A型肉毒毒素预防小鼠慢性偏头痛发作的机制研究

Preventive treatment of nitroglycerin-induced chronic migraine by peripheral single injection of Botulinum Neurotoxin A in mice

目的评价A型肉毒毒素(BoNT/A)预防小鼠慢性偏头痛(CM)发作的疗效并探讨其分子机制。方法24只C57BL/6雄性小鼠按随机数字表法分为对照组(n=8)、模型组(n=8)、BoNT/A预防组(n=8)。后2组小鼠均于第1、3、5、7、9天腹腔注射10 mg/kg硝酸甘油(NTG)建立CM模型,BoNT/A预防组小鼠于首次注射NTG前1 h先注射0.18 U/100μL BoNT/A,对照组注射等剂量生理盐水。第1、3、5、7、9天采用von Frey纤维丝测痛仪检测小鼠足底和口面部机械痛阈的基础值及NTG注射后2 h诱发的急性机械痛阈值;第1、3、5、7、9天注射NTG后2 h采用转棒实验测试小鼠的运动功能;第9天采用Western blotting检测小鼠三叉神经节(TG)、三叉神经脊束核尾核(TNC)中降钙素基因相关肽(CGRP)、裂解突触小体相关蛋白25(SNAP25)、胶质纤维酸性蛋白(GFAP)、瞬时受体电位通道蛋白,TNC中NOD样受体蛋白3(NLRP3)炎性因子通路相关蛋白的表达;实时荧光定量PCR(PT-qPCR)检测小鼠TNC中NLRP3炎性因子通路相关mRNA的表达;免疫荧光染色检测小鼠TNC中CGRP的表达。结果(1)与对照组比较,模型组小鼠第7、9天口面部机械痛阈的基础值降低;与模型组比较,BoNT/A预防组小鼠第7、9天口面部机械痛阈的基础值增加,差异均有统计学意义(P<0.05)。与对照组比较,模型组小鼠第5、9天口面部急性机械痛阈值降低;与模型组比较,BoNT/A预防组小鼠第5、9天口面部急性机械痛阈值增加,差异均有统计学意义(P<0.05)。与对照组比较,模型组小鼠第3、5、7、9天足底机械痛阈的基础值和急性机械痛阈值均降低;与模型组比较,BoNT/A预防组小鼠第3、5、7、9天足底机械痛阈的基础值和急性机械痛阈值均增加,差异均有统计学意义(P<0.05)。(2)转棒实验结果显示,3组小鼠在转棒上跑动时间的差异无统计学意义(P>0.05)。(3)Western blotting检测结果显示,与对照组比较,模型组小鼠TG中CGRP、SNAP25蛋白的表达增加;与模型组比较,BoNT/A预防组小鼠TG中CGRP、SNAP25蛋白的表达降低,差异均有统计学意义(P<0.05)。与对照组比较,模型组小鼠TNC中CGRP蛋白的表达增加;与模型组比较,BoNT/A预防组小鼠TNC中CGRP蛋白的表达降低,差异均有统计学意义(P<0.05)。与对照组比较,模型组小鼠TNC中NLRP3蛋白的表达增加;与模型组比较,BoNT/A预防组小鼠TNC中NLRP3蛋白的表达降低,差异均有统计学意义(P<0.05)。(4)RT-qPCR检测结果显示,与对照组比较,模型组小鼠TNC中IL-1βmRNA的表达增加;与模型组比较,BoNT/A预防组小鼠TNC中IL-1βmRNA的表达降低,差异均有统计学意义(P<0.05)。(5)免疫荧光染色检测结果显示,与对照组比较,模型组小鼠TNC中CGRP的表达增加;与模型组比较,BoNT/A预防组小鼠TNC中CGRP的表达降低,差异均有统计学意义(P<0.05)。结论BoNT/A可以降低TG中SNAP25的表达,减少CGRP在TG和TNC的释放,预防CM的发作;BoNT/A具有调控TNC中NLRP3水平的作用。

Objective To evaluate the effect of botulinum neurotoxin A(BoNT/A)on prevention of chronic migraine(CM)in mice and explore the potential mechanism.Methods Twenty-four male C57BL/6 mice were randomly divided into control group,nitroglycerin(NTG)group,and BoNT/A+NTG group(n=8).Mice in the latter two groups were intraperitoneally injected with 10 mg/kg NTG on the 1^(st),3^(rd),5^(th),7th and 9^(th) d of experiments to establish CM models.Mice in the BoNT/A+NTG group were injected with 0.18 U/100μL BoNT/A one h before the first injection of NTG.Mice in the control group were injected with the same dose of normal saline.Basal mechanical withdrawal threshold(MWT)and evoked MWT 2 h after NTG in the facial and hindpaw regions on the 1^(st),3^(rd),5^(th),7th and 9^(th) d of experiments were evaluated by von Frey filament test.The motor function of mice 2 h after NTG injection was tested by rotarod test on the 1^(st),3^(rd),5^(th),7th and 9^(th) d of experiments.On 9^(th) d of experiments,the mice were sacrified;the calcitonin gene-related peptide(CGRP),synaptosomal-associated protein 25(SNAP25),glial fibrillary acidic protein(GFAP)and TRP channel protein expressions in the trigeminal ganglia(TG)and trigeminal nucleus caudalis(TNC),and NOD-like receptor protein 3(NLRP3)inflammatory factor pathway-related protein expressions in TNC were detected by Western blotting;real-time quantitative PCR(RT-qPCR)was used to detect the NLRP3 inflammatory factor pathway-related mRNA expressions in TNC.The CGRP expression in TNC was detected by immunofluorescent staining.Results(1)As compared with the control group,the NTG group had significantly decreased basal facial MWT on the 7th and 9^(th) d of experiments(P<0.05);as compared with the NTG group,the BoNT/A+NTG group had significantly increased basal facial MWT on the 7th and 9^(th) d of experiments(P<0.05).As compared with the control group,the NTG group had significantly decreased evoked facial MWT on the 5^(th) and 9^(th) d of experiments(P<0.05);as compared with the NTG group,the BoNT/A+NTG group had significantly increased evoked facial MWT on the 5^(th) and 9^(th) d of experiments(P<0.05).As compared with the control group,the NTG group had significantly decreased basal and evoked MWT in the hindpaw regions on the 3^(rd),5^(th),7th and 9^(th) d of experiments(P<0.05);as compared with the NTG group,the BoNT/A+NTG group had significantly increased basal and evoked MWT in the hindpaw regions on the 3^(rd),5^(th),7th and 9^(th) d of experiments(P<0.05).(2)There was no significant difference in running time on rotarod among the three groups(P>0.05).(3)Western blotting results showed that as compared with those in the control group,the CGRP and SNAP25 protein expressions were significantly increased in TG of the NTG group(P<0.05);and those in the BoNT/A+NTG group were significantly decreased as compared with those in the NTG group(P<0.05).As compared with those in the control group,the CGRP and NLRP3 protein expressions were significantly increased in TNC of NTG group(P<0.05);and those in the BoNT/A+NTG group were significantly decreased as compared with those in the NTG group(P<0.05).(4)RT-qPCR results showed that as compared with that in the control group,the IL-1βmRNA expression in TNC of the NTG group was significantly increased(P<0.05),and that in the BoNT/A prevention group was statistically decreased as compared with that in the NTG group(P<0.05).(5)Immunofluorescent staining results showed that as compared with that in the control group,the CGRP expression in TNC of the NTG group was significantly increased,and that in the BoNT/A+NTG group was significantly decreased as compared with that in the NTG group(P<0.05).Conclusion BoNT/A can reduce the SNAP25 expression in TG,reduce the CGRP release in TG and TNC,and prevent CM onset;BoNT/A can regulate NLRP3 level in TNC.