The Study Of Ilex Pubescens Total Extract Antithrombotic Mechanism Based On The P2Y₁₂Receptor

ObjectiveThe issue was studied the antithrombotic effect of Ilex Pubescens total extract by three animal's thrombosis models initially,and it was studied the effect of Ilex Pubescens total extract on vascular endothelium by the carrageenan model;and it was researched the effect of Ilex Pubescens total extract on aGi subunit and ?? subunits of the P2Y₁₂ receptor by Ca²⁺-induced thrombosis model,and the antithrombotic mechanism of Ilex Pubescens total extract based on P2Y₁₂ receptor to be explored further.These study provide strong scientific basis for finding the target of the antithrombotic effect of Ilex Pubescens total extract and provide new idea for the cardiovascular diseases' prevention and treatment and drug research.Methods1.Collagen-epinephrine-induced thrombosis in mice,carrageenan-induced thrombosis in mice and FeCl₃-induced abdominal aorta thrombosis model in rat were used to study the antithrombotic effect of Ilex Pubescens total extract.1.1 The collagen-epinephrine-induced thrombosis in mice in vivo experimentKM mice were randomly divided into six groups,normal control group,model control group,aspirin group,the high,middle and low dose groups of Ilex Pubescens total extract,gavaged once 1 d and continuous 5 d.After the last gavaged 45 min at 5 d,in addition to the normal control group,the other five groups each mouse tail vein injection of collagen and epinephrine suspension,the normal control group was injected with the same volume of saline.After injection were observed and recorde the number of mice died within 5 min and the number of mice did not recover hemiplegia within 15 min,paralysis and loss of righting reflex in mice as the standard.1.2 The carrageenan-induced thrombosis in mice experimentKM mice were randomly divided into six groups,normal control group,model control group,aspirin group,the high,middle and low dose groups of Ilex Pubescens total extract,gavaged once 1 d and continuous 8 d.After gavaged 1 h at 5 d,the mice were injected subcutaneously 3%solution of carrageenan to induce thrombosis,subcutaneous injection of normal saline in the control group.The black-tailed length and black-tailed incidence was observed after 72 h of injecting carrageenan in mice.1.3 The FeC₁3-induced abdominal aorta thrombosis in rats experimentSD rats were randomly divided into six groups,normal control group,model control group,heparin group,the high,middle and low dose groups of Ilex Pubescens total extract.In addition to heparin group,the other group gavaged once Id and continuous 5 d.Before 12 h of the last gavaged subcutaneous injected heparin in the heparin group,and injected two times,at intervals of six hours.After 30 min the last gavaged,the rats were anesthetized by intraperitoneal injection of 10%chloral hydrate,the abdominal opened and 1.2 cm of the abdominal aorta isolated.Thereafter,a segment of the right carotid artery was exposed and injury was induced by the topical application of FeCl₃ for 50 minutes.When thrombus was formed,thrombus along with the exposed carotid was taken out and weighed.2.The effect of Ilex Pubescens total extract on carrageenan-induced thrombosis and vascular endothelial in mice in vivo experimentKM mice were randomly divided into thermostat and Postocclusion Reactive Hyperaemia,the two tranches were randomly divided into six groups,normal control group,model control group,nifedipine group,the high,middle and low dose groups of Ilex Pubescens total extract.Experimental operation in accordance with method "1.2"conducted.Observe the relative length of black-tail and the incidence of black tail length after modeling 72 h,at the same time using the PeriFlux system 5000 laser Doppler detected blood flow change of temperature and PORH.3.The effect of Ilex Pubescens total extract on FeCl₃-induced thrombosis and the aGi subunit and ?? subunits of P2Y₁₂ receptor in rats experimentSD rats were randomly divided into six groups,normal control group,model control group,clopidogrel group,the high,middle and low dose groups of Ilex Pubescens total extract.Experimental operation in accordance with method" 1.3",start experiment by solating carotid artery,and weight wet weight of thrombus,HE staining thrombosis morphological changes;detected platelet aggregation,coagulation?PT,APTT,TT,FIB?and ADP receptor(P2Y₁₂)expression levels;followed detected the function of subunits?aGi,???of P2Y₁₂ receptor:aGi subunits including cyclic adenosine monophosphate?cAMP?;?? subunits including thromboxane?TXB₂?,prostacyclin(6-Keto-PGF_1?),P-selection,and detected the platelet membrane glycoprotein?GP?b/?a?and Ca²⁺ of platelet cytosolic.Results1.In collagen-epinephrine-induced thrombosis in mice experiments,compared with model group,the number of death and hemiplegia of the high,middle and low dose groups of Ilex Pubescens total extract?1,0.5,0.25 g/kg?was decreased and protection rate was increased;in carrageenan-induced thrombosis in vivo experiment,compared with model group,the relative to black tail length of the high,middle and low dose groups of Ilex Pubescens total extract?1,0.5,0.25 g/kg?was reduced;in FeCI₃-induced abdominal aortic thrombosis experiment,compared with model group,the high,middle and low dose groups of Ilex Pubescens total extract?0.78,0.39,0.195 g/kg?reduce the wet weight of thrombus.2.In carrageenan-induced thrombosis in vivo experiment,compared with model group,the relative to black tail length of the high,middle and low dose groups of Ilex Pubescens total extract?125,62.5,31.25 mg/kg?were reduced;before modeling,with the temperature in each group changes,the percentage change in blood flow values were no differences;after modeling,compared with model group,the percentage change in the value of blood flow were increased;equally before modeling,the pressure before and after blocking,blood flow changes in the values were no differences in each group;after modeling,compared with model group,the pressure before and after blocking the blood flow changes in value decreased;it shows the endothelial injury reduced and Ilex Pubescens total extract has the function of improving endothelial.3.In FeCl₃-induced thrombosis in vivo experiment,the high,middle and low dose groups of Ilex Pubescens total extract?195,97.5,48.75 mg/kg?reduce the weight of thrombosis and endothelial extent of the damage;and ADP-induced platelet aggregation was reduced,and FIB and the P2Yi₂ receptor expression were reduced also,however it has no difference of the APTT,TT,PT.It increased expression levels of cAMP which the aGi subunit function;and it reduced the content of TXB₂,P-selectin levels which the ?? subunit function,but it didn't affect the 6-Keto-PGF_1?.Finally,it reduced the GP?b/?a expression and Ca²⁺ mean fluorescence of platelet cytosolic.Conclusion?1?The Ilex Pubescens total extract has the effect of anti-arterial,venous thrombosis.?2?The Ilex Pubescens total extract can improve the degree of endothelial injury and has the role of protectiving vascular endothelial.?3?The Ilex Pubescens total extract inhibits the platelet aggregation and thrombus formation by protecting vascular endothelium,finaliy.?4?The Ilex Pubescens total extract inhibits platelet aggregation and thrombus formation by affecting the expression aGi subunit and ?? subunit of P2Y₁₂ receptor and reducing the content of P2Y₁₂ receptor,finally.