Xanthohumol isolated from Humulus lupulus prevents thrombosis without increased bleeding risk by inhibiting platelet activation and mtDNA release

Abstract

Aim: As the global population has reached 7 billion and the baby boom generation reaches old age, thrombosis has become the major contributor to the global disease burden. It has been reported that, in moderate doses, beer may protect against thrombosis. Xanthohumol (XN), an antioxidant, is found at high concentrations in hop cones (Humulus lupulus L.) and is a common ingredient of beer. Here, the aim of the present work was to investigate the effects of XN on antithrombotic and antiplatelet activities, and study its mechanism.

Approach and results: Using ferric chloride-induced carotid artery injury, inferior vena cava ligation model, and platelet function tests, we demonstrated that XN uniquely prevents both venous and arterial thrombosis by inhibiting platelet activation. Interestingly, in tail bleeding time studies, XN did not increase bleeding risk, which is recognized as a major limitation of current antithrombotic therapies. We also demonstrated that XN induces Sirt1 expression and thereby decreases reactive oxygen species (ROS) overload, prevents mitochondrial dysfunction, and reduces activated platelet-induced mitochondrial hyperpolarization, respiratory disorders, and associated membrane damage at low concentrations. In mitochondrial function assays designed to detect amounts of extracellular mitochondrial DNA (mtDNA), we found that XN prevents mtDNA release, which induces platelet activation in a DC-SIGN-dependent manner.

Conclusions: XN exemplifies a promising new class of antiplatelet agents that are highly effective at inhibiting platelet activation by decreasing ROS accumulation and platelet mtDNA release without incurring a bleeding risk. This study has also provided novel insights into mechanisms of thrombotic diseases with possible therapeutic implications.

Keywords: Beer; Bleeding risk; Mitochondrial DNA; Thrombosis; Xanthohumol.

Conflict of interest statement

Conflict of interest statement

No conflicts of interest to declare.

Copyright © 2017. Published by Elsevier Inc.

Figures

Fig. 1

Xanthohumol (XN), a prenylated chalcone isolated from the hop plant. (A) The molecular structure and 1H NMR spectrum of xanthohumol (2′,4′,4-trihydroxy-6′-methoxy-3′-prenylchalcone). (B) MS spectrum of xanthohumol.

Fig. 2

XN inhibits formation of carotid arterial thrombosis and inferior vena cava thrombosis in animals. XN decreases weight (A, D) and length (B, E) of FeCl3-induced carotid arterial thrombosis and FeCl3-induced inferior vena cava thrombus in rats (pre-treated with 20 mg/kg/d XN for 7 d). XN decreases weight (G) and length (H) of partial inferior vena cava ligation-induced venous thrombosis in rats. (C, F) The average time to occlusive thrombosis in FeCl3-induced inferior vena cava and carotid arterial injury was longer in XN treated (XN: 20 mg/kg/d, 7 d) rats compared with untreated rats. (I) XN reduces mortality from pulmonary thromboembolism in mice (XN: 20 mg/kg/d, 7 d). (J) XN does not significantly prolong bleeding times in C57/BL6 mice (XN: 20 mg/kg/d, 7 d). (K) XN decreases the incidence of gastric ulcer in normal rats compared with the use of aspirin (XN: 20 mg/kg/d, 60 d. Aspirin: 30 mg/kg/d, 60 d). Data are expressed as mean ± SD. n = 9–10. *P < 0.05 vs Control.

Fig. 3

XN has insignificant effects on coagulation factor and thrombolysis system. Effects of XN treatment in rats (XN: 20 mg/kg/d, 7 d) on PT, APTT, TT, FIB (A), coagulation factors (B), and clot weight reduction (C). (D) Thromboelastogram of whole blood from rats treated with or without XN. Data are expressed as mean ± SD. n = 9–10. *P < 0.05 vs Control.

Fig. 4

XN inhibits platelet activation in vivo and in vitro. (A, B) Influence of XN on aggregation of platelets induced by ADP, AA, and thrombin in vitro (XN: 0.5 µM, 6 h; aspirin: 0.1 mM, 6 h) and in vivo (XN: 20 mg/kg/d, 7 d; aspirin: 30 mg/kg/d, 7 d). (C, D) αIIbβ3 expression in ADP-, AA-, and thrombin-activated platelets with XN in vitro and in vivo. (E, F) P-selectin expression in ADP-, AA-, and thrombin-activated platelets with treatment in vitro and in vivo. (G, H) Influence of XN on the elevation of cytosolic calcium levels in ADP-, AA-, and thrombin-activated platelets in vitro and in vivo. (I, J) Effect of XN on platelet adhesion to collagen-coated surfaces (phalloidin-labeled platelets). Data are expressed as mean ± SD. n = 9–10. *P < 0.05 vs Control.

Fig. 5

XN prevents ROS accumulation of activated platelets by increasing sirtuin 1 expression. (A, B) Dose and tome effects of XN on platelet ROS production. (C, D) Low concentrations of XN significantly decrease ROS levels of ADP-, AA-, and thrombin-activated platelets in vitro (XN: 0.5 µM, 6 h). (E) Transmission electronic microscopy imaging of platelets and associated membrane damage (black arrows) treated with XN or without. (F) XN inhibits ADP-, AA- and thrombin-activated platelets lipid oxidation (C11-BODIPY 581/591, lipid peroxidation sensor) (XN: 0.5 µM, 6 h). (G, H) XN at low concentrations increases Sirt 1 expression in a time-dependent manner. (I) EX-527, a Sirt 1 inhibitor, decreases inhibition of XN on ROS accumulation of activated platelets. (J) EX-527 decreases inhibition of XN on A DP-, AA- and thrombin-induced platelets aggregation. Data are expressed as mean ± SD. n = 8. *P < 0.05 vs Control, #P < 0.05 vs Agonist or XN.

Fig. 6

XN suppresses activated platelet-induced mtDNA release and mitochondrial dysfunction via inhibition of ROS generation. (A) mitochondrial hyperpolarization (JC-1 fluorescence ratio, FL2/FL1)ΔΨ, (B) mitochondrial routine respiration, (C) ATP, values in activated platelets treated with XN (XN: 0.5 µM, 6 h). (D, E) The effects of XN on mtDNA release at different concentrations and times. (F) XN inhibits mtDNA escape from activated platelets in vitro (XN: 0.5 µM, 6 h). (G) XN decreases the amounts of plasma mtDNA in FeCl3-induced carotid arterial thrombosis and partial inferior vena cava ligation-induced venous thrombosis (XN: 20 mg/kg/d, 7 d). (H) ROS (hydrogen peroxide, 6 h) induces mtDNA escape from platelets. (I) NAC reduces mtDNA release from platelets (NAC: 10 mM, 6 h). (J) EX-527 decreases inhibition of XN on platelet mtDNA release in vitro. Data are expressed as mean ± SD. n = 8. *P < 0.05 vs Control, #P < 0.05 vs ADP or Agonist.

Fig. 7

mtDNA induced platelet activation in vivo and in vitro. (A, C) mtDNA affects aggregation of platelets in vitro (mtDNA: 40 ng/µL, 6 h) and in vivo (mtDNA: 50 µg/kg/d, 7 d). (B, D) mtDNA influences αIIbβ3 level in vivo and in vitro. (E, F) DNase decreases inhibition of XN on platelet aggregation and αIIbβ3 level induced by ADP in vitro (Pre-incubated for 0.5 h at 20 µg/mL DNase I). Data are expressed as mean ± SD. n = 8. *P < 0.05 vs control, #P < 0.05 vs ADP.

Fig. 8

mtDNA induces platelet activation may through a DC-SIGN dependent pathway. (A and B) mtDNA increases DC-SIGN expression in vitro (mtDNA: 40 ng/µL, 6 h) and in vivo (mtDNA: 50 µg/kg/d, 7 d). C, XN reduces platelet DC-SIGN expression in FeCl3-induced carotid arterial thrombosis and partial inferior vena cava ligation induced venous thrombosis. D and E, Anti-DC-SIGN (Pre-incubated for 0.5 h at 25 µg/mL) agent decreases platelet aggregation and αIIbβ3 level induced by mtDNA in vitro. Data are expressed as mean ± SD. n = 8. *P < 0.05 vs Control, #P < 0.05 vs mtDNA.