Ligustrazine Suppresses Platelet Activation Via Regulating MiRNAs And Related Signaling Pathways

Objective:To evaluate the effect of ligustrazine(Ligustrazine Hydrochloride,LH)on anti-platelet activity in vitro and in vivo,and explore its underlying by determining the different expression of miRNA and their target genes,as well as activation of related signaling pathway.The current study will be helpful for us on understanding the underlying mechanism of ligustrazine on anti-platelet,and it will provide experimental evidences for clinical use of ligustrazine on anti-platelet.Methods:1.In vitro study:Rat platelet rich plasma(PRP)pre-treated with various concentrations of LH(1,2 or 3 mM)in vitro following by Adenosine diphosphate(ADP,5.5?M)stimulation was used in this study and platelet aggregometer was used to determine the aggregation of platelet;Flow Cytometer was performed to detect the intracellular Ca2+mobilization;Enzyme linked immunosorbent assay(ELISA)was used to evaluate the thromboxane B2(TXB2)secretion;Western-blot was used to determine the expression and phosphorylation of integrin ?IIb?3.In vivo study:Sprague-Dawley(SD)rat were pretreated with or without LH(80 mg/kg/d),followed by injection of adrenaline(1 mg/kg)to construct the platelet over-activated mouse model.A modified tail cutting method was used to evaluate the bleeding risk.PRP from each group was stimulated with ADP(5.5 ?M),and platelet aggregometer was used to determine the aggregation of platelet;Flow Cytometer was performed to detect the intracellular Ca2+ mobilization;ELISA assay was used to evaluate the TXB2 and 6-keto-PGF1? secretion.2.Western-blot was performed to determine the phosphorylation of AKT,ERK and p38MAPK in platelet pretreatned with various concentrations of LH(1,2 or 3 mM)treatment following by ADP(5.5 ?M)or phosphorylation of AKT in platelet pre-treated with LH(80 mg/kg/d)following by injection of adrenaline(1 mg/kg)and stimulation of ADP(5.5 ?M).AKT pathway activator(insulin-like growth factor 1,IGF-1;300 mM)was used to stimulate the PRP,which pretreated with or with various concentrations of LH(1,2 or 3 mM),and Western-blot was performed to determine the expression and phosphorylation of AKT in platelet;Platelet aggregometer was used to determine the aggregation of platelet;Flow Cytometer was performed to detect the intracellular Ca2+ mobilization;ELISA assay was used to evaluate the TXB2 secretion.3.miRNA TLDA was performed to screen the different expression of miRNA in platelet of SD rat pretreated with or without LH(80 mg/kg/d)followed by injection of adrenaline(1 mg/kg).Target gene prediction and signaling pathway analysis was performed to assess the activation of different expressed miRNA related signaling pathways;Western-blot was used to determine the expression of AKT pathway negative regulator PTEN,THEM4 and PHLPP on protein level.Result:1.In vitro:LH treatment significantly inhibited ADP-induced platelet aggregation,as well as TXB2 secretion,intracellular Ca2+ mobilization and integrin caIIb?3 phosphorylation.In vivo:LH treatment significantly rescued Adrenaline-induced shortening of bleeding time in vivo,and inhibited platelet aggregation,intracellular Ca2+ mobilization and TXB2 and secretion,as well as increased the 6-keto-PGF1? secretion.2.LH treatment profoundly inhibited ADP induced AKT,ERK and p38MAPK phosphorylation in platelet in vitro.Moreover,LH pretreatment significantly decreased the phosphorylation of AKT in platelet induced by adrenaline in ex vivo.Furthermore,LH treatment significantly inhibited Akt phosphorylation,platelets aggregation,TXA2 secretion and intracellular Ca2+mobilization of platelet induced by IGF-1.3.LH treatment significantly decreased the expression 39 miRNAs;PI3K/AKT signaling pathway was one of the significantly enriched signaling pathways;LH treatment significantly down-regulated the expression of miR-331-3p and miR-34a-5p,and up-regulated their target gene PHLPP expression on protein level,which is one of AKT pathway negative regulator.Conclusion:LH treatment inhibited platelet aggregation in vitro and in vivo,and by down-regualting the expression of 39 miRNAs,including miR-331-3p and miR-34a-5p,which further up-regulated their target gene PHLPP and regulated the activation of AKT pathway maybe its underlying mechanism on anti-platelet.