Study On The Hemostatic Mechanism Of 20(S)-panaxadiol

Objective:The study screened the hemostatic components in ginseng and explore the hemostatic mechanism of 20（S）-panaxadiol.Methods:1、The components of ginseng were extracted and separated from ginseng.The article carries out content determination and qualitative identification of each component of ginseng,and the hemostatic components were selected by measuring the effect on the platelet aggregation induced by ADP in rats.2、The tail amputation model and liver scratch model experiment were used to observe the bleeding time.Automatic blood analyzer was used to analyze of rat blood routine.The effects of PD on coagulation parameters were investigated by automatic coagulation analyzer.The effect of PD on the rate of platelet aggregation was observed by colorimetry.3、In vitro experimental,platelet aggregation rate and adenosine triphosphate（ATP）release were analyzed by platelet aggregometer.Flow cytometry analysis was applied to find out the effects of protopanaxadiol on the release of CD62P and expression of PAC-1.Cytation 5 multifunctional microplate reader was used to detect the concentration of[Ca²⁺]_iin washed human platelets.Enzyme-linked immunosorbent assay was used to detect the content of cyclic adenosine monophosphate（c AMP）and cyclic guanosine monophosphate（c GMP）in rat platelets.The protein expression levels of AKT,p-AKT,PI3K,p-PI3K,GSK3βand p-GSK3βin platelets were determined by immunoblotting.Result:1、Ginsenosides can promote platelet aggregation,and 20（S）-ginsengdiol has the most significant effect on promoting platelet aggregation.2、20（S）-panaxadiol shorted bleeding time on the mouse tail amputation and liver scratch models and mainly increased blood platelet count in the rats.PD significantly increased RDW-SD and RDW-CV of red blood cell count（RBC）,and markedly increased platelet count（PLT）and plateletcrit（PCT）,high-dose group markedly increased P-LCR.Meanwhile,PD decreased APTT,increased FIB content,but have no effect on PT and TT.3、In vitro experiment,in the absence of Ca²⁺,PD promotes the release of[Ca²⁺]i and ATP,the level of CD62P expression and PAC-1 binding in platelets.In addition,PD increased phosphorylation of phosphoinositide 3-kinase（PI3K）,protein kinase B（PKB or Akt）and glycogen synthase kinase 3β（GSK3β）in human platelets.Excitingly,PD-induced changes including platelet aggregation,decreased c AMP content,and the increases of ATP,CD62P and PAC-1 were significantly reversed by vorapaxar,which showed similar function as thrombin.Conclusion:1、Ginsenoside can promote platelet aggregation,meanwhile PD is an essential hemostatic ingredient in Panax ginseng for promoting platelet aggregation.2、PD has a certain hemostatic effect.On the one hand,PD activates the endogenous coagulation system of rats,increases the content of FIB,affects the parameters of platelets and red blood cells in the body,promotes platelet aggregation;On the other hand,PD can promote Ca²⁺influx in platelets,then activate platelets,decreased c AMP content,increase the number of ATP,CD62P and PAC-1（the effect is calciumion-dependent）,activate PI3K/Akt/GSK3βsignaling pathway,induce platelet aggregation,and then hemostasis.At the same time,the hemostatic effect of PD can be inhibited by VP.