Antithrombotic Activities And Potential Mechanisms Of Tanshinones And Phenolic Acids From The Roots Of Salvia Miltiorrhiza F.Alba

Thrombotic disorders,generally caused by two pathological processes of thromboembolism and thrombosis and divided into three main categories:venous thrombosis,arterial thrombosis,capillary thrombosis,could lead to serious cardiovascular and cerebrovascular events.The formation of thrombus is closely related with atherosclerosis(AS),thromboangiitis obliterans(TAO),acute myocardial infarction(AMI)and other relevant diseases.According to reports of World Health Organization(WHO),thrombotic disorders and its relevant diseases have become the world’s leading death and disabling diseases.Salvia miltiorrhiza f.alba C.Y.Wu&H.W.Li.is native to Laiwu,Zhangqiu in Shandong province.In clinical,the root of Salvia miltiorrhiza f.alba C.Y.Wu&H.W.Li.(RSMA)as a Chinese herbal medicine can be used not only for the treatment of cardiovascular and cerebrovascular diseases,but also for the treatment of chronic nephritis,chronic renal failure,type 2 diabetes,infectious diseases and so on,and it is usually used to substitute the root of S.miltiorrhiza Bunge(RSM).Researchers had already found active compounds of the RSM in early 1930s,and there were similar structures between RSMA and RSM.In the subsequent study,more compounds had been isolated from RSMA.In addition,the contents of components in the root,stem and leaf of RSMA were higher than those in the plant organs of RSM except for flower.RSMA was mainly used for the treatment of AS,AMI,hyperlipidemia,hypertension and especially for the treatment of thromboangiitis obliterans(TAO)for more than 100 years.Therefore,our research group aimed at studying the antithrombotic activities of RSMA,to provide a theoretical basis for the application in clinical practice and further utilization of the RSMA resources.In this study,we determined anti-thrombotic activities and its potential mechanisms of 12 tanshinones and phenolic acids isolated from RSMA using TNF-α-stimulated EA.hy926 cells.MTT assay or CCK-8 assay were used to measure the cell abilities of EA.hy926 cells;the protein level of PAI-1 and t-PA were measured by enzyme-linked immunosorbent assay(ELISA)assay and mRNA expression of them were determined by quantitative real-time polymerase chain reaction(qRT-PCR);chromogenic substrate methods were used to detect the activities of anti-FⅩa and anti-FⅡa activities;the production of p38,p-p38,p65,p-p65,JNK and pJNK were measured by Western Blot.The results showed that most of tanshinones inhibited the overexpression of PAI-1.And the tanshinones had a weekly effect on t-PA expression.Among them,deoxyneocryptotanshinone(7)significantly decreased the PAI-1 protein level and gene level and had a remarkable anti-FXa and anti-FIIa activities.The mechanism of anti-thrombotic activity may be associated with the NF-κB/JNK signaling pathway.In this study,we also found that lithospermic acid(J)has strong effects on PAI-1 protein level and gene level,and its abilities to inhibit FⅩa and FⅡa activities were stronger than those of phenolic acid B.The mechanism of anti-thrmbotic activity may be associated with the NF-κB/JNK/p38 MAPK signaling pathway.In summary,we found deoxyneocryptotanshinone and lithospermic acid had marked anti-thrombotic activities through the evaluation of tanshinones and phenolic acids on TNF-α stimulated EA.hy926 cells.These findings will provide the theoretical basis for further research of anti-thrombotic drugs,but also provides an evidence for the clinical treatment of cardiovascular diseases.