Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of Pharmacy, Nanjing University of Chinese Medicine, No.138 Xianlin Avenue, Qixia District, Nanjing, Jiangsu 210023, China.
Department of Pathogen Biology, School of Medicine and Holistic Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Phytomedicine. 2023 Mar;111:154657. doi: 10.1016/j.phymed.2023.154657. Epub 2023 Jan 11.
Cimifugin is one of the main bioactive components of Yu-Ping-Feng-San, a well-known traditional Chinese medicine, which can effectively relieve Allergic asthma (AA) and atopic dermatitis and reduce recurrence in clinic. However, the underlying mechanism of cimifugin on AA is still unknown.
In the present study, we aimed to investigate the effect and mechanism of cimifugin on AA.
In vivo and in vitro experimental studies were performed.
The effect of cimifugin on AA was demonstrated in vivo and in vitro. Sepiapterin reductase (SPR) was predicted as the most potent target of cimifugin in treating AA by reverse docking. Molecular docking and microscale thermophoresis (MST) were used to analyze the direct binding between cimifugin and SPR. Overexpression and interference of SPR were performed to verify whether targeting SPR is a key step of cimifugin in the treatment of AA. QM385, an inhibitor of SPR, was administrated in vivo and in vitro to evaluate the role of SPR in AA. Further, HPLC and cell-free direct hSPR enzyme activity assay were performed to research whether cimifugin regulated SPR by influencing the enzyme activity. Simultaneously, the inhibitors of protein degradation were used in vitro to explore the mechanism of cimifugin on SPR.
We found cimifugin effectively alleviated AA by reducing airway hyperresponsiveness, inhibiting type 2 cytokines-mediated airway inflammation, and restoring the expression of epithelial barrier proteins. Molecular docking predicted the direct binding ability of cimifugin to SPR, which was further verified by MST. Notably, the therapeutic effect of cimifugin on AA was dampened with SPR interfering, in contrast, the phenotypic features of AA were significantly alleviated with QM385 application both in vivo and in vitro. Interestingly, cimifugin showed no effect on the enzyme activity of SPR, as the level of its substrate sepiapterin was not affected with cimifugin treatment by cell-free enzyme activity assay. Furthermore, we found cimifugin could reduce SPR protein expression without affecting its mRNA expression probably through autophagosome pathway.
To our knowledge, we're reporting for the first time that cimifugin can suppresses type 2 airway inflammation to alleviate AA by directly binding to SPR and regulating its protein expression in a non-enzymatic manner.
毛蕊异黄酮是一种著名的中药-玉屏风散的主要生物活性成分之一，它可以有效缓解过敏性哮喘（AA）和特应性皮炎，并降低临床复发率。然而，毛蕊异黄酮治疗 AA 的潜在机制尚不清楚。
本研究旨在探讨毛蕊异黄酮对 AA 的作用及机制。
进行了体内和体外实验研究。
在体内和体外研究中观察了毛蕊异黄酮对 AA 的作用。通过反向对接预测蝶呤还原酶（SPR）为毛蕊异黄酮治疗 AA 的最有效靶点。采用分子对接和微量热泳动（MST）分析毛蕊异黄酮与 SPR 的直接结合。过表达和干扰 SPR 以验证靶向 SPR 是否是毛蕊异黄酮治疗 AA 的关键步骤。在体内和体外给予 SPR 抑制剂 QM385 来评估 SPR 在 AA 中的作用。进一步通过 HPLC 和无细胞直接 hSPR 酶活性测定来研究毛蕊异黄酮是否通过影响酶活性来调节 SPR。同时，在体外使用蛋白降解抑制剂来探讨毛蕊异黄酮对 SPR 的作用机制。
我们发现毛蕊异黄酮通过降低气道高反应性、抑制 2 型细胞因子介导的气道炎症和恢复上皮屏障蛋白的表达，有效缓解 AA。分子对接预测了毛蕊异黄酮与 SPR 的直接结合能力，这进一步通过 MST 得到了验证。值得注意的是，在 SPR 干扰时，毛蕊异黄酮对 AA 的治疗效果减弱，而在体内和体外应用 QM385 时，AA 的表型特征明显缓解。有趣的是，毛蕊异黄酮对 SPR 的酶活性没有影响，因为细胞游离酶活性测定中毛蕊异黄酮处理后其底物蝶呤的水平没有变化。此外，我们发现毛蕊异黄酮可以降低 SPR 蛋白表达，而不影响其 mRNA 表达，这可能是通过自噬体途径实现的。
据我们所知，这是首次报道毛蕊异黄酮可以通过直接与 SPR 结合并以非酶促方式调节其蛋白表达来抑制 2 型气道炎症，从而缓解 AA。
