The Pharmacodynamics And Mechanism Of Antiplatelet Aggregation Of The Effective Substance From Rostellularia Procumbens (L.) Nees

Objective:To lay a foundation for the development and utilization of medicinal materials in Rostellularia procumbens（L.）Nees（RP）,we explored the effective part of anti-platelet aggregation in RP and explain its mechanism.Methods:1.The RP was percolate extracted with ethanol and then decompressed to recover the ethanol.After that we got the extract of RP.To determine the effective part of RP（RPE）,the sections were separated by macroporous adsorption resin from the extract of RP,and they were screened by tail thrombus mice.The potential effective components of RPE were separated by HPLC.2.Lactate dehydrogenase（LDH）text determined whether RPE and its effective components are toxic to platelets.To demonstrate RPE and its effective components have the effect of anti-platelet aggregation,we examined their effects on rabbit platelet aggregation rate by turbidimetric method.Collagen-coated cover slip were prepared to simulate the collagen which exposed after normal vessel damaged.To demonstrate RPE and its effective components have the effect antiplatelet adhesion,their effects on platelet adhesion was observed under fluorescence microscope.3.To prove that RPE and its effective components has anti-thrombotic effect in vivo by the pulmonary embolism model and tail thrombosis model.4.To detect the effect of RPE and its effective components on the content of P-selectin,thromboxane B₂（TXB₂）and 6-keto-PGF1αin serum from tail thrombus rats by ELISA.To detect the effects of RPE and its effective components on the expression of integrinβ₃ in the platelets from tail thrombus rats by Western blot,and to detect the effects of RPE and its effective components on the phosphorylation of related proteins Akt,JNK,ERK and p38 in the Gi-PI3k-MAPKs signaling pathway.Results:1.Purifing RP by macroporous adsorption resin,and got the group A,B,C₁ and C₂.Then to screen potential effective parts by tail thrombosis test,and C₁ was determined to have great activity.We define C₁as RPE.Five kind of compounds was separated by HPLC from RPE,including 6’-Hydroxy justicidin B,Justiscidin B（JDB）,chinensinaphthol methyl ether（CME）,neojusticin B,neojusticin A.The contents of JDB and CME are relatively large.2.The result of LDH test showed that RPE,JDB and CME had not significant cytotoxicity,and the release rate was less than3%.In platelet aggregation test,compared with the Control,when thrombin was inducer,RPE-M,RPE-H and JDB has the function of platelet aggregation（P<0.05）.RPE,CME and JDB has the function of platelet aggregation when ADP was inducer（P<0.05）.RPE-M,RPE-H,CME and JDB had anti-platelet aggregation effects when collagen was inducer（P<0.05）.The results showed that RPE-M,RPE-H and JDB has an antiplatelet effect,and CME inhibited platelet aggregation induced by ADP and collagen.Observing the platelet adhesion by fluorescence microscopy,the results showed that RPE,CME and JDB had anti-platelet adhesion（P<0.05）.3.Compared with model,RPE-M,RPE-H,CME and JDB could reduce lung coefficient of pulmonary embolism mice（P<0.05）,HE slice showed that the clots accumulated in the blood vessels of the model mice’s lungs,but the mice who was treated was normal.The results showed that RPE-H and JDB had a protective effect on pulmonary embolism in mice.RPE,CME and JDB could reduce the rate of black-tail rate in tail thrombus rats.The results showed that JDB,CME and RPE had antithrombotic effect in vivo.The platelet aggregation rate of the anticoagulant blood from the treatment group rat was determined by turbidimetry,the results showed that the platelet aggregation rate of RPE-M,RPE-H and JDB decreased（P<0.05）,while CME had antiplatelet aggregation activity only when ADP was used as inducer（P<0.05）.The results showed that RPE-M,RPE-H and JDB has an antiplatelet effect in vivo,and the antiplatelet aggregation effect of CME was not obvious in vivo.4.Detecting the content of P-selectin in serum from tail thrombus rats,compared with model,RPE,CME and JDB could alleviate the content of P-selectin increased（P<0.05）.Detecting the content of TXB₂,compared with model,JDB,RPE-M and RPE-H could alleviate the content of TXB₂ increased（P<0.05）.Detecting the content of 6-keto-PGF1αin serum of tail thrombus rats,compared with the model,JDB,RPE-M and RPE-H could alleviate the content of 6-keto-PGF1αreduced（P<0.05）.In the platelet of tail thrombus rats,western blot showed that compared with model,ASP could significantly reduce integrinβ₃ expression（P<0.05）and reduce p-Akt,p-ERK,p-JNK and p-p38 expression（P<0.05）.JDB can significantly reduce integrinβ3 expression（P<0.05）and reduce p-JNK and p-p38expression（P<0.05）.CME can reduce p-Akt and p-ERK expression（P<0.05）.RPE-L can can reduce p-ERK and p-JNK expression（P<0.05）.RPE-M can can reduce p-ERK,p-JNK and p-p38 expression（P<0.05）.RPE-H can significantly reduce integrinβ₃ expression（P<0.05）and reduce p-Akt,p-ERK,p-JNK and p-p38 expression（P<0.05）.Conclusion:1.In vitro anti-platelet aggregation test,anti-platelet adhesion test,pulmonary embolism test in mice and tail thrombosis test in rats,showed that RPE,JDB and CME have a definite anti-platelet aggregation effect.2.The effective substance of RP have anti-platelet aggregation effect.Because they can reduce the expression of P-selectin in the serum of rats with tail thrombosis and prevent the release of platelet particles.And they can reduce the generation of TXA₂ in the serum of rats with tail thrombosis and increase the production of PGI₂.They also can reduce integrinαⅡbβ₃expression and reduce related proteins involved in the Gi-PI3k-MAPKs signaling pathway phosphorylation,such as p-Akt,p-JNK,p-ERK and p-p38.