The Study On The Effect And Mechanism Of Sanguinarine On The Formation Of Arterial And Deep Venous Thrombosis

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Sanguinarine attenuates collagen induced platelet activation and inhibits arterial thrombus formation and the underlying mechanismObjective:Injured vascular endothelial with exposed collagen can stimulate platelet activation and lead to arterial thrombosis.Inhibition of platelet activation is the core strategy of anti-arterial thrombosis.Sanguinarine(a benzophenanthridine alkaloid)has been reported to have an antiplatelet activity.But its antithrombotic effect and the mechanisms of platelet inhibition have not thoroughly been explored.The goal of this thesis is to investigate the effect and molecular mechanism of sanguinarine attenuating collagen induced platelet activation and inhibiting thrombosis.Methods:Occlusion time in the Fe Cl₃ induced carotid arterial injury thrombosis model was tested.The Doppler flow probe was used to measure the blood flow in the proximal carotid artery.Bioflux microfluidic system was used for the evaluation of sanguinarine effect on platelet adhesion to collagen matrix under high shear stress.Platelet aggregometer was used to observe the effect of sanguinarine on collagen and CRP induced platelet aggregation.Flow cytometry was used to detect the effects of sanguinarine on the expression of P-selectin and PAC-1 binding and intracellular Ca²⁺concentration([Ca²⁺]i)during collagen-induced platelet activation.Fluorescence microscopy was used to observe the effect of sanguinarine on platelet spreading on immobilized fibrinogen.Western blot was used to analyze the effect of sanguinarine on molecular phosphorylation in platelet signal pathway.Results:1)Sanguinarine inhibited the formation of arterial thrombus in vivo and in vitro in a dose-dependent manner.2)Sanguinarine inhibited platelet aggregation induced by collagen and CRP in a dose-dependent manner.3)Sanguinarine dose-dependently decreased the P-selectin expression and PAC-1 binding on the collagen-stimulated platelets.Moreover,it also reduced platelet intracellular Ca²⁺concentrations([Ca²⁺]i)in a dose-dependent manner.4)Sanguinarine inhibited platelet spreading on immobilized fibrinogen.5)Western blot analysis showed that sanguinarine significantly inhibited the phosphorylation of downstream signal molecules including Syk,PLC?2,PI3K,Akt and GSK3?in GPVI receptor pathway.Sanguinarine also significantly reduced the phosphorylation of?3 and Src in the"outside-in"signal pathway mediated by integrin?IIb?3.Conclusion:Current study clearly indicates that sanguinarine inhibited the formation of arterial thrombosis in a dose-dependent manner.Moreover,sanguinarine also inhibited platelet aggregation,release and spreading.The underlying mechanisms are as follows:1)Inhibiting the downstream signal molecules of collagen-specific GPVI receptor signal pathway,including the phosphorylation of Syk-PLC?2,PI3K-Akt-GSK3?;2)Inhibiting collagen-induced intracellular calcium mobilization;3)Negatively regulating the?IIb?3"outside-in"signal transduction.Chapter ? Sanguinarine inhibits deep venous thrombosis by dampening Platelet-derived HMGB1Objective: Deep venous thrombosis(DVT)is a high incidence disease worldwide,its complications constitute a high mortality rate or a major medical burden.DVT can be considered as an aberrant sterile inflammatory process,where innate immunity drives clot formation.Platelet-derived HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis.The purpose of this study was to explore the effect of sanguinarine on anti-DVT and inhibiting platelet derived HMGB1 in venous thrombosis.Methods: Mouse model of flow restriction in the IVC was established to evaluate the effect of sanguinarine on the thrombosis rate of DVT and the size of venous thrombus.The effects of sanguinarine on neutrophil recruitment and NETs formation in venous thrombus were observed by immunohistochemistry and immunofluorescence.The effects of sanguinarine on the expression of inflammatory cytokines and DAMPs in thrombus were observed by immunohistochemistry.The effects of sanguinarine on platelet recruitment and plateletderived HMGB1 expression in thrombus were observed by immunofluorescence triple staining.The megakaryocytes of control mice and treatment mice were separated and purified by Fluorescence activated Cell Sorting.The effect of sanguinarine on the m RNA expression of HMGB1 in megakaryocytes and platelets was detected by RT-PCR,and the expression of HMGB1 protein was detected by western blot.Flow cytometry was used to detect the effects of sanguinarine on the expression of P-selectin and JON/A binding in DVT mice.Moreover,the effect of sanguinarine on the co-localized expression of PS and HMGB1 on platelet surface activated by agonists was detected with flow cytometry.ELISA was used to detect the effect of sanguinarine on the release of HMGB1 from platelets induced by agonists.Molecular simulation software Autodock was used to speculate the molecular docking site and affinity of sanguinarine and TLR4 receptor.The mouse model of flow restriction in the IVC were established in TLR4-/-mice to evaluate the anti-DVT effect of sanguinarine as a potential TLR4 ligand.TLR4-/-mice were used to evaluate the regulation of HMGB1,MYD88 and NF?B p65 m RNA expression in megakaryocytes and platelets by sanguinarine.Meg-01 cells were used to evaluate the regulation of HMGB1 protein by sanguinarine through TLR4-NF?B pathway.Results: 1)Sanguinarine inhibited venous thrombosis in mice with IVC stenosis in a dosedependent manner.2)Sanguinarine reduced neutrophil infiltration and NETs formation in venous thrombosis.3)Sanguinarine decreased the expression of HMGB1 in venous thrombosis but had no effect on the expression of IL1 ?,IL6 and TNF?.4)Sanguinarine inhibited platelet-derived HMGB1 in venous thrombosis.5)Sanguinarine reduced the content of HMGB1 in platelets by inhibiting the m RNA and protein expression of HMGB1 in megakaryocytes.6)Sanguinarine inhibited the release of platelet HMGB1 induced by agonists.7)Sanguinarine was a potential TLR4 inhibitor,which participated in the inhibition of DVT.8)Sanguinarine inhibited the expression of HMGB1,MYD88 and NF?B m RNA in megakaryocytes and platelets of mice.9)Sanguinarine regulated the protein expression of HMGB1 in Meg-01 cells by inhibiting TLR4-NF?B signal pathway.Conclusion: Sanguinarine inhibits deep venous thrombosis via restraining platelet-derived HMGB1 in thrombus.It acts as a potential TLR4 inhibitor to regulate TLR4-MYD88-NF?B signal pathway,which inhibits HMGB1 m RNA and protein expression in megakaryocytes.