Study On The Effect And Mechanism Of Ligustrazine And Danshensu On Platelet Activation

Objective:Ischemic cardiovascular disease is one of the diseases that seriously threaten human health,and its morbidity and mortality are increasing year by year.Arterial thrombosis is the pathological basis of cardiovascular disease.Ischemia caused by vascular thrombosis can lead to tissue hypoxia,up-regulate the expression of platelet ERK5 in the environment rich in ROS,promote platelet activation,make platelets gather on a large scale,lead to vascular blockage,form thrombus,and aggravate myocardial infarction.Platelet ERK5 may be used as a redox sensor to accelerate the expansion of infarction area after acute myocardial infarction.Mitochondria are the main source of ROS production and play a central role in enhancing platelet activation.Increased platelet ROS in patients with heart failure,mitochondrial dysfunction and apoptosis activation,induced abnormal platelet activation,mitochondria can be used as a potential target for anti-platelet.Traditional Chinese medicine for promoting blood circulation and removing blood stasis has become the basic use of antiplatelet drugs by virtue of its multi-target and multi-pathway advantages.Ligusticum chuanxiong and Salvia miltiorrhiza are the two most commonly used traditional Chinese medicines for promoting blood circulation and removing blood stasis,and Ligustrazine and Danshensu are the main active components,respectively.Studies have shown that both ligustrazine and Danshensu have antiplatelet and antithrombotic effects,but their mechanism is not clear.The purpose of this study was to explore the antiplatelet and antithrombotic effects and molecular mechanism of ligustrazine and Danshensu,so as to provide theoretical and experimental basis for the clinical application of ligustrazine and Danshensu.Method:1.The model of common carotid artery thrombus induced by ferric chloride was established.The wet weight of thrombus was measured,the thrombosis was observed by HE staining,and the inhibitory effect of ligustrazine and Danshensu on thrombosis was detected.Platelet aggregation induced by adenosine diphosphate(Adenosine diphosphate,ADP),U46619 and TRAP-6 was detected by microwell method,platelet adhesion was detected by spreading on fibrinogen,and the expression of platelet P-selectin(Cluster of differentiation 62 platelet,CD62p)was detected by flow cytometry to determine the inhibitory effect of ligustrazine and Danshensu on platelet activation.2.The release of ROS from platelets was detected by CM-H2 DCFDA staining,and the expression of phosphorylated proteins of ERK5,P70S6 K and Rac1 was detected by Western blotting(Western blotting,WB)to clarify the mechanism of ligustrazine regulating the activation of hypoxic platelets.WB method was used to detect the protein expression of silent information regulator 1(Silent information regulator-1((Silent information regulator-1)and DC-SIGN,CM-H2 DCFDA staining was used to detect the release of platelet ROS,and mitochondrial DNA extraction kit was used to separate mt DNA,Nanodrop to detect DNA content to clarify the mechanism of Danshensu's anti-platelet activation.Result:1.Ligustrazine can inhibit platelet aggregation induced by ADP,U46619 and TRAP-6,inhibit platelet adhesion to fibrinogen and release of CD62 p induced by hypoxia,indicating that ligustrazine can inhibit platelet activation.By detecting the effect of ligustrazine on arterial thrombosis induced by ferric chloride,it is proved that ligustrazine can inhibit thrombosis.Danshensu can inhibit platelet aggregation induced by ADP,U46619 and TRAP-6,inhibit platelet adhesion to fibrinogen induced by ADP and release of CD62 p,indicating that Danshensu can inhibit platelet activation.By detecting the effect of Danshensu on arterial thrombosis induced by ferric chloride,it is proved that Danshensu can inhibit thrombosis without causing bleeding reaction.2.Ligustrazine inhibits the accumulation of platelet ROS and down-regulates the expression of phosphorylated proteins of ERK5,P70S6 K and Rac1.Ligustrazine may inhibit platelet activation and thrombosis by regulating ERK5 signal pathway.Danshensu can up-regulate the expression of SIRT1 protein,inhibit the accumulation of ROS and down-regulate the expression of DC-SIGN protein.The content of mitochondrial DNA showed that Danshensu inhibited the release of platelet mitochondrial DNA,suggesting that Danshensu can inhibit platelet activation by regulating the function of mitochondria.Conclusion:1.Ligustrazine can inhibit the expression of ERK5/P70S6K/Rac1 pathway protein and exert its antiplatelet and antithrombotic effect.2.Danshensu can up-regulate the expression of SIRT1 protein,inhibit the accumulation of ROS and the release of platelet mitochondrial DNA,up-regulate the expression of DC-SIGN protein,play the role of anti-platelet,inhibit platelet activation,and then inhibit thrombosis without causing bleeding.