Curdione Inhibits Thrombin-Induced Platelet Aggregation Via Regulating The AMP-Activated Protein Kinase-Vinculin/Talin-Integrin ??b?3 Signaling Pathway

The incidence of thrombotic diseases and disability caused by these diseases has been increasing yearly in China and has become the leading cause of death.Thrombosis is defined as a blood clot or deposit in the blood that forms a deposit on the inner membrane or blood vessel wall of the organ,causing the blood vessel to stenosis or obstruction,causing ischemia and infarction in various organs.Clinical manifestations arecerebrovasculardiseases,coronaryatherosclerosisandpulmonary thromboembolism.At present,there are many drugs for the treatment of thrombosis in the clinic.The anti-thrombotic effects are exerted by regulating different mechanisms,which are mainly divided into antiplatelet drugs,thrombolytic drugs and anticoagulant drugs,which are at different stages of the blood coagulation process and the formation of thrombus.However,antiplatelet drugs are an important method for the treatment of thrombotic diseases.And with the widely use of these drugs,the adverse reactions and bleeding events caused by them have received more and more attention.Therefore,exploring the pathological mechanism of platelet activation,finding new targets for prevention and treatment of platelet activation,and developing safe and effective drugs are urgent problems to be solved.They are very effective in solving the safety problems caused by antiplatelet drugs and preventing and treating thrombotic diseases.Modern medical research has found that curdione isolated from Curcuma arborescens oil,is a major effective component in Rhizoma Curcumae oil and is commonly used as a quality control indicator.Long-term research has found that curdione could inhibits platelet activation and aggregation when thrombin as an inducer.When exploring its target and specific mechanism,it was found that anti-platelet aggregation of curdione is related to vinculin/talin-mediated integrin signaling pathway.Moreover,the literatures reported that AMPK could regulate thrombin-induced platelet activation.Whether AMPK is involved in the vinculin/talin-mediated integrin signaling pathway,thereby promoting aggregation,and the specific anti-platelet molecular mechanism of curdione are still unclear and need further study.ObjectiveTo investigate whether curdione affects thrombin-induced platelet aggregation.The expression levels of the AMPK signaling molecule and integrin?IIb?3 signaling pathway-related proteins were examined using western blot and RT-PCR.The binding of vinculin and talin were studied using immunoprecipitation,immunofluorescence double staining and microscale thermophoresis.To clarify the mechanism of anti-platelet aggregation of curdione.MethodsThe venous blood of healthy volunteers was collected,and PRP was get by centrifugation.Then,the washed platelets were grouping in NS group,thrombin group,curdione group and tirofiban group.?1?Separation and extraction of curdione:20 g of zedoary oil was separated on silica gel using petroleum ether-acetic ether to yield three fractions.And recrystallized from petroleum ether to obtain curdione?>97%?.?2?Purity and structural analysis of curdione:The purity of curdione was determined by high-performance liquid chromatography?HPLC?using the ratio of the peak area and identified by its ¹H and ¹³C NMR spectra?Bruker Avance AV400?.The purity of curdione was greater than 97%.?3?Platelet aggregation studies:the washed platelets were added to the container and added with NS?vehicle?,tirofiban,or different concentrations of the experimental chemicals or curdione for the indicated times,and aggregation was induced by 0.02U/mL thrombin.At least three independent experiments were performed on platelets from different individuals.?4?Protein expression and purification:According to the amino acid sequence of the talin R3 subdomain fragments?TD,amino acids 795-911?and VD1?VD,amino acids1–258?,codon-optimized gene synthesis was performed in E.coli.Protein was produced in competent E.coli?DE3?.After centrifugation,the soluble fraction was loaded on the Ni-NTA resin.Finally,TD and VD proteins were further purified with S200 gel filtration chromatography.?5?Western blot experiments:the washed platelets were treated with normal saline?NS,vehicle?,tirofiban,or different concentrations of the experimental chemicals or curdione for the indicated times,and then induced by 0.02 U/mL thrombin to detect the expression of AMPK,vinculin,talin and integrin?IIb?3 protein.?6?RT-PCR experiments:the washed platelets were treated with normal saline?NS,vehicle?,tirofiban,or curdione for the indicated times,and then induced by 0.02 U/mL thrombin to detect the mRNA levels of AMPK,vinculin,talin and integrin?IIb?3.?7?Double immunofluorescence staining:the double immunofluorescent staining method was used observe the binding of talin and vinculin.?8?Immunoprecipitation and Western Blot:double-direction immunoprecipitation method was used validate the binding of talin and vinculin.And analysis was performed by Western blot.?9?Microscale Thermophoresis:double-direction fluorescently labeled constant concentration of TD,titrated with different concentrations of VD,protein binding was detected by fluorescence intensity curve.Results?1?The results of HPLC analysis showed that the purity of curdione was more than 97%.?2?The structures of curdione identified by ¹H and ¹³C NMR spectra.?3?The results of platelet aggregation showed that the platelet aggregation rate decreased after incubation with curdione,and it showed the same trend as the positive drug control group tirofiban.?4?Western blot experiments results showed that the domain fragment VD and TD were successfully synthesized.?5?Western blot experiments results showed that curdione inhibited the expression of talin and vinculin and phosphorylation of AMPK and integrin?IIb?3 at protein level.?6?RT-PCR experiments results showed that curdione inhibited the expression of talin and vinculin at mRNA level.?7?Double immunofluorescence staining research showed that curdione inhibited the co-localization of talin and vinculin in washed platelets.?8?Immunoprecipitation experiments showed that curdione inhibited the binding of talin and vinculin in washed platelets.?9?MST experiments showed that curdione inhibited the binding of talin and vinculin in washed platelets.ConclusionsIn present study,we explored the mechanism of inhibition of platelet aggregation by curdione.Our findings demonstrated that the thrombin-induced platelet aggregation was mediated by the AMPK-vinculin/talin-integrin?IIb?3 signaling pathway.Our investigation also demonstrated that curdione not only could inhibit the phosphorylation of AMPK and down-regulate the expressions of talin/vinculin,but also could inhibit the binding of vinculin and talin,and the phosphorylation of integrin?IIb?3.Our study may clarify the mechanism of curdione,as a national medicine,the effects of inhibition of aggregation.