Study On Molecular Pharmacology And Metabolomics Of The Chinese Formula XXT Based On "Dual Therapy Of Blood And Vessel” Theory

According to the Chinese theory “Dual Therapy of Blood and Vessel”,modern molecular pharmacology and metabolomics were applied to investigate the activities of the Chinese medicine formula Xueshuan Xinmaining Tablet(XXT)from the two aspects “Treat Blood” and “Treat Vessel”,and to study the action mechanisms of XXT at gene,protein and metabolism levels.The results showed that XXT can ameliorate the status of blood flow via regulating F13a1,Car1 and Tbxa2 r genes,and thus reduce intrinsic resistance of blood circulation.Besides the results also demonstrated that XXT can treat vascular endothelial damage via up-regulating gene and protein expression in Nrf2 pathway,such as Keap1,Nrf2,HMOX1,GCLM and NQO1.Further investigation by metabolomics revealed that XXT exhibited the “Dual Therapy of Blood and Vessel” activity via normalizing the disturbed steroid hormone biosynthesis,arachidonic acid metabolism and sphingolipid metabolism.Finally six Q-markers of XXT that possess anti-FXa activity were detected in anti-coagulation assays.The achieved new findings are as follow.1.It is the first study on the “Treat Blood” activity of XXT by pharmacodynamics.The results showed that XXT significantly decreased hemorheological and coagulation function parameters including whole blood viscosity,plasma viscosity,hematocrit,erythrocyte aggregation index,prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT)and Fibrinogen(FIB),indicating that XXT possesses significant bio-activities against intrinsic resistance of blood circulation.2.It is the first study on the action mechanism of ameliorating blood status by PCR Array technique and by clustering analysis via Matlab software.The results demonstrated that compared to Normal Control group(NC),373 genes were up-regulated significantly and 62 genes were down-regulated significantly in Blood Stasis Model group(BM),and that compared to BM,307 genes were significantly up-regulated and 194 genes were significantly down-regulated in XXT-treated group(XXT).These differentially expressed genes are involved in celluar signaling transduction pathways including apoptosis,celluar migration and proliferation,and immune regulation.The current study also revealed that F13a1,Car1 and Tbxa2 r gene expression was elevated significantly in BM group,and was decreased after XXT pretreatment,suggesting that XXT can ameliorate blood flow status via modulating these genes.3.MTT chromatometry was applied to determine survival rates of Human Umbilical Vein Endothelial Cells(HUVECs),and FACS Calibur cytometry was performed to assess the level of intracellular reactive oxygen species(ROS),which demonstrated the bioactivity of XXT against vascular endothelial oxidative damage for the first time.4.It is the first study on the action mechanism of the bioactivity of XXT against vascular endothelial oxidative damage by PCR Array,Real-Time PCR and Western Blotting.The results showed that XXT significantly modulated the transcription regulation of Nrf and its downstream genes in a dose-dependent manner,and that XXT exhibited anti-oxidative activity in vascular endothelial cells via increasing the expression of proteins including Keap1,Nrf2,HMOX1,GCLM and NQO1 in Nrf2 signaling pathway.5.It is the first metabolomics study on XXT by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)combined with multivariable analysis,revealing the efficacy of XXT by modulating endogenous metabolite pathways including steroid hormone biosynthesis,arachidonic acid metabolism and Sphingolipid metabolism.It is also the first time to observe that blood stasis syndrome can cause the disturbance of steroid hormone biosynthesis in vivo,and Chinese formula can mitigate the syndrome via modulating the pathway,which provides a new therapeutic target and new insight for the treatment of blood stasis syndrome.6.It is the first time to select six bio-components with high anti-FXa activity including Rutin,Salvianolic acid B,cholic acid,Rg2,Rg3 and PPT from fourteen Q-markers and bio-ingredients of the Chinese formula XXT by chromogenic substrate method.Besides molecular docking was performed by Glide XP of Schr?dinger software to predict the interaction modes and accurate binding sites between anti-FXa inhibitors and FXa protein.These innovative findings comprehensively and efficiently revealed the molecular pharmacological effects and action mechanisms of “Dual Therapy of Blood and Vessel” of XXT,and lay a firm foundation for secondary development of the Chinese formula XXT.