Avancha Aneesh, Hoyt Helen, Bhave Kieran, Medeiros Madyson, Cho Daniel, Brown Lauren E, Fernández González Davinia, Porco John A, Forman Stuart A
From the Department of Anesthesia Critical Care & Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts.
Department of Anesthesiology, Weill Cornell Medical Center, New York, New York.
Anesth Analg. 2024 Nov 21. doi: 10.1213/ANE.0000000000007318.
R-Glabridin is a major flavonoid of licorice (Glycyrrhiza glabra) root and known to modulate GABAA receptors, which are targets of many clinical hypnotics. However, R-glabridin hypnotic activity has not been reported in animals.
Inverted photomotor responses (IPMRs) were used to assess the hypnotic effects of natural R-glabridin and synthetic R/S-glabridin in wild-type zebrafish larvae and transgenic larvae lacking functional GABAA receptor β3 subunits (β30/0). Two-electrode voltage-clamp electrophysiology in Xenopus oocytes heterologously expressing ion channels quantified the effects of R-glabridin on wild-type and mutated human α1β3γ2L GABAA receptors, NR1B/NR2A N-methyl-D-aspatate (NMDA) receptors, and α4β2 neuronal nicotinic (nnACh) receptors.
IPMRs in wild-type zebrafish larvae identified R/S-glabridin as an inhibitor (IC50 = 7.5 µM; 95% confidence interval [CI], 5.9-9.3 µM) that was about half as potent as R-glabridin (IC50 = 4.4. µM; 95% CI, 3.6-5.4 µM). In β30/0 zebrafish larvae, R-glabridin inhibited IPMRs with IC50 = 7.5 µM (95% CI, 5.6-10.0 µM). Electrophysiologic studies revealed that R-glabridin directly activated and positively modulated α1β3γ2L GABAA receptors. Modulation was significantly reduced by α1L232W and β3N265M mutations in the β+/α- transmembrane intersubunit sites where etomidate binds, but not by 5 other point mutations in 4 other transmembrane modulator binding sites. NMDA and nnACh receptors were inhibited by R-glabridin.
DISCUSSION/CONCLUSIONS: Our findings in zebrafish larvae indicate that IPMR inhibition by R-glabridin is more potent than S-glabridin and that β3-containing GABAA receptors contribute significantly to this behavioral effect. Molecular studies show that R-glabridin modulates at least 3 known anesthetic-sensitive ion channels, suggesting that it is a multimodal hypnotic.
光甘草定是甘草根中的一种主要黄酮类化合物，已知其可调节γ-氨基丁酸A型（GABAA）受体，而该受体是许多临床催眠药的作用靶点。然而，光甘草定的催眠活性在动物中尚未见报道。
采用反向光运动反应（IPMR）评估天然光甘草定和合成的R/S-光甘草定对野生型斑马鱼幼虫以及缺乏功能性GABAA受体β3亚基（β30/0）的转基因幼虫的催眠作用。在异源表达离子通道的非洲爪蟾卵母细胞中进行双电极电压钳电生理实验，以量化光甘草定对野生型和突变型人α1β3γ2L GABAA受体、NR1B/NR2A N-甲基-D-天冬氨酸（NMDA）受体以及α4β2神经元烟碱型（nnACh）受体的影响。
野生型斑马鱼幼虫的IPMR实验确定R/S-光甘草定为一种抑制剂（半数抑制浓度[IC50]=7.5 μM；95%置信区间[CI]，5.9 - 9.3 μM），其效力约为光甘草定（IC50 = 4.4 μM；95% CI，3.6 - 5.4 μM）的一半。在β30/0斑马鱼幼虫中，光甘草定抑制IPMR的IC50 = 7.5 μM（95% CI，5.6 - 10.0 μM）。电生理研究表明，光甘草定可直接激活并正向调节α1β3γ2L GABAA受体。在依托咪酯结合的β+/α-跨膜亚基位点发生的α1L232W和β3N265M突变可显著降低这种调节作用，但在其他4个跨膜调节剂结合位点的5个其他点突变则无此作用。NMDA和nnACh受体受到光甘草定的抑制。
讨论/结论：我们在斑马鱼幼虫中的研究结果表明，光甘草定对IPMR的抑制作用比S-光甘草定更强，且含β3的GABAA受体对这种行为效应有显著贡献。分子研究表明，光甘草定可调节至少3种已知的麻醉敏感离子通道，提示其为一种多模式催眠药。
