Study On The Intervention Of Xiang-Qi-Tang On RAECs Injury With Toxin-Heat And Blood Stasis Syndrome Based On PC-EPCR Pathway

Background: Toxin-heat and blood stasis syndrome is a Traditional Chinese Medicine(TCM)syndrome type with fever,blood stasis and bleeding of various organs as the main pathological symptoms.Endothelial protein C receptor(EPCR)is a transmembrane protein mainly present on aortic endothelial cells,which plays an anticoagulant role in protein C(PC)signaling pathway.The soluble EPCR indicates the injury of vascular endothelium,and it also indicates that the appearance of petechia is related to the injury of vascular endothelium.Xiang-Qi-Tang(XQT)is a traditional Chinese herbal compound which is effective on promoting blood circulation to remove blood stasis,so it may have a good therapeutic effect on this syndrome.Aims: We studied the effect of XQT in rat aortic endothelial cells(RAECs)mediated by serum of rat with toxin-heat and blood stasis syndrome through PC-EPCR signaling pathway.Then we elaborate the interventional mechanism of this pathway by exploring the expression of protein and gene related with the signaling pathway.Methods: Vascular endothelial cells were treated with serum of rat with toxin-heat and blood stasis syndrome.The morphology of the cells was observed by transmission electron microscope,and the cell viability was detected by CCK-8.We use ELISA,q RT-PCR and Western Blot to detect EPCR,soluble EPCR(s EPCR),Thrombomodulin(TM),soluble TM(s TM),inflammatory factors IL-1β and TNF-α,apoptosis-related factors Bcl-2 and BAX,and cell barrier protective factors SPHK1 and S1PR1.Basing on the changes of the above target proteins,we assess the optimal serum concentration to establish the toxin-heat and blood stasis syndrome cell model.Then the same method was used to detect the cells treated with XQT and its components.We investigated the effects of XQT on cell morphology and the intervention of the above-mentioned target proteins to explore the therapeutic effects of XQT on the cell model of heat toxicity and blood stasis syndrome.Based on the above studies,we use ELISA,q RT-PCR and Western Blot to detect the effect of XQT on the activation efficiency of APC.After blocking the EPCR on endothelial cells by EPCR-blocking antibody,we explore the intervention mechanism through the effect of XQT and its components on the expression of proteins and genes regulated by PC-EPCR signaling pathway.Results: 1.Using 5%,10%,15% serum of rat with toxin-heat and blood stasis syndrome to stimulate endothelial cells leads to the reducing of cell viability.It can also destroy the cell morphology and organelle structure,promote the shedding of EPCR and TM,increase inflammatory factors IL-1β and TNF-α.Compared with the blank group,the anti-apoptotic factor Bcl-2 was inhibited and the protein and gene expression of apoptotic factor BAX was promoted,while the protein and gene expression of cell barrier related factors SPHK1 and S1PR1 were inhibited.After comprehensive investigation,we selected 10% serum of rat with toxin-heat and blood stasis syndrome to stimulate endothelial cells for 24 hours to build the cell model of toxin-heat and blood stasis syndrome.2.XQT and its components have certain therapeutic effects on the toxin-heat and blood stasis syndrome cell model mediated by 10% serum of rat with heat toxicity and blood stasis syndrome.XQT could restore the histology structure of damaged cells.Further studies showed that XQT and its components could inhibit the shedding of EPCR and TM,reduce the secretion of inflammatory factors IL-1β and TNF-α,maintain Bcl-2,downregulate the protein and gene expression of BAX,and promote the gene and protein expression of SPHK1 and S1PR1 in cell model of toxin-heat and blood stasis syndrome.3.XQT and its components not only inhibit EPCR shedding,but also promote its intracellular expression to a certain extent.XQT and its components can improve the activation efficiency of APC.After blocking the PC-EPCR signaling pathway,we found that XQT could not effectively reduce the secretion of IL-1β.it could play a role in antiapoptosis and cell barrier protection by increasing Bcl-2 and down-regulating the gene and protein expression of BAX,and increasing the gene and protein expression of SPHK1 and S1PR1.Conclusions: We could build the cell model of toxin-heat and blood stasis syndrome by stimulated by 10% serum of rat with toxin-heat and blood stasis syndrome.XQT and its components have certain therapeutic effects on the cell model of heat toxicity and blood stasis syndrome and may have an effect on anti-inflammation,anti-apoptosis and cell barrier protection by interfering with PC-EPCR signaling pathway.It indicates that XQT has pharmacological effect on improving the cell injury of toxin-heat and blood stasis syndrome.This study enriches the pharmacodynamic study of XQT,and has certain reference value for the treatment of toxin-heat and blood stasis syndrome in clinic.