胃给药两次,观察大鼠挠头、爬笼、咬尾、往返运动等情况,判断造模成功与否；Ⅰ组大鼠均乙醚麻醉后取颈外静脉血采用ELISA检测血浆中CGRP、5-HT、NF-κB含量,心脏灌注后取全脑剥离TCC部位采用IHC染色检测CGRP、5-HT、NF-κB阳性表达；Ⅱ组大鼠均乙醚麻醉,其中18只心脏灌注后取全脑剥离TCC部位采用IHC染色检测CGRP、CRLR、RAMP1阳性表达,余18只直接取全脑剥离TCC部位并平分两份,一份组织采用Real-Time PCR检测CGRP、 CRLR、RAMP1mRNA表达,另一份组织采用Western-Blot检测CGRP.CRLR.RAMP1蛋白表达。实验结果(1)Ⅰ、Ⅱ组大鼠行为学评分比较：造模前30min内,各组大鼠行为学无显著性差异。造模后30min内,与造模前及KB组比较,各组大鼠行为学评分均明显升高(P0.05),说明动物造模成功。给药后30min内,与同期M组比较,芎麻汤药效部位各剂量组、Y组行为学评分明显降低(P0.05)。(2)Ⅰ组大鼠：(①血浆ELISA:与KB组比较,M组大鼠血浆中CGRP、5-HT、NF-κB浓度显著升高(P0.01)。与M组比较,Z、G组,Y组血浆中CGRP浓度显著降低(P0.01),D组明显降低(P0.05)；芎麻汤药效部位各剂量组、Y组5-HT显著降低(P0.01); NF-κB在各组之间无显著性差异(P0.05)。②TCC部位IHC染色：SABC法检测发现,各组大鼠TCC部位均可见胞体被染成棕色的CGRP、5-HT、NF-κB阳性细胞。与KB组比较,M组大鼠TCC内CGRP免疫阳性染色平均光密度值显著升高(P0.01);5-HT、NF-κB明显升高(P0.05)。与M组比较,芎麻汤药效部位各剂量组、Y组TCC内CGRP免疫阳性染色平均光密度值显著降低(P0.01)；Z、G组5-HT明显降低(P0.05),Y组显著降低(P0.01)；Z组NF-κB明显降低(P0.05)。(3)Ⅱ组大鼠：①IHC染色：SABC法检测发现,各组大鼠TCC部位均可见胞体被染成棕色的CGRP、CRLR、RAMP1阳性细胞。与KB组比较,M组大鼠TCC内CGRP、CRLR、RAMP1免疫阳性细胞数显著升高(P0.01)。与M组比较,芎麻汤药效部位各剂量组TCC内CGRP免疫阳性细胞数显著降低(P0.01),Y组明显降低(P0.05)：Z组CRLR显著降低(P0.01),D、Y组明显降低(P0.05),G组无显著性差异(P0.05)；芎麻汤药效部位各剂量组、Y组RAMP1显著降低(P0.01)。②mRNA表达：Rea1-Time PCR检测发现,各组大鼠TCC部位均可见CGRP、CRLR、 RAMP1mRNA的表达。与KB组比较,M组TCC内的CGRP、RAMP1mRNA的表达量明显升高(P0.05); CRLR显著降低(P0.01)。与M组比较,芎麻汤药效部位各剂量组TCC内CGRP mRNA的表达量均显著降低(P0.01),Y组明显降低(P0.05)；G组CRLR显著升高(P0.01)；D、Y组RAMP1显著降低(P0.01),Z、G组明显降低(P0.05)。③蛋白表达：SDS-PAGE凝胶电泳检测发现,各组大鼠TCC内均可见CGRP、CRLR、RAMP1蛋白表达,且在各组之间存在明显差异。与KB组比较,M组TCC内CGRP蛋白表达下降；CRLR、RAMP1明显下降。与M组比较,芎麻汤药效部位各剂量组TCC内CGRP蛋白表达明显下降；Z、G组CRLR蛋白表达明显升高；芎麻汤药效部位各剂量组RAMP1蛋白表达明显升高。最终结论(1)芎麻汤药效部位降低NTG型偏头痛大鼠外周血中CGRP、5-HT浓度,从而改善偏头痛发作时血管活性物质和神经递质水平失常达到缓解偏头痛症状。(2)芎麻汤药效部位下调NTG型偏头痛大鼠TCC内CGRP、5-HT、NF-κB表达,从而改善偏头痛发作时三叉神经血管系统激活后引起的神经源性炎症反应,达到缓解偏头痛症状。但与芎麻汤药效部位治疗偏头痛相关性最强的指标为CGRP。(3)芎麻汤药效部位通过降低NTG型偏头痛大鼠TCC内CGRP、CRLR、RAMP1免疫阳性细胞数和下调CGRP、RAMP1mRNA表达来下降偏头痛发作时三叉神经血管系统激活后RAMP1过表达引起的三叉神经节对CGRP高度敏感现象,通过下调CGRP蛋白表达,上调CRLR、RAMP1蛋白表达,进一步导致CGRP释放减少、表达下降,干扰CGRP的正负反馈回路,最终实现治疗偏头痛的作用原理。
[Abstract]:Objective to establish a nitroglycerin (NTG) migraine model, and observe the effect of Xiong Ma Decoction (ethyl acetate and n-butanol extract) on the CGRP, 5-HT, NF- kappa B in the peripheral blood and the trigeminal cervical myelin complex (TCC) during the migraine attack, and determine the correlation between the indexes and the efficacy sites of Xiong Ma Tang, and the CGRP-CRLR/RAMP in TCC. 1 the role of signal transduction pathway in the treatment of migraine.
The experimental methods used the different polar organic solvents (petroleum ether, ethyl acetate and n-butanol) for the gradient extraction of Xiong Ma Tang, and obtained the pharmacodynamic parts of Xiong Ma Tang (ethyl acetate and n-butanol extract mixture). 96 healthy SD male rats were randomly divided into two groups (group I, 60, and 36 in group II). Group (group KB), model blank group (group M), Xiong Ma Tang pharmacodynamic site low, middle, high dose group (D, Z, G group), Sumatriptan Succinate Tablets positive control group (Y group), except the KB group, M group, the rest of the group corresponding to the drug pretreatment 7d, subcutaneous injection of NTG copy hemicalgia model, after the process of 30120min, gavage two times, observe the rat's head, the observation of the head, cages, the rats, the observation of the head, cages, the rats. In the group I rats, the content of CGRP, 5-HT, NF- kappa B was detected by ELISA in the external jugular blood of the rats after ether anesthesia, and the positive expression of CGRP, 5-HT, NF- kappa B was detected by IHC staining after cardiac perfusion, and the group II rats were all anaesthetized with ether after cardiac perfusion, and 18 hearts were perfused after 18 heart perfusion. The positive expression of CGRP, CRLR and RAMP1 was detected by IHC staining in the TCC site of total brain dissection. The remaining 18 were directly removed from the TCC site of the whole brain and divided into two parts. The tissues were detected CGRP, CRLR, RAMP1mRNA expression by Real-Time PCR, and the other tissue was expressed by Western-Blot detection.
The results of the experiment (1) I, group II rats behavior score comparison: there was no significant difference in behavior in each group before the model 30min. After the model 30min, the behavior score of the rats in each group was significantly increased (P0.05), indicating that the animal model was successful. After 30min, the pharmacodynamic parts of Xiong Ma Decoction were compared with the same period of M group. (2) group I rats: (2) the concentration of CGRP, 5-HT and NF- kappa B in the plasma of M group was significantly higher than that in group KB (P0.01). There was no significant difference (P0.01), and there was no significant difference between NF- kappa B (P0.05). (2) IHC staining in TCC site. The SABC method found that the TCC parts of each group were found to be stained with brown CGRP, 5-HT, NF- kappa B positive cells. High (P0.05). Compared with group M, the average light density of CGRP immunoreactive staining in group Y was significantly decreased (P0.01) in group Y, Z, G group 5-HT decreased significantly (P0.05), Y group decreased significantly (P0.01). (3) Compared with the KB group, the number of CGRP, CRLR, and RAMP1 positive cells in the M group was significantly higher than that in the KB group (P0.01). Compared with the M group, the number of positive cells in each dose group of the Xiong Ma Decoction group was significantly lower than that in the M group. There was no significant difference in the group (P0.05), and there was no significant difference in the G group (P0.05), and the RAMP1 in the group Y was significantly reduced (P0.01). (P0.01). (2) mRNA expression: Rea1-Time PCR detection found that TCC parts of each group showed CGRP, CRLR, and expression. Significantly lower (P0.01). Compared with group M, the expression of CGRP mRNA in each dose group of Xiong Ma Decoction group decreased significantly (P0.01), Y group decreased significantly (P0.05), CRLR significantly increased in G group (P0.01), D, significantly decreased (P0.01) in group G (P0.01). The expression of CGRP, CRLR, RAMP1 protein was obviously different between each group. Compared with the KB group, the expression of CGRP protein in M group decreased and CRLR, RAMP1 decreased obviously. Compared with the M group, the expression of TCC CGRP protein in the dosage group of Xiong Ma Decoction decreased obviously; The expression of RAMP1 protein in the group was significantly increased.
The final conclusion (1) Xiong Ma Decoction effective site to reduce the concentration of CGRP and 5-HT in peripheral blood of NTG migraine rats, thus improving the level of vasoactive substances and neurotransmitters to relieve migraine symptoms during migraine attack. (2) the effect site of Xiong Ma decoction can reduce the expression of CGRP, 5-HT, NF- kappa B in TCC of NTG type migraine rats, thus improving the migraine hair. The neurogenic inflammatory reaction caused by the activation of the trigeminal vascular system was made to alleviate migraine symptoms, but the strongest index of the relationship with Xiong Ma Decoction in the treatment of migraine was CGRP. (3) Xiong Ma decoction, which reduced the number of CGRP, CRLR, RAMP1 immunoreactive cells and CGRP, RAMP1mRNA in the TCC of NTG migraine rats. Expression to decrease the sensitivity of trigeminal ganglia to CGRP induced by RAMP1 overexpression of the trigeminal vascular system during migraine attacks, by downregulating the expression of CGRP protein, up regulating the expression of CRLR and RAMP1 protein, further leading to the reduction of CGRP release, the decrease of expression, and the interference of CGRP in the positive and negative feedback loops of CGRP, and finally realizing the treatment of migraine. Use the principle.
【学位授予单位】：湖南中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R277.7

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