The Role And Mechanisms Of CD36/NOX2 Signaling Pathway In The Effects Of Sulforaphane On Oxidized Low Density Lipoprotein-induced Platelet Activation

BackgroundThe incidence of cardiovascular diseases（CVDs）is increasing,and it has gradually become one of the diseases that affect human life and health.Hyperlipidemia is one of the important risk factor of CVDs.Atherosclerosis and thrombosis are important cardiovascular events in the pathogenesis of many CVDs.Platelets are mainly derived from megakaryocytes in bone marrow and lungs.They have no nucleus but complete plasma membrane Studies have shown that platelets not only play a key role in the normal physiological process of hemostasis and coagulation,but also participate in the process of atherosclerosis and thrombosis formation in hyperlipidemia.Platelet CD36 plays a key role in the process of platelet activation,aggregation,adhesion and thrombosis mediated by hyperlipidemia.High levels of oxidized low density lipoprotein（ox-LDL）in the circulation of patients with hyperlipidemia trigger a series of intracellular signaling events mainly by recognizing and binding to platelet CD36 receptor,These signaling pathways further activates ultimately increases platelet surface expression of CD62P and CD63 and release of soluble proteins such as PF4 and CCL5.These markers promote platelet aggregation and adhesion,and further participate in the formation of atherosclerosis and thrombosis.Dietary intervention is one of the important ways to prevent development of CVDs.Sulforaphane（SFN）are isothiocyanate phytochemicals and abundant in cruciferous vegetables such as broccoli,cauliflower,cabbage,kale and turnips.SFN has a variety of biological effects,such as anti-tumor and regulation of immune metabolism.In recent years,studies have shown that SFN can also inhibit platelet aggregation and activation,thereby inhibiting thrombosis.However,the effect of SFN on platelet activation and aggregation in hyperlipidemic environment is still unclear.This study investigated the effects of SFN on ox-LDL-induced platelet aggregation and activation,as well as to explore its possible molecular mechanism.Our findings may have important theoretical and applied implications for the prevention and treatment of CVDs by SFN in the way of nutritional diet.Objective1.To investigate the effect of SFN on platelet activation and aggregation in healthy adults induced by ox-LDL in vitro.2.To further investigate whether the effects of SFN on ox-LDL-induced platelet aggregation and activation through CD36-mediated signaling pathway.Our study may provide important theory and application value for SFN in the prevention and treatment of CVDs.Research objects and methods1.To investigate the effect of SFN on platelet aggregation and activation induced by ox-LDL in vitroPurified healthy human platelets were incubated with different concentrations of SFN（1μM,2.5μM,and 5μM）or solvent control（0.05%DMSO）for 40 min in vitro.Platelets were further incubated with ox-LDL（50μg/m L）for 20 min.The platelet aggregation was measured by platelet aggregometer.The expression of CD62P on platelet surface was dectected by Flow cytometry.The release levels of platelet PF4and CCL5 were detected by ELISA.2.To investigate whether the effect of SFN on ox-LDL-induced platelet hyperreactivity was through CD36 mediated signaling pathwayGp91-ds-tat,an inhibitor of NOX2,was used to investigate whether SFN regulated platelet function through NOX2/ROS/ERK5 signaling pathway.PKA specific inhibitor H89 was used to investigate whether SFN regulated platelet function through PDE3A/c AMP/PKA signaling pathway.Src family kinase inhibitor PP2 was used to investigate whether SFN regulated platelet function through Src/Syk signaling pathway.CD36 inhibitor FA6-152 was used to investigate whether SFN regulated platelet function through CD36-mediated signaling pathway.Results1.In vitro,we found that SFN inhibited ox-LDL-induced platelet aggregation in healthy adults and suppressed platelet surface CD62P expression,and release of PF4and CCL5.2.SFN significantly inhibited ox-LDL-induced activation of NOX2/ROS/ERK5signaling pathway in vitro.For example,SFN inhibited the phosphorylation of p47^phoxand ERK5,total intracellular ROS production.Using NOX2 specific inhibitor gp91ds-tat,we found that gp91ds-tat significantly inhibited phosphorylation of p47^phoxand ERK5,total ROS production,platelet aggregation and CD62P expression.There was no synergistic inhibitory effects on the above indexes when gp91ds-tat combined with SFN.These results suggest that SFN attenuates ox-LDL-induced platelet activation and aggregation through NOX2/ROS/Erk5 signaling pathway.3.Further mechanistic research found that,under the stimulation of ox-LDL,SFN can up-regulate the c AMP/PKA signaling pathway,such as increasing the c AMP content in platelets and up-regulating the phosphorylation of VASP(Ser¹⁵⁷).Using a specific inhibitor of PKA H89,we found that H89 down-regulated VASP(Ser¹⁵⁷)phosphorylation,promoted platelet aggregation and CD62P expression.H89 reversed the inhibitory effect of SFN on platelet aggregation and platelet CD62P expression.These results indicated that SFN could up-regulate the c AMP/PKA signaling pathway and inhibit platelet aggregation and activation induced by ox-LDL.4.In addition,SFN also inhibited platelet activation and aggregation by down-regulating Src/Syk signaling pathway.For example,SFN inhibited the phosphorylation of Src(Tyr⁴¹⁶)and Syk(Tyr³²³).By using the Src family kinase inhibitor PP2,we found that PP2 inhibited ox-LDL-induced phosphorylation of Src(Tyr⁴¹⁶)and Syk(Tyr³²³)in human platelets.PP2 combined with SFN did not show synergistic effects.These results suggest that SFN attenuates platelet function through down-regulating Src/Syk signaling pathway.Furthermore,the role of Src/Syk signaling pathway in the ox-LDL-regulated c AMP/PKA signaling pathway and NOX2/ROS/ERK5 signaling pathway was investigated.The results showed that PP2could increase VASP(Ser¹⁵⁷)phosphorylation,inhibit p47^phoxphosphorylation,ROS production,and ERK5 phosphorylation,and inhibit platelet aggregation and platelet CD62P exppression.PP2 had no synergistic effect when combined with SFN.These results indicate that SFN can inhibit platelet function by down-regulating Src/Syk signaling,thus up-regulating c AMP/PKA signaling pathway and inhibiting NOX2/ROS/ERK5 pathway.5.Finally,we found that SFN inhibited Src/Syk signaling pathway,upregulated platelet c AMP/PKA signaling pathway,and inhibited NOX2/ROS/ERK5 signaling,mainly through down-regulating CD36.For example,CD36 inhibitor FA6-152 could down-regulate the phosphorylation of Src and Syk,up-regulate the phosphorylation of VASP(Ser¹⁵⁷),and inhibit the phosphorylation of p47^phoxand ERK5 and ROS generation in human platelets induced by ox-LDL.FA6-152 inhibited platelet aggregation and activation.However,FA6-152 had no synergistic inhibitory effect on when combined with SFN.These results suggest that SFN inhibits platelet aggregation and activation mainly by down-regulating CD36-mediated Src/Syk signaling pathway,up-regulating the c AMP/PKA signaling pathway,thereby down-regulating the NOX2/ROS/ERK5 signaling pathway.Conclusions1.SFN inhibited ox-LDL-induced platelet aggregation and activation in human platelets in vitro.2.SFN inhibited ox-LDL-induced platelet function mainly through down-regulating CD36-mediated Src/Syk signaling,which in turn upregulated c AMP/PKA signaling pathway and inhibited NOX2/ROS/ERK5 signaling pathway.