Inhibitory Effect Of Salvianolic Acid A On Platelet And Neutrophil Activation Through Complement Action

Background and Objective:Myocardial infarction is one of the cardiovascular diseases that seriously threaten human health.In cardiovascular disease,vascular dysfunction is the initial link,and platelets play an important role in vascular homeostasis,especially arterial and arteriolar thrombosis.In addition,neutrophils are recruited to myocardial ischemic area after myocardial injury,which leads to acute inflammatory reaction through phagocytosis,rapid degradation and degranulation,and forms neutrophil-platelet aggregates with platelets to enhance migration ability,in order to increase the recruitment of ischemic areas and play an anti-inflammatory role.Complement system is a group of proteins with enzyme activity after activation in the serum and tissue fluid of human and vertebrates.As an important part of innate immunity,complement system plays an important role in tissue injury caused by myocardial ischemia.Complement is activated in the process of tissue injury,mediating inflammatory response and tissue re-injury.Salvia miltiorrhiza is a traditional medicine for promoting blood circulation and removing blood stasis.Its water-soluble phenolic acid compounds have antithrombotic and antiplatelet aggregation effects.Therefore,this study explored the inhibition of platelet and neutrophil degranulation by regulating complement action of Salvianolic acid A.Methods:1.The acute myocardial infarction model was established by ligating the left anterior descending coronary artery in C57BL/6 mice,and the cardiac function was detected by ultrasonic Doppler system,the morphological changes of ischemic myocardium were observed by hematoxylin-eosin staining,and the effect of Salvianolic acid A on the release of platelet activation marker CD62p in peripheral blood of myocardial ischemic mice was detected by flow cytometry.Immunohistochemical CD42c staining was used to detect the effect of Salvianolic acid An on platelet recruitment in myocardial ischemic area of myocardial ischemic mice,immunohistochemical CD45 staining was used to detect the effect of Salvianolic acid An on leukocyte infiltration in myocardial ischemic area of myocardial ischemic mice,immunohistochemical Ly-6G staining was used to detect the effect of Salvianolic acid An on neutrophil infiltration in myocardial ischemic area of myocardial ischemic mice,immunohistochemical staining was used to detect the effect of Salvianolic acid An on the expression of C3a R in myocardial ischemic area of mice with myocardial ischemia,and.mmunofluorescence staining was used to detect the effect of Salvianolic acid An on the release of CXCL1 in myocardial ischemic area of mice with myocardial ischemia.The tail shearing method was used to detect the time of tail bleeding.2.Platelets were prepared from whole blood of SD rats,and the aggregation rate of platelets induced by adenosine diphosphate and C3a was detected by turbidimetry,the expression of CD62p and C3a R induced by ADP were detected by flow cytometry,and the platelet adhesion to fibrinogen was detected by platelet staining with rhodamine-labeled cyclopeptide.Western-blotting was used to detect the phosphorylated expression of vasodilator-stimulated phosphatidylinositol,phosphatidylinositol 3 kinase and serine threonine kinase protein;neutrophils were extracted from C57BL/6 mice with mouse neutrophil extraction kit,and neutrophils hypoxia model was established under the condition of hypoxia?1%O₂ for 4 h?.The release of neutrophil myeloperoxidase was detected by colorimetry,and the release of neutrophil elastase,matrix metalloproteinase-9 and lactoferrin was detected by enzyme-linked immunosorbent assay.Results:Three days after operation,compared with the model group,Salvianolic acid A could improve the cardiac function of myocardial ischemic mice and promote the repair of ischemic tissue injury;Salvianolic acid A could significantly reduce the release of platelet activation marker CD62p,inhibit the recruitment of activated platelets in the ischemic area,inhibit the infiltration of leukocytes and neutrophils in the ischemic area,and the release of inflammatory chemokine CXCL1 and CXCL2,and inhibit the expression of complement C3a R in the ischemic area without serious bleeding and other side effects.Salvianolic acid A can effectively inhibit ADP-induced platelet activation,inhibit PI3K/Akt and VASP phosphorylation,Salvianolic acid A can effectively inhibit ADP-induced C3a R expression,Salvianolic acid A can inhibit C3a-induced platelet aggregation,C3a R inhibitor can inhibit ADP-induced platelet aggregation and adhesion release.Salvianolic acid An and C3a R inhibitors could inhibit the release of MPO,NE,MMP9 and LF during mouse neutrophil degranulation induced by 1%O₂.Conclusion:Salvianolic acid A can inhibit the expression of complement factor C3a R in the complement pathway,inhibit the activation of platelets and neutrophils,thus play the role of anti-platelet and anti-inflammation,and finally promote the recovery of myocardial ischemic tissue and improve myocardial ischemic injury.