| Background:Migraine is an episodic, pulsating, mostly laterality, moderate-to-severe primary headache, which is often accompanied by nausea, vomiting, photophobia, phonophobia and visual, sensory, speech, movement defect or irritation. It is a common clinical chronic neurovascular disorders. Migraine prevalence in different countries is not same. The prevalence of women is roughly3.3%to32.6%, and the males’prevalence is roughly0.7%to16.1%[1]. It attacks frequently, and seriously affects patient’s physical and mental health, life, work and study [2]; and it also brings a heavy economic burden to the country and patients [3]. The World Health Report2001, released by the World Health Organization (WHO), arranged the common diseases according to years lived with disability (YLD), and migraine was arranged for the19th. It thought severe migraine was one of the most disabling chronic diseases, and was similar to dementia, quadriplegia and severe mental illness [4].Tibetan medicine is one of the treasures of the Chinese medicine. After the accumulation of experience in medical practice and the sublimation of the numerous Tibetan medical scientists for thousands of years, it gradually forms a set of unique theory system of diagnosis and treatment of migraine. Due to rich clinical experience and exact curative effect in the treatment of migraine, Ershiwuwei Shanhuwan is very worthy of promotion and application. However, at present the research work of Ershiwuwei Shanhuwan in the treatment of migraine mainly focus on the clinical field, and basic research is basically a blank state, especially the modern pharmacology and pharmacodynamics research did not carry out. At the same time, the modern medical pathogenesis of migraine is not clear, and various theories have different limitations, they can only explain the pathogenesis of migraine on the one hand. Therefore, in order to explore the mechanism and further dig the advantage of Ershiwuwei Shanhuwan in the treatment of migraine, it is necessary to carry out this research work.Methods:1. Effect of Ershiwuwei Shanhuwan on the central pain of miceA total of80female Kunming mice of SPF level were used. Put them on a hot plate nociceptive tester, and recorded the first licking foot or jump time of each mouse (in seconds). The mice whose first licking foot or jump time was less than5seconds, and more than30seconds were abandoned.64qualified mice were randomly divided into eight groups. These groups were blank group, Ershiwuwei Shanhuwan low dose group, Ershiwuwei Shanhuwan middle dose group, Ershiwuwei Shanhuwan high dose group, Zhengtianwan group, flunarizine hydrochloride capsule group, Ershiwuwei Shanhuwan plus Zhengtianwan group and Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule group. The blank group was given normal saline one time per day and the administration groups were administered one time per day. All of them were continuously lavaged for ten days. After one and two hour of the last administration, put the mice on a hot plate nociceptive tester, and recorded the first licking foot or jump time of each mouse (in seconds). If the time was more than60seconds, immediately stopped the test and the final time was recorded as60seconds. 2. Effect of Ershiwuwei Shanhuwan on the peripheral pain of miceA total of80Kunming mice of SPF level, half male and half female, were randomly divided into eight groups. These groups were similar with the effect of Ershiwuwei Shanhuwan on the central pain of mice. The blank group was given normal saline one time per day and the administration groups were administered one time per day. All of them were continuously lavaged for ten days. After one hour of the last administration, each mouse was intraperitoneally injected0.6%acetic acid solution at the dose of O.lml/lOg. The writhing number and first writhing time of each mouse were record in the15minutes.3. Effect of Ershiwuwei Shanhuwan on NO, NOS, Mg2+and ET in plasma of rats with migraineA total of54SD rats of SPF level, half male and half female, were randomly divided into nine groups. These groups were blank group, model group, Ershiwuwei Shanhuwan low dose group, Ershiwuwei Shanhuwan middle dose group, Ershiwuwei Shanhuwan high dose group, Zhengtianwan group, flunarizine hydrochloride capsule group, Ershiwuwei Shanhuwan plus Zhengtianwan group and Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule group. The blank group and model group were given normal saline one time per day and the administration groups were administered one time per day. All of them were continuously lavaged for ten days. After0.5hour of the last administration, the blank group was subcutaneously injected the same volume of saline, and the other groups were subcutaneously injected nitroglycerin injection at the dose of10mg/kg to establish the migraine rat model. The appearance and behavior change of each rat were observed. After4hours of the last administration, each group rats were anaesthetized by10%chloral hydrate solution, and abdominal aortic blood was collected by vacuum blood tube. Abdominal aortic blood was mixed and centrifuged, and then the plasma was separated from it and stored in the refrigerator. The NO and NOS in plasma of rats with migraine were tested by nitrate reductase method. The Mg2+in plasma of rats with migraine was tested by spectrophotometry. The ET in plasma of rats with migraine was tested by enzyme-linked immunosorbent assay.4. Effect of Ershiwuwei Shanhuwan on c-Fos protein in mesencephalon and hypothalamus of rats with migraineA total of54SD rats of SPF level, half male and half female, were randomly divided into nine groups. These groups were similar with the effect of Ershiwuwei Shanhuwan on NO, NOS, Mg2+and ET in plasma of rats with migraine. The blank group and model group were given normal saline one time per day and the administration groups were administered one time per day. All of them were continuously lavaged for ten days. After0.5hour of the last administration, the blank group was subcutaneously injected the same volume of saline, and the other groups were subcutaneously injected nitroglycerin injection at the dose of10mg/kg to establish the migraine rat model. The appearance and behavior change of each rat was observed. After4hours of the last administration, each group rats were anaesthetized by10%chloral hydrate solution. The rats were perfused through heart by PBS contained4%paraformaldehyde. The brains of rats were removed intact and fixed with PBS contained4%paraformaldehyde for24to48hours. Then removed the mesencephalon and hypothalamus and made paraffin sections at4μm. HE staining was used to observed wether the organizational structure was broken. The c-Fos protein in mesencephalon and hypothalamus was tested by immunohistochemical method.5. Statistical analysisStatistical software SPSS17.0is used to analyze the experimental data, and the test results are expressed as mean±standard deviation (x±s). One-way ANOVA is used to analyze pairwise comparisons between various sets of data. When the variance is homogeneous, Least-significance difference(LSD)is applied to test, otherwise Dunnett T3method is used. A=0.05is the significance level of test. When the P value is less than0.05, the test results are statistical significance.Results:1. Effect of Ershiwuwei Shanhuwan on the central pain of miceAfter1hour and2hours of the last administration, the first licking foot or jump time of the administration groups, in addition to the Ershiwuwei Shanhuwan low dose group, compared with the blank group have statistical significance (P<0.05or P<0.01). After1hour and2hours of the last administration, the first licking foot or jump time of the administration groups compared with the Ershiwuwei Shanhuwan middle dose group, the Ershiwuwei Shanhuwan high dose group and Ershiwuwei Shanhuwan plus Zhengtianwan group have statistical significance (P<0.05) However, the Zhengtianwan group, flunarizine hydrochloride capsule group and Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule group have no statistical significance (P>0.05). After1hour of the last administration, the first licking foot or jump time of the Ershiwuwei Shanhuwan low dose group compared with the Ershiwuwei Shanhuwan middle dose group has no statistical significance (P>0.05). However, after2hours of the last administration, it has statistical significance (P<0.05)2. Effect of Ershiwuwei Shanhuwan on the peripheral pain of miceThe writhing number and first writhing time in15minutes of the administration groups, in addition to the Ershiwuwei Shanhuwan low dose group, compared with the blank group have statistical significance (P<0.05or P<0.01). The writhing number and first writhing time in15minutes of the administration groups compared with the Ershiwuwei Shanhuwan middle dose group, the Ershiwuwei Shanhuwan high dose group and Ershiwuwei Shanhuwan plus Zhengtianwan group have statistical significance(P<0.05or P<0.01). However, the Zhengtianwan group and flunarizine hydrochloride capsule group have no statistical significance (P>0.05). The writhing number in15minutes of the Ershiwuwei Shanhuwan low dose group compared with the Ershiwuwei Shanhuwan middle dose group has no statistical significance (P>0.05). However, the first writhing time has statistical significance (P<0.05). The writhing number in15minutes of the Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule group compared with the Ershiwuwei Shanhuwan middle dose group has statistical significance(P<0.05). However, the first writhing time has no statistical significance (P>0.05)3. Effect of Ershiwuwei Shanhuwan on NO, NOS, Mg2+and ET in plasma of rats with migraineAfter all the groups were subcutaneously injected the saline or nitroglycerin injection, the blank group’s ears didn’t turn red and the phenomenon of scratching head and pawing cage could only be seen occasionally. However, the other groups’ ears turn a little red and the phenomenon of scratching head and pawing cage becomes a little more. Especially, the phenomenon of scratching head and pawing cage of the model group is very frequently. The phenomenon of scratching head and pawing cage lasts about1.5to3hours, and then accidie, curled up and reduced activity could only be seen.The level of NO, NOS, Mg2+and ET in plasma of all the groups, in addition to the Ershiwuwei Shanhuwan low dose group, compared with the model group have statistical significance (P<0.05or P<0.01). The level of NO, NOS, Mg2+and ET in plasma of the administration groups compared with the Ershiwuwei Shanhuwan middle dose group, the Ershiwuwei Shanhuwan plus Zhengtianwan group has statistical significance (P<0.05or P<0.01). However, the Ershiwuwei Shanhuwan low dose group, Zhengtianwan group and flunarizine hydrochloride capsule group have no statistical significance (P>0.05). The level of NO and ET in plasma of the Ershiwuwei Shanhuwan high dose group compared with the Ershiwuwei Shanhuwan middle dose group has statistical significance (P<0.05or P<0.01). However, the level of NOS and Mg2+in plasma of the Ershiwuwei Shanhuwan high dose group has no statistical significance (P>0.05). The level of NO, NOS and Mg2+in plasma of Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule group compared with the Ershiwuwei Shanhuwan middle dose group has statistical significance (P<0.05). However, the level of ET in plasma of the Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule group has no statistical significance (P>0.05)4. Effect of Ershiwuwei Shanhuwan on c-Fos protein in mesencephalon and hypothalamus of rats with migraineThe appearance and behavior change of each rat were similar with the effect of Ershiwuwei Shanhuwan on NO, NOS, Mg2+and ET in plasma of rats with migraine.The total number and total area of positive cells in mesencephalon and hypothalamus and integral optical density of positive cells in hypothalamus of all the groups, in addition to the Ershiwuwei Shanhuwan low dose group, compared with the model group have statistical significance (P<0.05or P<0.01). The integral optical density of positive cells in mesencephalon of all the groups, in addition to the Ershiwuwei Shanhuwan low dose group and flunarizine hydrochloride capsule group, compared with the model group have statistical significance (P<0.05or P<0.01) The total number, total area and integral optical density of positive cells in mesencephalon of the administration groups compared with the Ershiwuwei Shanhuwan middle dose group, the Ershiwuwei Shanhuwan plus Zhengtianwan group and Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule group have statistical significance (P<0.05or P<0.01). However, the Ershiwuwei Shanhuwan low dose group, Ershiwuwei Shanhuwan high dose group, Zhengtianwan group and flunarizine hydrochloride capsule group have no statistical significance (P>0.05). The total number, total area and integral optical density of positive cells in hypothalamus of the administration groups, compared with the Ershiwuwei Shanhuwan middle dose group, the Ershiwuwei Shanhuwan plus Zhengtianwan group and Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule group have statistical significance (P<0.05or P<0.01). However, the Ershiwuwei Shanhuwan low dose group, Zhengtianwan group and flunarizine hydrochloride capsule group have no statistical significance (P>0.05). The total number and total area in hypothalamus of the Ershiwuwei Shanhuwan high dose group compared with the Ershiwuwei Shanhuwan middle dose group has statistical significance (P<0.05). However, the integral optical density of positive cells in hypothalamus has no statistical significance (P>0.05)Conclusion:1. Ershiwuwei Shanhuwan can reduce the central and peripheral pain of mice. It can lower the level of NO, NOS and ET in plasma and elevate the level of Mg2+in plasma of rats. It also can lower the expression of c-Fos protein in mesencephalon and hypothalamus of rats with migraine. However, the dose-effect relationship is not obvious.2. The effect of Ershiwuwei Shanhuwan, Zhengtianwan and flunarizine hydrochloride capsule on central and peripheral pain of mice has no significant difference. The effect of Ershiwuwei Shanhuwan, Zhengtianwan and flunarizine hydrochloride capsule on NO, NOS, Mg2+and ET in plasma and c-Fos protein in mesencephalon and hypothalamus of rats with migraine also has no significant difference.3. The effect of Ershiwuwei Shanhuwan plus Zhengtianwan on central and peripheral pain of mice compared with Ershiwuwei Shanhuwan is obvious. The effect of Ershiwuwei Shanhuwan plus Zhengtianwan on NO, NOS, Mg2+and ET in plasma and c-Fos protein in mesencephalon and hypothalamus of rats compared with Ershiwuwei Shanhuwan is also obvious. The effect of Ershiwuwei Shanhuwan plus Flunarizine Hydrochloride Capsule on NO, NOS and Mg2+in plasma and c-Fos protein in mesencephalon and hypothalamus of rats compared with Ershiwuwei Shanhuwan is obvious. |
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