Naringenin Inhibits Platelet Activation and Arterial Thrombosis Through Inhibition of Phosphoinositide 3-Kinase and Cyclic Nucleotide Signaling

Figures

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  FIGURE 1. Effects of naringenin and naringin on platelet aggregation. Chemical structure of naringin (A) and naringenin (B). The effects of naringin (C) or naringenin (D) on PRP were measured following stimulation with 10 μmol/L ADP. Rat washed platelet aggregation performed with or without different concentrations of naringin (E) or naringenin (F) was recorded after stimulation with 10 μmol/L ADP. Similarly, Washed platelet aggregation performed with vehicle or various concentrations of naringenin was recorded after stimulation with 0.2 U/mL thrombin (G) or 2 μg/ml collagen (H). WP represents washed platelets. Data are expressed as means ± SD (n = 6), the level of aggregation obtained at 5 min with vehicle-treated samples (0 μM) was taken as 100%. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control group.

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  FIGURE 2. Naringenin inhibits platelet granule secretion, integrin inside-out signaling and calcium mobilization. The effects of naringenin on P-selectin exposure (A) and fibrinogen binding (B) upon stimulation with ADP (10 μmol/L) were determined in washed platelets. Fluo-3 AM dye-labeled washed platelets were stimulated with ADP (10 μmol/L) in the absence or presence of different concentrations of naringenin, and the intracellular calcium levels were detected by flow cytometry (C). R represents the levels of P-selectin, fibrinogen binding or calcium mobilization in resting platelets. Data are expressed as means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control group.

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  FIGURE 3. Naringenin inhibits integrin outside-in signaling and platelet adhesion on collagen-coated surfaces. Washed platelets were preincubated with various concentrations of naringenin or vehicle for 10 min at 37°C, then allowed to spread on fibrinogen-coated surface. (A) Representative images from one of three independent experiments with similar results. The percentages of the average surface area of individual platelets were plotted (B). PRP was incubated with naringenin (200, 400 and 800 μmol/L) or vehicle for 10 min at 37°C. Fibrinogen (400 μg/ml) was added and fibrin clot formation was initiated by the addition of thrombin (1 U/mL). Photographs of the clots were taken 15 min later. Similar results were obtained in three independent experiments (C). Washed platelets were preincubated with various concentrations of naringenin or vehicle for 10 min at 37°C, then allowed to adhere on collagen-coated coverslips. (D) Representative images from one of three independent experiments with similar results. The percentages of the average surface area of individual platelets were plotted (E). Data are expressed as means ± SD (n = 3). **p < 0.01, ***p < 0.001 compared with the vehicle control…

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  FIGURE 4. Naringenin inhibits PI3K-mediated signaling in ADP-induced platelets. Washed rat platelets were preincubated without or with naringenin at 37°C for 5 min, and then stimulated with ADP (10 μmol/L) under stirring for 5 min. Platelet proteins were then extracted and analyzed by immunoblotting using PI3Kp110β antibody (A) and phospho-specific antibodies such as AktSer473 (B), AktThr308 (C), ERK1/2Thr202/Tyr204 (D) and GSK3βSer9 (E). Total level of GAPDH was measured on each sample as a loading control. The blots shown are representative of three separate experiments. The relative protein expression levels were quantified by ImageJ 1.47v software. The level of relative protein expression obtained with vehicle control (0 μM) was taken as 1. R represents the level of relative protein expression in resting platelets. Data are expressed as means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control group.

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  FIGURE 5. Effects of naringenin combined with PI3K pan inhibitor LY294002 on platelet aggregation and Akt phosphorylation. Washed rat platelets were preincubated with naringenin (200 μmol/L) alone, the PI3K pan inhibitor LY294002 (5 μmol/L) alone or both for 5 min prior to ADP (10 μmol/L) activation. After stimulation with ADP, platelet aggregation was quantified and expressed as a percentage (A). Data are expressed as means ± SD (n = 6). ***p < 0.001 compared with the ADP-treated group, ### p < 0.001 compared with naringenin alone or LY294002 alone. Platelet proteins were then extracted and specific antibodies were used to measure the level of total and phosphorylated Akt (B). The blots shown are representative of three separate experiments. The relative protein expression levels were quantified by ImageJ 1.47v software. The level of phosphorylation obtained in the ADP-treated group was taken as 1. Data are expressed as means ± SD (n = 3). ***p < 0.001 compared with the ADP-treated group, ### p < 0.001 compared with naringenin alone or LY294002 alone.

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  FIGURE 6. Naringenin elevates platelet cGMP levels and inhibits phosphodiesterase activity. The levels of cAMP (A) and cGMP (B) in washed platelets preincubated with vehicle or naringenin (100, 200, 400, and 800 μmol/L) were determined after stimulation with ADP (10 μmol/L) using ELISA kits. Similarly, the levels of cAMP (C) and cGMP (D) were determined in resting platelets incubated with naringenin (200, 400, and 800 μmol/L) or vehicle for 5 min. R represents the levels of cAMP or cGMP in resting platelets. Data are expressed as means ± SD (n = 4). *p < 0.05, ***p < 0.001 compared with the vehicle control group. The effects of naringenin on phosphodiesterase activity were measured by HPLC on the hydrolysis of cAMP (E) and cGMP (F). The level of phosphodiesterase activity obtained with vehicle control (0 μM) was taken as 100%. Data are expressed as means ± SD (n = 3). *p < 0.05, ***p < 0.001 compared with the vehicle control group.

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  FIGURE 7. Naringenin promotes VASP phosphorylation. The effects of naringenin on phosphorylation of VASP (at positions Ser 157 and Ser239) were measured in washed platelets upon stimulation with ADP (10 μmol/L) by western blotting (A). The blots shown are representative of three separate experiments. The phosphorylation levels were quantified by ImageJ 1.47v software. The level of phosphorylation obtained with vehicle control (0 μM) was taken as 1 (B,C). R represents the phosphorylation level in resting platelets. Data are expressed as means ± SD (n = 3). *p < 0.05, ***p < 0.001 compared with the vehicle control group. VASP phosphorylation at Ser239 was also analyzed in washed platelets incubated with 5 μmol/L PKA (H89), 30 μmol/L PKG (Rp-8-Br-PET-cGMPS), or 5 μmol/L PKC (GF109203X) inhibitors before treatment with naringenin (D). Washed platelets were incubated with vehicle or naringenin (200 μmol/L) at 37°C in the presence or absence of 5 μmol/L PKA (H89) or 30 μmol/L PKG (Rp-8-Br-PET-cGMPS) inhibitors. After treatment, platelets were stimulated with ADP (10 μmol/L) to trigger aggregation (E). Data are expressed as means ± SD (n = 6). ***p < 0.001 compared with the ADP-treated group, # p…

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  FIGURE 8. Intragastric administration of naringenin inhibits ADP-induced rat platelet aggregation and platelet PI3K signaling phosphorylation. Rats were orally administered naringenin (200, 400, and 800 mg/kg), clopidogrel (7.875 mg/kg) or vehicle (0.5% CMC-Na) for 7 days. Blood was collected and PRP was obtained after the last treatment. Platelet aggregation was induced by ADP (5 μmol/L) (A). Data are expressed as means ± SD (n = 6). After ADP stimulation, platelet proteins were then extracted and analyzed by immunoblotting using PI3Kp110β antibody (B) and phospho-specific antibodies such as AktSer473 (C), AktThr308 (D) and ERK1/2Thr202/Tyr204 (E). Total level of GAPDH was measured on each sample as a loading control. The blots shown are representative of three separate experiments. The relative protein expression levels were quantified by ImageJ 1.47v software. The level of relative protein expression obtained with vehicle control was taken as 1. R represents the level of relative protein expression in resting platelets. Data are expressed as means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle control group (Con).

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  FIGURE 9. Naringenin inhibits arterial thrombosis formation without bleeding risk. Arterial thrombosis was induced by 10% FeCl3 on the common carotid artery of rats that were orally treated with naringenin (200, 400, and 800 mg/kg), clopidogrel (7.875 mg/kg) or vehicle (0.5% CMC-Na) for 7 days. Time to occlusion of the carotid artery after FeCl3 injury for 5 min was measured using a Transonic Model TS420 flowmeter (A). Representative sections of H&E-stained carotid artery from rats treated as labeled (B). Magnification: 10×20. Naringenin (280, 560, and 1,120 mg/kg), clopidogrel (11.375 mg/kg) or vehicle (0.5% CMC-Na) was administered orally to mice for 7 days. Sixty minutes after the last administration, the bleeding time were measured in a mouse tail transection model (C). Data are expressed as means ± SD (n = 6). The significance between control and treated groups was calculated by nonparametric Kruskal-Wallis global test using GraphPad Prism. **p < 0.01, ***p < 0.001 compared with the model group.