The Role And Mechanisms Of CD36-Mediated Signaling In The Effects Of Panax Notoginseng Saponins On Oxidized Low Density Lipoprotein-Induced Platelet Apoptosis

ObjectiveCardiovascular diseases(CVDs)pose a grave threat people’s lives and health.Hyperlipidemia,one of important risks for CVDs,is associated with an increased chance of serious complications,including atherosclerosis(AS)and thrombosis.Oxidized low-density lipoprotein(ox-LDL)in the circulating blood of hyperlipidemic patients can induce platelet aggregation and activation,which plays an essential role in the formation and development of thrombosis.Platelet apoptosis contributes to the multiple progresses of CVDs;nevertheless,the mechanism by how hyperlipidemia induces platelet apoptosis and thus contributes to thrombosis remains unknown.Panax notoginseng saponins(PNS)is the primary active component extracted from the roots of Panax notoginseng,and it has a variety of pharmacological effects.Recent research has shown that PNS can inhibit platelet aggregation and activation induced by physiological agonists(e.g.,ADP,thrombin,etc.)in vitro.Nonetheless,the effect of PNS on platelet apoptosis in hyperlipidemic conditions has not been previously reported.In this in vitro study,we investigated the efficacy of PNS on ox-LDL-induced platelet apoptosis in platelets prepared from healthy subjects and clarified its possible molecular mechanisms.Our findings may have important theoretical and applied implications for the use of PNS in the prevention of CVDs.MethodsHuman washed platelets from healthy adults were pre-incubated with different concentrations of PNS(1,10,100 μg/mL)or the solvent control(0.05%DMSO)for 40 min,followed by stimulation by ox-LDL(50 μg/mL)or native LDL(n-LDL,50 μg/mL)for additional 60 min in vitro.Subsequently,the levels of Bak,Bax,and Bcl-2 expression,as well as caspase-3 and caspase-9 activation were detected by using Western blotting(WB).Depolarization of mitochondrial membrane potential(ΔΨm)and phosphatidylserine(PS)exposure were measured by flow cytometry.We used gp91ds-tat(a specific inhibitor of NOX2)and NAC(a ROS scavenger)to investigate whether PNS regulated ox-LDL-induced platelet apoptosis via regulating NOX2/ROS/ERK5 signaling pathway.Moreover,a PKA inhibitor H89 was used to investigate whether PNS modulated ox-LDL-induced platelet apoptosis through cAMP/PKA signaling pathway.Furthermore,we used a Src inhibitor PP2 to investigate whether PNS regulated ox-LDL-induced platelet apoptosis through Src/Syk signaling pathway.In addition,the CD36 inhibitor FA6-152 was used to investigate whether the action of PNS was through CD36 mediating down-regulation of Src/Syk signaling pathway,up-regulation of cAMP/PKA signaling pathway,inhibition of NOX2/ROS/ERK5 signaling pathway,and thus attenuating ox-LDL-induced platelet apoptosis.Results1.Our in vitro study found that PNS exhibited significant inhibition of ox-LDL-induced platelet apoptosis,including inhibiting ΔΨm depolarization,caspase-3 and caspase-9 activation,and PS exposure.Furthermore,in platelets stimulated by ox-LDL,PNS exhibited a significant decrease in the expression of pro-apoptotic proteins Bak and Bax,while significant increase in the expression of anti-apoptotic protein Bcl-2.2.Our study clarified that PNS significantly down-regulated the NOX2/ROS/ERK5 signaling pathway activated by ox-LDL.This down-regulation was observed through decreases in platelet p47phox phosphorylation,total reactive oxygen species(ROS)and superoxide production,as well as ERK5 phosphorylation.Moreover,ox-LDL increased platelet p47phox phosphorylation,total ROS generation,ERK5 phosphorylation,ΔΨm depolarization,caspase-9 activation,and PS exposure were considerably suppressed via gp91ds-tat(a selective inhibitor of NOX2).Furthermore,the combination of PNS and gp91ds-tat failed to demonstrate a synergistic inhibition on these markers.Furthermore,ROS generation,ERK5 phosphorylation and the activation of caspase-9 triggered by ox-LDL was suppressed by N-acetylcysteine(NAC),a reactive oxygen species scavenger.Moreover,the combination of PNS and NAC did not exhibit a synergistic inhibitory impact on ROS generation,ERK5 phosphorylation and caspase-9 activation.The data indicate that the inhibitory effects of PNS on ox-LDL-induced platelet apoptosis mainly through down-regulating the NOX2/ROS/ERK5 signaling pathway.3.In addition,PNS activated cAMP/PKA signaling pathway in platelets in response to ox-LDL,including elevating intracellular cAMP concentration and VASP(Ser157)phosphorylation.H89,a specific inhibitor for PKA,was found to completely reverse the increased VASP(Ser157)phosphorylation that was mediated by PNS.Additionally,H89 reversed the effects of PNS on ox-LDL-induced platelet p47phox phosphorylation,ROS generation,ERK5 phosphorylation,ΔΨm depolarization,and PS exposure.The data indicate that the inhibitory effects of PNS on ox-LDL-induced platelet apoptosis were also mediated by up-regulating the cAMP/PKA signaling pathway,resulting in down-regulating NOX2/ROS/ERK5 signaling pathway in response to ox-LDL.4.Additional research showed that PNS dramatically reduced the levels of Src and Syk phosphorylation activated by ox-LDL.A specific Src inhibitor PP2 reversed ox-LDL-induced phosphorylation of Src,Syk,intracellular cAMP level,VASP(Ser157)phosphorylation,ROS generation,ERK5 phosphorylation,ΔΨPm depolarization,caspase-9 activation,and PS exposure.the combination of PNS and PP2 failed to demonstrate a synergistic impact on these markers.The data indicate that via downregulating Src/Syk signaling pathway,promoting the cAMP/PKA signaling,and inhibiting the NOX2/ROS/ERK5 signaling pathway,PNS plays an important role in attenuating ox-LDL-induced platelet apoptosis.Finally,we demonstrated that no synergistic impact on the aforementioned indicators when PNS combined with an anti-CD36 antibody FA6-152.Collectively,our in vitro study indicates PNS attenuates ox-LDL-induced platelet apoptosis mainly through reducing CD36-mediated Src/Syk signaling,upregulating cAMP/PKA signaling pathway,and decreasing NOX2/ROS/ERK5 signaling pathway.ConclusionsOur in vitro study demonstrate that PNS can inhibit ox-LDL-induced platelet apoptosis by downregulating CD36-mediated Src/Syk signaling pathway and upregulating the cAMP/PKA signaling pathway,and thus inhibiting the NOX2/ROS/ERK5 signaling pathway.