State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, 300353, PR China.
State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Key Laboratory of Quality Markers of Traditional Chinese Medicine, Tianjin Institute of Pharmaceutical Research, Tianjin, 300462, PR China.
J Ethnopharmacol. 2024 Aug 10;330:118217. doi: 10.1016/j.jep.2024.118217. Epub 2024 Apr 17.
The natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive.
The objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel.
The therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches.
The administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca influx through the channel opening and ultimately relieving pain.
Lig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.
天然阿片样物质 Ligustilide（Lig）源自当归（Angelica sinensis（Oliv.）Diels）和川芎（Ligusticum chuanxiong Hort.），传统上用于治疗痛经、偏头痛和类风湿性关节炎疼痛的镇痛特性。尽管已有关于 TRP 通道与 Lig 的镇痛作用之间相关性的报道，但对其潜在作用机制仍知之甚少。
本研究旨在阐明 Lig 对镇痛靶标 TRPA1 通道的作用机制。
在大鼠急性软组织损伤模型中评估 Lig 的治疗效果。通过在动物水平上竞争性抑制 TRP 通道激动剂来确定镇痛靶标，然后在过表达 TRP 蛋白的活细胞上进行 Fluo-4/Ca 成像。通过胶内成像、用 Lig 衍生的分子探针进行共定位以及药物亲和力反应靶标稳定性测定来验证潜在靶标。通过蛋白质谱鉴定 Lig 的结合位点，并进一步通过分子对接、定点突变和多学科方法进行分析。
Lig 的给药可有效改善软组织损伤大鼠的疼痛，并减轻氧化应激和炎症反应。此外， Lig 的镇痛作用特异性归因于 TRPA1。机制研究表明， Lig 通过与 TRPA1 的预 S1 区连接域相互作用直接激活 TRPA1。通过代谢转化，6,7-环氧当归内酯（EM-Lig）在高浓度和延长暴露时间下与 TRPA1 的 Cys703 形成共价键。这种不可逆的结合阻止内源性亲电产物进入 TRPA1 的配体结合口袋的半胱氨酸活性中心，从而通过通道开放抑制 Ca 内流，最终缓解疼痛。
Lig 通过非亲电性/亲电性代谢转化以双模态方式选择性调节 TRPA1 通道。氧化代谢中间体 EM-Lig 通过不可逆抑制感觉神经元中 TRPA1 的激活发挥镇痛作用。这些发现不仅突出了 Lig 的镇痛机制，还为在预 S1 区域开发 TRPA1 拮抗剂提供了新的亲核攻击位点。
