The Molecule Mechanisam Of Xiang-qi-tang And Its Mainly Compounds On The EPCR Shedding

Background:Endothelial cell protein receptor C(EPCR)is an important member of PC anticoagulation system.EPCR is a membrane protein,and when it detaches from the cell membrane called EPCR shedding,could cause anticoagulation system dysfunctiom.Besides,EPCR also participates in many other pathological processes,such as anti-inflammatory,anti-apoptotic,autoimmunity and cancer.So research on EPCR shedding is meaningful.Xiang-Qi-Tang is a traditional Chinese medicine compound,mainly composed by Rhizoma Cyperi,Astragalus membranaceus and Andrographis paniculata.In our previous study on animal models of chicken,we found that the Xiang-Qi-Tang and its effective components can inhibit the EPCR shedding.But so far,there has been no evidence that they inhibit EPCR shedding in human umbilical vein endothelial cells(HUVECs).Aims:To establish the Phorbol-12-myristate-13-acetate(PMA)-induced EPCR shedding model in HUVECs,and investigate the inhibitive effect of Xiang-Qi-Tang and its main active component on EPCR shedding.Method:HUVECs were treated with different concentrations of PMA.The expression of EPCR was detected by Western-blot.The EPCR shedding was detected by ELISA,thus screening out the best concentration of PMA.Then,the proteins level of TACE,MAPK family and PKC isozymes were further detected by Western-blot.Based on the above data,the EPCR shedding model would be established.In this model,the cells were pretreated with Xiang-Qi-Tang or its main active component before stimulated with PMA.Then the mechanism of those drugs on EPCR shedding was investigated.Result:We found that HUVECs treated with 2μM PMA for 1 hour can release EPCR to the greatest extent,and it could also induce MAPK phosphorylation and PKC translocation and phosphorylation.For the first time,EPCR shedding is proven to be regulated by PKC pathway.Xiang-Qi-Tang could inhibit EPCR shedding in HUVECs.Furthermore,three monomers may be the main active component of Xiang-Qi-Tang,inhibiting EPCR shedding through different pathway.α-Cyperone could prevent PKC translocation to restrain EPCR shedding;Astragaloside IV could down-regulate the phosphorylation of MAPK and PKC activation in inhibition of EPCR shedding;Andrographolide could only reduce the phosphorylation of MAPK and then inhibit EPCR shedding.Conclusion:In this study,we established the PMA-induced EPCR shedding model in HUVECs.In addition,we verified the anti-EPCR function of Xiang-Qi-Tang in HUVECs.a-Cyperone,Astragaloside IV and Andrographolide were proven to exhibit their inhibiting ability for EPCR shedding.These results revealed pharmacological effects of these three components,and also provided a theoretical basis for the future treatment of EPCR-related diseases.