| Purpose To explore the bioactive parts of Xiongma Tang (parts of ethyl acetate and n-butanol extract) impact CGRP,5-HT, and NF-kappa B in plasma and trigeminocervical complex on animal model of migraine with nitroglycerin, then determine the correlation of the bioactive parts of Xiongma Tang and each index; To explore the bioactive parts of Xiongma Tang impact the CGRP-CRLR/RAMP1signal transduction pathway, finally hope to clarify the therapeutic mechanism of it on migraine.Methods Using different polar organic solvents (petroleum ether, ethyl acetate, butanol) gradient extraction of Xiong Ma Tang, had the effective parts (ethyl acetate and n-butanol parts).96SD male rats were randomly divided into two groups (group I60, group Ⅱ36. Each big group including blank control group, model control group, the low, medium and high dose of the bioactive parts of Xiongma Tang group, positive control group). Except blank control and model control groups, the rest groups pretreatment after7d with corresponding drugs, then to copy the model of migraine by injection of NTG (10mg/kg). After making model30,120min, and intragastric administration for two times, to observe rat scratching, climbing cage, tail biting, reciprocating movement and so on, to judge the model are successful or not. After ether anesthesia, all rats in group I were taking the external jugular vein blood to detection the content of CGRP,5-HT, NF-kappa B in plasma with ELISA, and the expression of CGRP,5-HT, NF-kappa B on TCC with immunohistochemistry after the heart perfusion. All rats in group II were anesthetized with diethyl ether, and of which only18rats were observed the positive expression of CGRP, CRLR, RAMP1on TCC with immunohistochemistry after the heart perfusion. The remaining18rats directly from the whole brain stripping TCC site and divide two parts, detection of CGRP, CLR, RAMP1mRNA expression in a tissue by Real-Time PCR, detected CGRP, CLR, RAMP1protein in the other tissue by Western-Blot.Results (1) The behavior score comparison of group Ⅰ and Ⅱ:Within30min before modeling, no differences in behavior. Within30min after modeling, compared with before modeling and blank control group, the behavioral score were significantly increased (P<0.05). And suggesting that all the animal were modeled successfully. Within30min after administration, compared with model control group, the bioactive parts of Xiongma Tang and Sumatriptan group was significantly reduced (P<0.05).(2) Rats in group1:①ELISA:compared with the blank control group, the plasma concentration of CGRP,5-HT, and NF-kappa B in model group were increased significantly (P<0.01). Compared with model control group, the plasma concentration of CGRP in the high and medium dose group and Sumatriptan group were decreased significantly (P<0.01), and low dose group was decreased (P<0.05);5-HT in each dose group and Sumatriptan group were significantly decreased (P<0.01). NF-kappa B in all rats were no significant difference (P>0.05).②IHC staining:each group were visible the positive cells brown of CGRP,5-HT, and NF-kappa B. Compared with the blank control group, the immunoreactive staining average optical density of CGRP in model group was significantly increased (P<0.01);5-HT, NF-kappa B were increased (P<0.05). Compared with model control group, CGRP in each dose group and Sumatriptan group were significantly reduced (P<0.01);5-HT in medium and high dose were decreased (P<0.05), Sumatriptan group decreased significantly (P<0.01); NF-kappa B in medium dose was decreased (P<0.05).(3) Rats in group Ⅱ:①IHC:each group were visible the positive cells brown of CGRP, CRLR and RAMP1. Compared with the blank control group, the number of CGRP, CRLR and RAMP1immunoreactive cells in model control group were significantly increased (P<0.01). Compared with model control group, CGRP in each dose group and Sumatriptan group were decreased (P<0.05); CRLR in middle dose group was decreased significantly (P<0.01), while low dose group and Sumatriptan group were decreased (P＜0.05), and the high dose was no significant difference (P>0.05); RAMP1in each dose group and Sumatriptan group were decreased significantly (P<0.01).②The expression of mRNA:each group were visible the expression of CGRP, CRLR and RAMP1mRNA. Compared with the blank control group, the expression of CGRP and RAMP1mRNA were increased (P<0.05); CRLR was significantly decreased (P<0.01). Compared with model control group, CGRP mRNA in each dose group were significantly decreased (P<0.01), and Sumatriptan group was decreased (P<0.05); CRLR in the high dose group was increased significantly (P＜0.01); RAM P1in low dose group and sumatriptan group was significantly decreased (P＜0.01), while the middle and high dose group were decreased (P<0.05).③The expression of protein:each group were visible the expression of CGRP, CRLR and RAMP1protein. Compared with the blank control group, the expression of CGRP, CRLR and RAMP1protein were decreased. Compared with model control, the expression of CGRP protein in each dose group were significantly decreased; the expression of CRLR protein in middle and high dose group were significantly increased; the expression of RAM PI protein in each dose group were significantly increased.Conclusions (1) The bioactive parts of Xiongma Tang reduced the plasma concentration of CGRP,5-HT on migraine model rats, thus relieve the symptoms of migraine by improving the vasoactive substances and neurotransmitters disorder.(2) The bioactive parts of Xiongma Tang regulated the expression of CGRP,5-HT, NF-kappa B, so as to improve the neurogenic inflammation reaction caused by activation of the trigeminovascular system after the migraine attack, relieve migraine symptoms. And the strongest correlation index with the bioactive parts of Xiongma Tang in treating migraine was CGRP.(3) Th bioactive parts of Xiongma Tang reduced the number of CGRP, CRLR and RAMP1immunoreactive cells, cut the expression of CGRP and RAMP1mRNA, thus decreased the phenomenon that trigeminal ganglion was highly sensitive to CGRP induced by the over expression of RAMP1; Cut the expression of CGRP protein and raised the expression of CRLR and RAMP1protein, and further lead to reduce CGRP release and expression, interference the positive and negative feedback loop of CGRP, to realize the treatment of migraine. |
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