Study On The Mechanism For The Anti-platelet Aggregation Of The Active Substance In Rostelluaria Procumbens(L.)

Object: Explores the target protein of anti-platelet aggregation effect of the active substance in Rostelluaria Procumbens(L.),and lay the foundation for the study of its mechanism.Method: Seven kinds of inducers were used to induce platelet aggregation,and the maximum inhibitory rate of antiplatelet aggregation of monomeric compounds was determined by optical nephelometry,suspected active ingredients were initially identified.Analyzed and compared the suspected active ingredients with the rat blood component.The luminescent properties of lignans in Rostelluaria Procumbens(.Lin)Nees were determined by thin layer chromatography.Isolation the platelet total protein using natural gel electrophoresis technology,get the protein band,and incubation with a suspected active compound having a fluorescent property,the target protein band is determined by fluorescence properties.Cut the target protein band,enzymatic hydrolysis of the gelatinous protein,and the LC-MS / MS technique was used to analyze the information such as charge,peak and so on.Identify the protein in the gel and determine the suspected target protein.The expression of suspected target protein was verified by Western Blot method,and the mechanism of antiplatelet aggregation was speculated.Result: The results of optical turbidimetry show that justicidin B,neojusticidin A,neojusticidin B,6?-hydroxy justicidin B and chinensinaphthol methyl ether had different inhibitory effects on platelet aggregation induced by different inducers.When platelet aggregation was induced by AA,the anti-aggregation effect of justicidin B was the strongest,it's MIR was 97.04 ± 0.85%,which was higher than that of aspirin 93.43 ± 2.81%.When platelet aggregation was induced by ADP,the anti-aggregation effect of justicidin B,neojusticidin A,neojusticidin B,6?-hydroxy justicidin B and chinensinaphthol methyl were weaker than aspirin.In the case of PMA-induced platelet aggregation,the anti-agglutination effect of 6'-hydroxylithinein B is comparable to aspirin.When platelet aggregation was induced by PAF,the anti-aggregation effect of justicidin B and chinensinaphthol methyl were higher than aspirin.When thrombin induces platelet aggregation,the anti-agglutination effect of the justicidin B is comparable to aspirin.Rat blood composition analysis,justicidin B,6?-hydroxy justicidin B and chinensinaphthol methyl ether were the prototype ingredients.Justicidin B,neojusticidin A,neojusticidin B and chinensinaphthol methyl ether have obvious blue fluorescence under the UV lamp 365 nm,and 6?-hydroxy justicidin B no fluorescence.In native-PAGE test,the target protein band was determined by the fluorescence properties of the suspected active compound.LC-MS / MS technique was used to identify a number of proteins from the target protein band,and Integrin ? 3(Protein name:Integrin beta;Gene name:ITGB3;The biological source is:Oryctolagus cuniculus)was found to be a suspected target protein.Western Blot test results showed that the expression level of Integrin ? 3 of justicidin B and aspirin was significantly down-regulated(P <0.05)compared with model group,and was significant difference with it.Conclusion: Integrin ? 3 protein is one of the target proteins of Rostelluaria Procumbens(.Lin)Nees to play its antiplatelet aggregation effect.Rostelluaria Procumbens(.Lin)Nees may achieve its antiplatelet aggregation by inhibiting the expression of Integrin ? 3 protein.