Naringenin Inhibits Platelet Activation And Arterial Thrombosis Via P2Y₁₂ Receptor Signal Ing Pathway

ObjectiveCoronary heart disease(CHD)is one of the leading causes of morbidity and mortality worldwide,and it is the first in the composition of residents disease death in China Platelet activation and the consequent intravascular arterial thrombogenesis is the common pathological basis of CHD.Platelet adhesion and aggregation triggered by atherosclerotic plaque disruption or endothelium injury initiates the formation of coronary artery thrombosis.Therefore,antiplatelet therapy has been established as a corner stone in the treatment of arterial thrombotic diseases.There are various antiplatelet drugs used in clinical,including cyclooxygenase inhibitor aspirin,P2Y,2 receptor antagonist clopidogrel,phosphodiesterase(PDE)inhibitor cilostazol,GP ?b/?a receptor antagonist abciximab and so on.Although these drugs are effective in treatment of arterial thrombotic diseases,they have more adverse effects,such as internal bleeding and drug resistance.Thus,there is need to develop more effective and safe antiplatelet drugs.In recent years,many traditional Chinese medicine compounds,active ingredients and monomers have been proved to have antiplatelet and antithrombotic effects.Because of multi-target and multi-channel effects of traditional Chinese medicine in prevention of platelet aggregation and thrombosis,it is of great significance to seek safe and effective anti platelet drugs from traditional Chinese medicine for the prevention and treatment of arterial thrombotic diseases.Exocarpium Citri Grandis(ECG)is the dried ripe peel of Citrus grandis Osbeck and its cultivars,and is one of the most versatile Chinese herbal drugs.It has been used in clinically to treat and prevent respiratory,digestive and cardiovascular diseases.ECG is rich in flavonoids,of which naringenin as a dihydrogen flavonoid compound,is the one of the major active ingredients and'resolving phlegm' active ingredients.Modern pharmacological researches have revealed that naringenin possesses a variety of pharmacological activities,such as anti-inflamnatory,anti-oxidation,hypolipidemic,anti-arrythmic and anti-atherosclerosis.Naringenin plays an important role in the prevention and treatment of cardiovascular diseases and can be applied to medicine,food and other fields.At present,the antiplatelet and antithrombotic activities of naringenin have not been reported.Our previous study found that naringenin could significantly inhibited adenosine diphosphate(ADP)-induced platelet aggregation,but its mechanism is unclear.Thus,taking P2Y12 receptor signaling pathway as the breakthrough point in the present study,we studied the effects of naringenin on platelet function,signal transduction and arterial thrombosis to explore the potential antiplatelet target of naringenin.Methods1.The antiplatelet effects of naringeninPlatelet-rich plasma and washed platelets were prepared.Different concentrations of naringenin and naringin on ADP-induced platelet aggregation in PRP or washed platelets were detected by aggregational detector.Similarly,the effects of naringenin on washed platelets aggregation induced by thrombin or collagen were also detected.The lactate dehydrogenase(LDH)leakage from platelets was measured to evaluate the potential cytotoxicity of naringenin.The effects of naringenin on P-select in expression and fibrinogen binding in ADP-stimulated platelets were measured by flow cytometry.Moreover,Fluo-3 AM dye-labeled washed platelets were stimulated with 10 ? M ADP in the presence or absence of different concentrations of naringenin,and the intracellular calcium levels were measured by flow cytometry.We also measured the effects of naringenin on platelet spreading and adhesion to immobilized fibrinogen and collagen by fluorescence microscope.Finally,the effect of naringenin on fibrin clot retraction was evaluated.2.The antiplatelet mechanism of naringeninWashed platelets were stimulated with 10 ?M ADP and then were split after incubation with different concentrations of naringenin for 5 min.The expression of PI3Kp110? the phosphorylation of Akt(Ser473/Thr308),ERK1/2(Thr202/Tyr204)and GSK3?(Ser9)as well as the phosphorylation of Akt when giving naringenin together with pan PI3K inhibitor LY294002 were analyzed by western blot.Moreover,the effects of naringenin combined with LY294002 on platelet aggregation waere detected by aggregational detector.The effects of naringenin on intracellular cyclic adenosine monophosphate(cAMP)and cyclic guanosine monophosphate(cGMP)levels in ADP-stimulated platelets or resting platelets were detected using ELISA kits.The effects of various concentrations of naringenin on PDE activity were also measured by HPLC on the hydrolysis of cAMP and cGMP.Moreover,the effects of various concentrations of naringenin on phosphorylation of vasodilator stimulated phosphoprotein(VASP)(at positions pS157and pS239)were analyzed in washed platelets on stimulation with ADP by immunoblotting.VASP phosphorylation at S239 was also analyzed in the presence of protein kinase A(PKA),protein kinase G(PKG),and protein kinase C(PKC)inhibitors before incubation with naringenin.Platelet aggregation was measured in the presence of PKA and PKG inhibitors before incubation with naringenin,after stimulation with ADP.Similarly,the effects of naringenin combined with P2Y1 antagonist(MRS2179)or P2Y12 antagonist(MRS2395)on ADP induced rat platelet aggregation were measured.3.the effects of naringenin on thrombosis and coagulation system in vivoSD rats were orally administrated with naringenin(200,400,800 mg/kg),clopidogrel(7.875 mg/kg)or 0.5%CMC-Na for 7 days.Blood was collected 60 min after last treatment.PRP was obtained by centrifugation of the blood sample at 1000 rpm for 10 min.Platelet aggregation was induced by 5 ?M ADP and detected by aggregational detector.After the platelet aggregation assay was terminated,platelet proteins were then extracted and analyzed by immunoblotting using PI3Kp110 ? antibody and phospho-specific antibodies such as Akt(Ser473),Akt(Thr308)and ERK1/2(Thr202/Tyr204).Arterial thrombosis was induced by 10%FeCl3 on common carotid artery in rats orally administrated with naringenin(200,400,800 mg/kg),clopidogrel(7.875 mg/kg)or 0.5%CMC-Na for 7 days.The carotid arterial occlusion time was measured with a miniature Doppler flowprobe(1 mm diameter)using a Transonic Model TS420 flowmeter.At the end of the experiment,the left carotid artery was dissected.Hematoxylin and eosin stain was performed on the artery.Moreover,the effects of naringenin on mouse tail bleeding time and on plasma coagulation parameters in rats were determined.Results1.The antiplatelet effects of naringenin? We found that naringenin(100-800 ? M)or naringin(400-800 ? M)pretreatment substantially inhibited ADP(10 ? M)-induced platelet aggregation in PRP.Similarly,naringenin(100-800 ?M)or naringin(100-800 ?M)significantly inhibited ADP-induced platelet aggregation in washed platelets.In PRP or washed platelets,the antiplatelet activity of naringenin on ADP-induced platelet aggregation was significantly higher than that of naringin.And the antiplatelet effects of naringenin or naringin in washed platelets were significantly increased compared with that in PRP.Moreover,naringenin effectively inhibited thrombin(0.2 U/mL)-and collagen(2 ?u g/mL)-induced washed platelets aggregation,and exerted strongest inhibitory effects on ADP-induced platelet aggregation in the same concentration range(100-800 ?M).?Naringenin(100-1600 ?M)did not show cytotoxic effects on platelets.?Naringenin inhibited ADP induced platelet P-selectin expression.?Naringenin(400-800 ? M)significantly decreased intracellular calcium level in washed platelets stimulated with ADP(10 ?M).?Naringenin(400-800 ?M)reduced ADP-induced fibrinogen binding,which indicated that naringenin inhibited ? ?b ? 3-mediated inside-out signaling in platelets.?Meanwhile,naringenin reduced platelet spreading on fibrinogen and clot retraction,which indicated that naringenin inhibited ?a ?b ? 3-mediated outside-in signaling in platelets.?Moreover,naringenin decreased platelet adhesion on collagen surfaces.2.The antiplatelet mechanism of naringenin?Western blot analysis showed that naringenin markedly suppressed not only the exptression of PI3Kp110 ?,but also the phosphorylation of Akt(Ser473/Thr308)and ERK1/2(Thr202/Tyr204).Moreover,naringenin and LY294002 demonstrated an additive inhibition on Akt phosphorylation and platelet aggregation when combined.On the other hand,naringenin did not significantly affect GSK3? phosphorylation.? Naringenin(100-800 ? M)significantly increased cGMP levels but not cAMP levels in a dose-dependent manner in both ADP-stimulated platelets and resting platelets.? Naringenin(200-800 ? M)showed inhibitory effects on PDE activity on the hydrolysis of either cAMP or cGMP.?Immunoblot analysis showed that naringenin increased the phosphorylation of VASP at S239 in a dose-dependent manner on stimulation with ADP but only modest changes in phosphorylation of position S157.Increase in phosphorylation of VASP at S239 position was inhibited when platelets were treated with PKA inhibitor before incubation with naringenin.Incubation of platelets with either PKG or PKC inhibitors before treatment with naringenin did not affect the phosphorylation of VASP at S239,?Similarly,the prior treatment of platelets with PKA inhibitor significantly elevated the naringein-mediated reduction of platelet aggregation levels in ADP-stimulated platelets,but the preincubation of PKG inhibitor before treatment of naringenin did not significantly affect the final extent of platelet aggregation.?Naringenin combined with P2Y1 antagonist(MRS2179)or P2Y12 antagonist(MRS2395)showed an additive inhibition on ADP-induced platelet aggregation.3.The effects of naringenin on thrombosis and coagulation system in vivo?Naringenin administered orally inhibited ADP-induced platelet aggregation in a dose-dependent manner.? Western blot analysis showed that naringenin administered orally suppressed Akt(Ser473/Thr308)and ERK1/2(Thr202/Tyr204)phosphorylation in platelets stimulated with ADP,and hardly had any effect on PI3Kp110 ? expression.? Naringenin(800 mg/kg)significantly extended the carotid arterial occlusion time in FeCl3-induced carotid arterial thrombus model in rats.Meanwhile,H&E-stained carotid artery showed that naringenin could reduce thrombosis and vascular endothelial injury.? Naringenin did not significantly prolong the bleeding time of mice at all the concentrations used.?Naringenin did not affect the coagulation time in rats,while clopidogrel could significantly prolong clotting time and reduce fibrinogen content,showing potent anticoagulant effects.ConclusionNaringenin has potent antiplatelet and antithrombotic effects without any bleeding diathesis.Naringenin inhibits platelet activation via blockade of PI3Kt pathway along with the increased cGMP generation and PKA-mediated VASP phosphorylation.Based on signal pathway,we suppose that the antiplatelet target of naringenin might be P2Y12 receptor.Our data suggest that naringenin may be developed as a novel,effective and safe antiplatelet drug.