Bejing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, 100029, Beijing, China.
College of Traditional Chinese Medicine, Beijing University of Chinese Medicine, 100029, Beijing, China.
J Ethnopharmacol. 2023 Dec 5;317:116781. doi: 10.1016/j.jep.2023.116781. Epub 2023 Jun 12.
Shaoyao Gancao Decoction (SGD) is well known as an effective prescription for analgesia composed of two herbs, and is noted as traditional Chinese medicine morphine. It is widely used in various conditions causing pain, including migraine. However, there is currently no research exploring the mechanism of action in the treatment of migraines.
The current research was devised to determine the underlying regulatory mechanism of SGD, by verifying its role in the NGF/TRPV1/COX-2 signal pathway.
The active components in SGD were identified by UHPLC-MS. A migraine model was prepared by subcutaneous (s.c.) injection of nitroglycerin (NTG) into the neck to detect migraine-like behavior, orbital hyperalgesia threshold changes, and the therapeutic effect of SGD. The mechanism of SGD in remedying migraine was studied through transcriptome sequencing (RNA-seq), which was further validated utilizing Elisa, Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) experiments.
In the SGD chemical composition analysis, 45 components were identified including gallic acid, paeoniflorin and albiforin. In the behavioral experiments, SGD treatment significantly decreased the score of migraine-like head scratching in the NTG-induced migraine model (Mod) rats, while the hyperalgesia threshold increased outstandingly on days 10, 12, and 14 (P < 0.01, P < 0.001 or P < 0.0001). In migraine biomarkers experiment, compared with the Mod group, the 5-hydroxytryptamine (5-HT) contents were outstandingly enhanced by SGD treatment, while nitric oxide (NO) contents were markedly declined (P < 0.01). In the RNA-seq test, the down-regulated genes of SGD inhibiting hyperalgesia migraine included the neurotrophic factor (NGF) and transient receptor potential vanillic acid subfamily protein 1 receptor (TRPV1). The down-regulation pathway is the inflammatory mediator regulation of TRP channels. In gene set enrichment analysis (GSEA), SGD decreased the over-expression of protooncogene tyrosine-protein kinase Src (SRC) and TRPV1 in this pathway, and the two genes clustered at its lower end, with similar functions. PPI network results show that NGF interacts with TRPV1. Further verification shows that when compared with Mod group, the plasma cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) protein expression levels and the dura mater calcitonin gene-related peptide (CGRP), extracellular signal-regulated kinase (ERK), p-ERK, SRC and NGF protein expression levels in the SGD group were remarkably decreased (P < 0.01, P < 0.001 or P < 0.0001), and the expression level of TRPV1 protein showed a downward trend (P = 0.06). The expression levels of COX-2, NO, CGRP, TRPV1, SRC and NGF mRNA in the dura mater was overtly down-regulated (P < 0.05, P < 0.01 or P < 0.001).
SGD has a significant inhibitory effect on the NGF/TRPV1/COX-2 signaling pathway that mediates central hyperalgesia migraine, thus suggesting the molecular mechanism of SGD in improving the symptoms of migraine may be related to the central hyperalgesia neurotransmitter that regulates the pathogenesis of migraine.
芍药甘草汤（SGD）是一种由两味草药组成的有效止痛处方，被誉为中药吗啡。它被广泛用于各种引起疼痛的情况，包括偏头痛。然而，目前尚无研究探索其在偏头痛治疗中的作用机制。
本研究旨在通过验证 SGD 在神经生长因子（NGF）/瞬时受体电位香草酸亚型 1 受体（TRPV1）/环氧化酶-2（COX-2）信号通路中的作用，确定 SGD 的潜在调节机制。
通过 UHPLC-MS 鉴定 SGD 的活性成分。通过颈部皮下（s.c.）注射硝化甘油（NTG）制备偏头痛模型，以检测偏头痛样行为、眶内痛觉阈值变化以及 SGD 的治疗效果。通过转录组测序（RNA-seq）研究 SGD 纠正偏头痛的机制，进一步通过 Elisa、逆转录定量聚合酶链反应（RT-qPCR）和 Western blot（WB）实验进行验证。
在 SGD 化学成分分析中，鉴定出 45 种成分，包括没食子酸、芍药苷和白芍苷。在行为学实验中，SGD 治疗显著降低了 NTG 诱导的偏头痛模型（Mod）大鼠偏头痛样头部搔抓的评分，同时在第 10、12 和 14 天痛觉阈值明显升高（P<0.01、P<0.001 或 P<0.0001）。在偏头痛生物标志物实验中，与 Mod 组相比，SGD 治疗显著增强了 5-羟色胺（5-HT）含量，而显著降低了一氧化氮（NO）含量（P<0.01）。在 RNA-seq 测试中，抑制偏头痛高敏性的 SGD 下调基因包括神经生长因子（NGF）和瞬时受体电位香草酸亚型 1 受体（TRPV1）。下调通路是炎症介质对 TRP 通道的调节。在基因集富集分析（GSEA）中，SGD 降低了该通路中原癌基因酪氨酸蛋白激酶Src（SRC）和 TRPV1 的过表达，并且这两个基因在其下游聚类，具有相似的功能。PPI 网络结果表明 NGF 与 TRPV1 相互作用。进一步验证表明，与 Mod 组相比，SGD 组血浆环氧化酶-2（COX-2）、前列腺素 E2（PGE2）蛋白表达水平和硬膜促甲状腺素释放激素相关肽（CGRP）、细胞外信号调节激酶（ERK）、p-ERK、Src 和 NGF 蛋白表达水平显著降低（P<0.01、P<0.001 或 P<0.0001），而 SGD 组 TRPV1 蛋白表达呈下降趋势（P=0.06）。硬膜 COX-2、NO、CGRP、TRPV1、Src 和 NGF mRNA 的表达水平明显下调（P<0.05、P<0.01 或 P<0.001）。
SGD 对介导中枢性偏头痛高敏性的 NGF/TRPV1/COX-2 信号通路具有显著的抑制作用，提示 SGD 改善偏头痛症状的分子机制可能与调节偏头痛发病机制的中枢性高敏性神经递质有关。
