背景:丹皮酚(PAE)和甘草酸(GLY)是PiantongtangNo.1,这是一种治疗慢性偏头痛的中药处方,副作用最小。丹皮酚和甘草酸均具有镇痛作用,单独的神经保护和抗炎特性。我们先前的研究强调了它们在改善偏头痛症状方面的联合作用(PAE+GLY)。然而,目前尚无任何研究探索PAE+GLY治疗偏头痛的作用机制。
目的:本研究旨在确定PAE+GLY改善大鼠复发性硝酸甘油样偏头痛表型的机制。
方法:通过颈部皮下注射使用硝酸甘油诱导的偏头痛模型,我们评估了PAE+GLY对偏头痛样症状的影响。采用行为测试和生物标志物分析,与转录组测序(RNA-seq)一起。利用逆转录定量PCR(RT-qPCR)进一步验证了机理见解,蛋白质印迹(WB),ELISA和免疫荧光(IF)技巧。
结果:用PAE+GLY治疗后,痛觉过敏阈值和5-羟色胺(5-HT)水平增加,和偏头痛样的头部抓挠,组胺和降钙素基因相关肽(CGRP)水平降低。RNA-Seq实验表明,PAEGLY上调谷氨酸脱羧酶2(GAD2)和γ-氨基丁酸B型受体亚基2(GABBR2)基因的表达。这种上调激活了GABA能突触通路,有效抑制偏头痛发作。进一步验证表明,PAE+GLY治疗后脑脊液中γ-氨基丁酸(GABA)含量增加,同时硬脑膜GAD2、GABBR2和瞬时受体电位通道M8(TRPM8)的表达增加。因此,这种抑制硬脑膜cAMP依赖性蛋白激酶催化亚基α(PRKACA)和瞬时受体电位通道1型(TRPV1)的表达,随后下调p-ERK1/2、p-AKT1、IL-1β和TNF-α。
结论:我们的发现强调了PAE+GLY通过上调抑制性神经递质和调节GABBR2/TRPM8/PRKACA/TRPV1通路改善炎性痛觉过敏偏头痛。
BACKGROUND: Paeonol (PAE) and glycyrrhizic acid (GLY) are predominate components of 14 blood-entering ones of Piantongtang No. 1, which is a traditional Chinese medicine prescription for chronic migraine with minimal side effects. Both paeonol and glycyrrhizic acid exhibit analgesic, neuroprotective and anti-inflammatory properties individually. Our previous research has highlighted their combined effect (PAE + GLY) in ameliorating migraine symptoms. However, there are not yet any studies exploring the mechanism of action of PAE + GLY in the treatment of migraine.
OBJECTIVE: This research aimed to determine the mechanism of PAE + GLY in ameliorating the recurrent nitroglycerin-induced migraine-like phenotype in rats.
METHODS: Using a nitroglycerin-induced migraine model via subcutaneous injection in the neck, we evaluated the effect of PAE + GLY on migraine-like symptoms. Behavioural tests and biomarkers analysis were employed, alongside transcriptome sequencing (RNA-seq). Mechanistic insights were further verified utilising reverse transcription quantitative PCR (RT-qPCR), Western blot (WB), ELISA and immunofluorescence (IF) techniques.
RESULTS: Following treatment with PAE + GLY, hyperalgesia threshold and 5-hydroxytryptamine (5-HT) levels increased, and migraine-like head scratching, histamine and calcitonin gene-related peptide (CGRP) levels were reduced. RNA-Seq experiments revealed that PAE + GLY upregulated the expression of Glutamate decarboxylase 2 (GAD2) and γ-aminobutyric acid type B receptor subunit 2 (GABBR2) genes. This upregulation activated the GABAergic synapse pathway, effectively inhibiting migraine attacks. Further validation demonstrated an increase in γ-aminobutyric acid (GABA) content in cerebrospinal fluid post PAE + GLY treatment, coupled with increased expression of dural GAD2, GABBR2 and transient receptor potential channel M8 (TRPM8). Consequently, this inhibited the expression of dural cAMP-dependent protein kinase catalytic subunit alpha (PRKACA) and transient receptor potential channel type 1 (TRPV1), subsequently downregulating p-ERK1/2, p-AKT1, IL-1β and TNF-α.
CONCLUSIONS: Our findings underscore that PAE + GLY ameliorates inflammatory hyperalgesia migraine by upregulating inhibitory neurotransmitters and modulating the GABBR2/TRPM8/PRKACA/TRPV1 pathway.