头痛宁对偏头痛大鼠硬脑膜及三叉神经节IL-1β表达影响

摘要

目的:研究头痛宁对反复发作性偏头痛大鼠硬脑膜及三叉神经节IL-1β(interleukin-1beta)表达的影响.方法:选择健康成年雄性SD大鼠60只,初始质量180~220 g.先取20只随机分为4组,每组5只,分别为生理盐水7天组(sham)、致炎剂(inflammatory soup,IS)1天组(IS1)、致炎剂3天组(IS3)和致炎剂7天组(IS7),建立反复发作性偏头痛大鼠模型;再取25只随机分为5组,每组5只,分别为致炎剂7天对照组(IS7)、0.375 g/kg头痛宁干预组(T1)、0.75 g/kg头痛宁干预组(T2)、1.5 g/kg头痛宁干预组(T3)和3.0 g/kg头痛宁干预组(T4),取硬脑膜及三叉神经节进行IL-1β蛋白免疫印迹分析(western blot)及IL-1βmRNA实时荧光定量PCR检测.最后取15只,随机分为3组,每组5只,分别为生理盐水7天组(sham)、致炎剂7天组(IS7)和3.0 g/kg头痛宁干预组(T4),灌流固定后各组分别取大鼠硬脑膜及三叉神经节进行IL-1β蛋白免疫荧光检测.结果:(1)与生理盐水7天组比较,致炎剂7天组IL-1β的表达在硬脑膜(P<0.01)及三叉神经节(P<0.01)均明显升高;(2)与致炎剂7天组相较,在大鼠硬脑膜/三叉神经节,0.75 g/kg头痛宁干预组(P<0.01/P<0.01)、1.5 g/kg头痛宁干预组(P<0.01/P<0.05)和3.0g/kg头痛宁干预组(P<0.01/P<0.01)IL-1β表达均明显降低,0.375 g/kg头痛宁干预组IL-1β表达下降不明显;(3)与致炎剂7天组相较,3.0 g/kg头痛宁干预组三叉神经节神经元细胞胞质内免疫荧光斑点数明显减少,大鼠硬脑膜未能成功染色;(4)与致炎剂7天组相较,3.0 g/kg头痛宁干预组在硬脑膜(P<0.01)及三叉神经节(P<0.05)IL-1βmRNA表达均减少.结论:头痛宁抑制硬脑膜及三叉神经节IL-1β基因转录,下调IL-1β的表达可能是其治疗偏头痛的机制之一.%Objective:To investigate the effect of Toutongning on the expression of IL-1β (interleukin-1beta) both in dura and trigeminal ganglion of the recurrent migraine rat model.Methods:Totally 60 healthy adult male SD rats were selected. Twenty SD rats were randomly divided into 7-day saline control group and 1, 3, 7-day inflammatory soup (IS) groups (5 in each group). The recurrent migraine rat model was created. Twenty-five adult male SD rats were randomized into 7-day IS control group and 0.375 g/kg, 0.75 g/kg, 1.5 g/kg, 3.0 g/kg Toutongning groups (5 in each group). Expression of IL-1β in dura matter and trigeminal ganglion of above was detected by Western blot and quantified realtime-PCR. At last, fifteen adult male SD rats were randomized into 7-day saline control group, 7-day IS control group and 3.0 g/kg Toutongning group. Expression of IL-1β in dura matter and trigeminal ganglion of this 3 groups was detected by immunofluorescence after being perfused with paraformaldehyde.Results:(1)IL-1β levels of 7-day IS group was significantly higher than that of saline control group both in dura (P < 0.01) and trigeminal ganglion (P< 0.01). (2) Compared with 7-day IS control group, the expression of IL-1β in dura/trigeminal ganglion sharply decreased in 0.75 g/kg (P < 0.01/P< 0.01), 1.5 g/kg (P < 0.01/P < 0.05) and 3.0 g/kg Toutongning group (P < 0.01/P < 0.01). However, there was no difference in 0.375 g/kg Toutongning group. (3) The number of fluorescence spots in the neuron plasma of 3.0 g/kg Toutongning group was greatly reduced compared with 7-day IS control group. The immunofluorescence of dura was unsuccessful. (4) The IL-1β mRNA levels of 3.0 g/kg Toutongning group was significantly lower than 7-day IS control group both in dura (P < 0.01) and trigeminal ganglion (P < 0.05). Conclusion:Toutongning may down-regulate the expression of IL-1β by repressing transcription which may be one of the underlying mechanisms for the treatment of migraine.