Li Liang, Wu Xiaorong, Gong Jili, Wang Zhuqiang, Dai Weibo, Qiu Li, Zuo Hongyuan, Yi Mengqin, Yuan Hui, Hu Mei, Gao Zhaobing, Tian Fuyun
Pharmacology Laboratory, Zhongshan Traditional Chinese Medicine Hospital, Guangzhou University of Chinese Medicine, Zhongshan, China.
School of Pharmaceutical Sciences, Guizhou Medical University, Guiyang, China.
Front Pharmacol. 2024 May 23;15:1389768. doi: 10.3389/fphar.2024.1389768. eCollection 2024.
Huanglian Wendan Decoction (HWD) is a traditional Chinese medicine (TCM) prescribed to patients diagnosed with insomnia, which can achieve excellent therapeutic outcomes. As positively modulating the γ-aminobutyric acid (GABA) type A receptors (GABAARs) is the most effective strategy to manage insomnia, this study aimed to investigate whether the activation of GABAARs is involved in the anti-insomnia effect of HWD. We assessed the metabolites of HWD using LC/MS and the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and tested the pharmacological activity and using whole-cell patch clamp and insomnia zebrafish model. In HEK293 cells expressing α1β3γ2L GABAARs, HWD effectively increased the GABA-induced currents and could induce GABAAR-mediated currents independent of the application of GABA. In the LC-MS (QToF) assay, 31 metabolites were discovered in negative ion modes and 37 metabolites were found in positive ion modes, but neither three selected active metabolites, Danshensu, Coptisine, or Dihydromyricetin, showed potentiating effects on GABA currents. 62 active metabolites of the seven botanical drugs were collected based on the TCMSP database and 19 of them were selected for patch-clamp verification according to the virtual docking simulations and other parameters. At a concentration of 100 μM, GABA-induced currents were increased by (+)-Cuparene (278.80% ± 19.13%), Ethyl glucoside (225.40% ± 21.77%), and β-Caryophyllene (290.11% ± 17.71%). In addition, (+)-Cuparene, Ethyl glucoside, and β-Caryophyllene could also serve as positive allosteric modulators (PAMs) and shifted the GABA dose-response curve (DRC) leftward significantly. In the PCPA-induced zebrafish model, Ethyl glucoside showed anti-insomnia effects at concentrations of 100 μM. In this research, we demonstrated that the activation of GABAARs was involved in the anti-insomnia effect of HWD, and Ethyl glucoside might be a key metabolite in treating insomnia.
黄连温胆汤（HWD）是一种用于治疗失眠症患者的中药，能取得良好的治疗效果。由于正向调节γ-氨基丁酸（GABA）A型受体（GABAARs）是治疗失眠最有效的策略，本研究旨在探讨GABAARs的激活是否参与黄连温胆汤的抗失眠作用。我们使用液相色谱/质谱联用仪（LC/MS）和中药系统药理学（TCMSP）数据库评估了黄连温胆汤的代谢产物，并使用全细胞膜片钳和失眠斑马鱼模型测试了其药理活性。在表达α1β3γ2L GABAARs的HEK293细胞中，黄连温胆汤有效地增加了GABA诱导的电流，并且可以在不施加GABA的情况下诱导GABAAR介导的电流。在液相色谱-质谱联用（QToF）分析中，在负离子模式下发现了31种代谢产物，在正离子模式下发现了37种代谢产物，但所选的三种活性代谢产物，丹参素、黄连碱或二氢杨梅素，均未显示出对GABA电流的增强作用。根据TCMSP数据库收集了七种植物药的62种活性代谢产物，并根据虚拟对接模拟和其他参数选择了其中19种进行膜片钳验证。在浓度为100μM时，（+）-蒈烯（278.80%±19.13%）、葡萄糖苷乙酯（225.40%±21.77%）和β-石竹烯（290.11%±17.71%）增加了GABA诱导的电流。此外，（+）-蒈烯、葡萄糖苷乙酯和β-石竹烯还可作为正变构调节剂（PAMs），并使GABA剂量反应曲线（DRC）显著左移。在对氯苯丙氨酸（PCPA）诱导的斑马鱼模型中，葡萄糖苷乙酯在浓度为100μM时显示出抗失眠作用。在本研究中，我们证明了GABAARs的激活参与了黄连温胆汤的抗失眠作用，葡萄糖苷乙酯可能是治疗失眠的关键代谢产物。
