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. 2022 Sep 27;23(19):11372.
doi: 10.3390/ijms231911372.

Glabridin, a Bioactive Flavonoid from Licorice, Effectively Inhibits Platelet Activation in Humans and Mice

Chi-Li Chung  1 , Jui-Hsuan Chen  2 , Wei-Chieh Huang  2 , Joen-Rong Sheu  2 , Chih-Wei Hsia  2 , Thanasekaran Jayakumar  3 , Chih-Hsuan Hsia  4 , Kuan-Rau Chiou  5 , Shaw-Min Hou  2   6   7
Affiliations

Glabridin, a Bioactive Flavonoid from Licorice, Effectively Inhibits Platelet Activation in Humans and Mice

Chi-Li Chung et al. Int J Mol Sci. .

Abstract

Platelets are crucial for hemostasis and arterial thrombosis, which may lead to severe cardiovascular diseases (CVDs). Thus, therapeutic agents must be developed to prevent pathological platelet activation. Glabridin, a major bioalkaloid extracted from licorice root, improves metabolic abnormalities (i.e., obesity and diabetes) and protects against CVDs and neuronal disorders. To the best of our knowledge, no studies have focused on glabridin's effects on platelet activation. Therefore, we investigated these effects in humans and mice. Glabridin exhibited the highest inhibitory effects on collagen-stimulated platelet aggregation and moderate effects on arachidonic-acid-stimulated activation; however, no effects were observed for any other agonists (e.g., thrombin or U46619). Glabridin evidently reduced P-selectin expression, ATP release, and intracellular Ca2+ ([Ca2+]i) mobilization and thromboxane A2 formation; it further reduced the activation of phospholipase C (PLC)γ2/protein kinase C (PKC), phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β), mitogen-activated protein kinase (MAPK), and NF-κB. In mice, glabridin reduced the mortality rate caused by acute pulmonary thromboembolism without altering bleeding time. Thus, glabridin effectively inhibits the PLCγ2/PKC cascade and prevents the activation of the PI3K/Akt/GSK3β and MAPK pathways; this leads to a reduction in [Ca2+]i mobilization, which eventually inhibits platelet aggregation. Therefore, glabridin may be a promising therapeutic agent for thromboembolic disorders.

Keywords: MAPK; PI3K/Akt/GSK3β; PLCγ2/PKC; glabridin; microvascular thrombosis; platelet; pulmonary thromboembolism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Inhibitory effects of glabridin on platelet aggregation stimulated by different agonists in human platelets. (A) Chemical structure of glabridin (C20H20O4). (B) Washed human platelets (3.6 × 108 cells/mL) were preincubated with a solvent control (0.1% DMSO) or glabridin (10–100 μM) and subsequently treated with collagen (1 μg/mL), thrombin (0.01 U/mL), U46619 (1 μM), or arachidonic acid (AA; 60 μM) to stimulate platelet aggregation. (C) Concentration–response histograms of glabridin’s effects on platelet aggregation triggered by various agonists (%). Data are presented as mean ± standard error of the mean (n = 4). ** p < 0.01 and *** p < 0.001 vs. 0.1% DMSO-treated group.
Figure 2
Figure 2
Inhibitory effects of glabridin on ATP release, relative [Ca2+]i mobilization, P-selectin surface expression, and thromboxane B2 formation in human platelets. Washed platelets (3.6 × 108 cells/mL) were preincubated with DMSO (0.1%) or glabridin (25 and 40 µM), followed by the addition of collagen (1 μg/mL) or arachidonic acid (AA; 60 μM) to trigger (A) ATP release (arbitrary unit [AU]), (B) relative [Ca2+]i mobilization, and (C) P-selectin surface expression (mean fluorescence intensity [MFI]) ((a) Tyrode’s solution, (b) collagen-stimulated platelets, (c) glabridin 25 µM, and (d) 40 µM). (D) Thromboxane B2 (TxB2) formation. Respective statistical analyses are indicated in the bar diagrams. Data are presented as the mean ± standard error of the mean (n = 4). (A,B) ** p < 0.01 and *** p < 0.001 vs. 0.1% DMSO + collagen group. (C,D) *** p < 0.001 vs. resting platelets (in Tyrode’s solution); # p < 0.05 and ## p < 0.01 vs. 0.1% DMSO + collagen group.
Figure 3
Figure 3
Effects of glabridin on the activation of phospholipase Cγ2 and protein kinase C in platelets. DMSO (0.1%) or glabridin (25 and 40 µM) were preincubated in washed platelets, and they were subsequently treated with collagen (1 µg/mL) or phorbol 12,13-dibutyrate (PDBu, 150 nM) to stimulate (A) phospholipase Cγ2 (PLCγ2) activation, (B) protein kinase C (PKC) activation (p–p47), or (C) platelet aggregation. Data are shown as the mean ± standard error of the mean (n = 4). *** p < 0.001 vs. resting platelets (in Tyrode’s solution); ### p < 0.001 vs. 0.1% DMSO + collagen group. Diagram in (C) represents four independent experiments.
Figure 4
Figure 4
Regulatory activity of glabridin on phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β) and mitogen-activated protein kinase (MAPK) pathways in platelets. Washed platelets were preincubated with DMSO (0.1%) or glabridin (25 and 40 µM) and treated with collagen (1 μg/mL) for the immunoblotting of (A) PI3K, (B) Akt, (C) GSK3β, (D) ERK1/2, (E) JNK1/2, and (F) p38 MAPK. Data are shown as the mean ± standard error of the mean (n = 4). ** p < 0.01 and *** p < 0.001 vs. resting platelets (in Tyrode’s solution); # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. 0.1% DMSO + collagen group.
Figure 5
Figure 5
Effects of glabridin on the activation of NF-κB in human platelets. Washed platelets were preincubated with DMSO (0.1%) or glabridin (25 and 40 µM) and then treated with collagen (1 μg/mL) for the immunoblotting of (A) IκBα and (B) p65 phosphorylation, or (C) IκBα degradation, and confocal microscopic assessment (1000× magnification) of (D) phosphorylated NF-κB (p65) (green fluorescence) and α-tubulin (blue fluorescence) using goat anti-rabbit CFTM 488A and anti-mouse CFTM 405M dyes, respectively. Data are expressed as the mean ± standard error of the mean (n = 4). *** p < 0.001 vs. resting platelets (in Tyrode’s solution); # p < 0.05 and ### p < 0.001 vs. 0.1% DMSO + collagen group. The confocal images represent four independent experiments. Bar: 2.5 μm.
Figure 6
Figure 6
Effects of glabridin on the degree of acute pulmonary thromboembolism and time of tail vein bleeding. (A) The development of acute pulmonary thrombosis in mice was induced by ADP (700 mg/kg) through tail veins injection after they were treated DMSO (0.1%) or glabridin (6 and 12 mg/kg) through intraperitoneal route. (B) Bleeding time was measured via the tail vein transection model after 30 min of the intraperitoneal administration of DMSO (0.1%), glabridin (6 and 12 mg/kg), or aspirin (1 mg/kg). Data are expressed as the mean ± standard error of the mean (n = 10). *** p < 0.001 vs. 0.1% DMSO-treated group. Data in (A) are presented as mortality rates. (C) Proposed scheme of the mechanisms underlying the inhibitory effects of glabridin on platelet activation in humans. Glabridin inhibits platelet activation associated with signaling cascades (e.g., PLCγ2/PKC, PI3K-Akt-GSK3β, and MAPKs), followed by the regulation of [Ca2+]i mobilization, which eventually inhibits platelet aggregation.
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