本文選題：芎麻湯藥效部位 + 偏頭痛��； 參考：《湖南中醫(yī)藥大學》2015年博士論文

【摘要】：研究目的建立硝酸甘油(NTG)型偏頭痛大鼠模型,觀察芎麻湯藥效部位(乙酸乙酯及正丁醇提取部位)對偏頭痛發(fā)作時外周血及三叉神經(jīng)頸髓復合體(TCC)內CGRP、5-HT、NF-κB的影響,確定各指標與芎麻湯藥效部位的相關性；及對TCC內CGRP-CRLR/RAMP1信號轉導通路的干預作用,以期闡明芎麻湯藥效部位治療偏頭痛的作用原理。 實驗方法采用不同極性有機溶劑(石油醚、乙酸乙酯、正丁醇)對芎麻湯進行梯度提取,得芎麻湯藥效部位(乙酸乙酯、正丁醇提取部位混合物)。健康SD雄性大鼠96只,按體質量隨機分為Ⅰ、Ⅱ兩大組(Ⅰ組60只,Ⅱ組36只。各大組內包括空白對照組(KB組),模型空白組(M組),芎麻湯藥效部位低、中、高劑量組(D、Z、G組),琥珀酸舒馬普坦片陽性對照組(Y組)),除KB組,M組外,其余組均對應藥物預處理7d后,皮下注射NTG復制偏頭痛模型,造模后的第30、120min時,灌胃給藥兩次,觀察大鼠撓頭、爬籠、咬尾、往返運動等情況,判斷造模成功與否；Ⅰ組大鼠均乙醚麻醉后取頸外靜脈血采用ELISA檢測血漿中CGRP、5-HT、NF-κB含量,心臟灌注后取全腦剝離TCC部位采用IHC染色檢測CGRP、5-HT、NF-κB陽性表達；Ⅱ組大鼠均乙醚麻醉,其中18只心臟灌注后取全腦剝離TCC部位采用IHC染色檢測CGRP、CRLR、RAMP1陽性表達,余18只直接取全腦剝離TCC部位并平分兩份,一份組織采用Real-Time PCR檢測CGRP、 CRLR、RAMP1mRNA表達,另一份組織采用Western-Blot檢測CGRP.CRLR.RAMP1蛋白表達。 實驗結果(1)Ⅰ、Ⅱ組大鼠行為學評分比較：造模前30min內,各組大鼠行為學無顯著性差異。造模后30min內,與造模前及KB組比較,各組大鼠行為學評分均明顯升高(P0.05),說明動物造模成功。給藥后30min內,與同期M組比較,芎麻湯藥效部位各劑量組、Y組行為學評分明顯降低(P0.05)。(2)Ⅰ組大鼠：(①血漿ELISA:與KB組比較,M組大鼠血漿中CGRP、5-HT、NF-κB濃度顯著升高(P0.01)。與M組比較,Z、G組,Y組血漿中CGRP濃度顯著降低(P0.01),D組明顯降低(P0.05)；芎麻湯藥效部位各劑量組、Y組5-HT顯著降低(P0.01); NF-κB在各組之間無顯著性差異(P0.05)。②TCC部位IHC染色：SABC法檢測發(fā)現(xiàn),各組大鼠TCC部位均可見胞體被染成棕色的CGRP、5-HT、NF-κB陽性細胞。與KB組比較,M組大鼠TCC內CGRP免疫陽性染色平均光密度值顯著升高(P0.01);5-HT、NF-κB明顯升高(P0.05)。與M組比較,芎麻湯藥效部位各劑量組、Y組TCC內CGRP免疫陽性染色平均光密度值顯著降低(P0.01)；Z、G組5-HT明顯降低(P0.05),Y組顯著降低(P0.01)；Z組NF-κB明顯降低(P0.05)。(3)Ⅱ組大鼠：①IHC染色：SABC法檢測發(fā)現(xiàn),各組大鼠TCC部位均可見胞體被染成棕色的CGRP、CRLR、RAMP1陽性細胞。與KB組比較,M組大鼠TCC內CGRP、CRLR、RAMP1免疫陽性細胞數(shù)顯著升高(P0.01)。與M組比較,芎麻湯藥效部位各劑量組TCC內CGRP免疫陽性細胞數(shù)顯著降低(P0.01),Y組明顯降低(P0.05)：Z組CRLR顯著降低(P0.01),D、Y組明顯降低(P0.05),G組無顯著性差異(P0.05)；芎麻湯藥效部位各劑量組、Y組RAMP1顯著降低(P0.01)。②mRNA表達：Rea1-Time PCR檢測發(fā)現(xiàn),各組大鼠TCC部位均可見CGRP、CRLR、 RAMP1mRNA的表達。與KB組比較,M組TCC內的CGRP、RAMP1mRNA的表達量明顯升高(P0.05); CRLR顯著降低(P0.01)。與M組比較,芎麻湯藥效部位各劑量組TCC內CGRP mRNA的表達量均顯著降低(P0.01),Y組明顯降低(P0.05)；G組CRLR顯著升高(P0.01)；D、Y組RAMP1顯著降低(P0.01),Z、G組明顯降低(P0.05)。③蛋白表達：SDS-PAGE凝膠電泳檢測發(fā)現(xiàn),各組大鼠TCC內均可見CGRP、CRLR、RAMP1蛋白表達,且在各組之間存在明顯差異。與KB組比較,M組TCC內CGRP蛋白表達下降；CRLR、RAMP1明顯下降。與M組比較,芎麻湯藥效部位各劑量組TCC內CGRP蛋白表達明顯下降；Z、G組CRLR蛋白表達明顯升高；芎麻湯藥效部位各劑量組RAMP1蛋白表達明顯升高。 最終結論(1)芎麻湯藥效部位降低NTG型偏頭痛大鼠外周血中CGRP、5-HT濃度,從而改善偏頭痛發(fā)作時血管活性物質和神經(jīng)遞質水平失常達到緩解偏頭痛癥狀。(2)芎麻湯藥效部位下調NTG型偏頭痛大鼠TCC內CGRP、5-HT、NF-κB表達,從而改善偏頭痛發(fā)作時三叉神經(jīng)血管系統(tǒng)激活后引起的神經(jīng)源性炎癥反應,達到緩解偏頭痛癥狀。但與芎麻湯藥效部位治療偏頭痛相關性最強的指標為CGRP。(3)芎麻湯藥效部位通過降低NTG型偏頭痛大鼠TCC內CGRP、CRLR、RAMP1免疫陽性細胞數(shù)和下調CGRP、RAMP1mRNA表達來下降偏頭痛發(fā)作時三叉神經(jīng)血管系統(tǒng)激活后RAMP1過表達引起的三叉神經(jīng)節(jié)對CGRP高度敏感現(xiàn)象,通過下調CGRP蛋白表達,上調CRLR、RAMP1蛋白表達,進一步導致CGRP釋放減少、表達下降,干擾CGRP的正負反饋回路,最終實現(xiàn)治療偏頭痛的作用原理。
[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.
【學位授予單位】：湖南中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R277.7

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