[关键词]
[摘要]
目的 观察愈风宁心滴丸对硝酸甘油诱导的偏头痛小鼠的影响,探索愈风宁心滴丸预防性治疗血管痉挛性偏头痛的作用及机制。方法 采用脂多糖(lipopolysaccharide,LPS)刺激BV2神经小胶质细胞,以愈风宁心滴丸(10、50、100 μmol/L)及葛根素(50 μmol/L)进行干预,收集细胞。将野生型C57BL/6J小鼠随机分成照组、模型组以及愈风宁心滴丸低、中、高剂量(200、360、600 mg/kg)组和葛根素组(37.8 mg/kg),给予药物进行干预,于第3、5、7、9、11天ip硝酸甘油溶液(10 mg/kg)建立小鼠偏头痛模型,第3天开始进行痛觉敏感(机械压痛和热敏耐受)行为学实验;实验结束后,取小鼠血清和脑组织。检测各组BV2细胞活性氧含量;采用Western blotting法检测各组细胞超氧化物歧化酶1(superoxide dismutase 1,SOD1)、SOD2蛋白表达以及各组小鼠三叉神经脊束尾核(trigeminal nucleus caudalis,TNC)区降钙素基因相关肽(calcitonin gene-related peptide,CGRP)、c-Fos、磷酸化细胞外信号调节激酶(phosphorylated extracellular regulated protein kinases,p-ERK)及促炎细胞因子白细胞介素-6(interleukin-6,IL-6)蛋白表达;采用qRT-PCR法检测各组细胞SOD1、SOD2、IL-1β、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)mRNA表达以及小鼠TNC区IL-1β、TNF-α mRNA表达;采用免疫荧光检测各组小鼠TNC区CGRP、c-Fos表达;采用生化分析仪检测各组小鼠血清中丙氨酸转氨酶(alanine aminotransferase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)和碱性磷酸酶(alkaline phosphatase,ALP)活性。结果 愈风宁心滴丸显著上调BV2细胞抗氧化酶SOD1、SOD2 mRNA和蛋白表达水平(P<0.05、0.01、0.001),降低活性氧水平(P<0.01、0.001),抑制炎症因子IL-1β和TNF-α mRNA表达(P<0.001)。与对照组相比,模型组小鼠基础痛阈值显著降低(P<0.05、0.001),TNC区中CGRP、c-Fos、p-ERK蛋白表达水平显著升高(P<0.01、0.001),TNC区IL-1β、TNF-α mRNA和IL-6蛋白表达水平显著升高(P<0.01);与模型组相比,各给药组小鼠基础痛阈值显著升高(P<0.05、0.01、0.001),TNC区中CGRP、c-Fos、p-ERK蛋白表达水平显著降低(P<0.05、0.01、0.001),TNC区IL-1β、TNF-α mRNA和IL-6蛋白表达水平显著降低(P<0.05、0.01、0.001),且呈剂量相关性;愈风宁心滴丸对偏头痛小鼠血清中ALT、AST和ALP活性无显著性影响。结论 愈风宁心滴丸可能通过抑制血管扩张、抑制炎症反应、促进抗氧化酶表达等多重途径发挥预防性治疗血管痉挛性偏头痛的作用。
[Key word]
[Abstract]
Objective To observe the effect of Yufeng Ningxin Dropping Pills (愈风宁心滴丸) on nitroglycerin-induced migraine mice, and explore the effect and mechanism of Yufeng Ningxin Dropping Pills on preventive treatment of vasospasm migraine. Methods Lipopolysaccharide (LPS) was used to stimulate BV2 neuroglial cells, Yufeng Ningxin Dripping Pills (10, 50, 100 μmol/L) and puerarin (50 μmol/L) were used for intervention, and cells were collected. Wild-type C57BL/6J mice were randomly divided into control group, model group, low-, medium-, and high-dose (200, 360, 600 mg/kg) Yufeng Ningxin Dripping Pill group and puerarin (37.8 mg/kg) group, drugs were given for intervention, migraine model of mice was established with ip nitroglycerin solution (10 mg/kg) on 3rd, 5th, 7th, 9th and 11th day, hyperalgesia tolerance behavioral experiment (mechanical tenderness and heat sensitivity) were started on 3rd day; After experiment, the serum and brain tissue of mice were taken. The content of reactive oxygen species in BV2 cells of each group was detected; Western blotting was used to detect the expressions of superoxide dismutase 1 (SOD1) and SOD2 protein in BV2 cells and calcitonin gene-related peptide (CGRP), c-Fos, phosphorylated extracellular regulated protein kinases (p-ERK) and pro-inflammatory cytokine interleukin-6 (IL-6) protein expressions in trigeminal nucleus caudalis (TNC) region in mice of each group; qRT-PCR method was used to detect SOD1, SOD2, IL-1β, tumor necrosis factor-α (TNF-α) mRNA expressions in BV2 cells and IL-1β and TNF-α mRNA expressions in TNC of mice; Immunofluorescence was used to detect the expressions of CGRP and c-Fos in TNC area of mice in each group; Automatic biochemical analyzer was used to detect the alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities in serum of mice. Results Yufeng Ningxin Dripping Pills significantly increased antioxidant enzyme SOD1 and SOD2 mRNA and protein expression levels in BV2 cells (P<0.05, 0.01, 0.001), reduced reactive oxygen species levels (P<0.01, 0.001), inhibited the inflammatory factor IL-1β and TNF-α mRNA expression (P<0.001). Compared with control group, basic pain threshold of mice in model group was significantly reduced (P<0.05, 0.001), expressions of CGRP, c-Fos and p-ERK proteins in TNC area were significantly increased (P<0.01, 0.001). The expressions of IL-1β, TNF-α mRNA and IL-6 protein in TNC area were significantly increased (P<0.01); Compared with model group, basic pain threshold of mice in each administration group were significantly increased (P<0.05, 0.01, 0.001), expressions of CGRP, c-Fos and p-ERK proteins in TNC area were significantly reduced (P<0.05, 0.01, 0.001), expressions of IL-1β, TNF-α mRNA and IL-6 proteins in TNC area were significantly reduced (P<0.05, 0.01, 0.001), and showed a dose-related relationship; Yufeng Ningxin Dripping Pills had no significant effect on activities of ALT, AST and ALP in serum of migraine mice. Conclusion Yufeng Ningxin Dripping Pills may play a preventive effect on vasospasm migraine by inhibiting vasodilation, inhibiting inflammation and promoting the expression of antioxidant enzymes.
[中图分类号]
R285.5
[基金项目]
安徽省重点研究和开发计划项目(201904a07020007);中国博士后科学基金资助项目(2020M681914)