The inhibitory effect of isoliquiritigenin on human platelets in vitro.

Background: Platelets play important roles in several physiological and pathological processes. Multiple antiplatelet drugs have been developed for clinical practice. The active components of traditional Chinese medicine with antithrombotic effects are promising drugs to modulate platelet function. In our study, the antiplatelet effect of isoliquiritigenin (ILTG) and its mechanisms were examined. Methods: Human platelet-rich plasma and a washed platelet suspension were prepared. Platelets were stimulated using collagen, thrombin, or adenosine diphosphate (ADP). The platelet lumi-aggregometer was applied to detect the aggregation of platelets and the release of adenosine triphosphate (ATP). The expression of P-selectin and the activation of integrin αIIbß3 were detected using flow cytometry. The spreading of platelets on a fibrinogen-coated surface was visualized using immunofluorescent staining. The mechanisms of the antiplatelet effect were investigated using Western blotting. Results: In this study, ILTG inhibited collagen- and thrombin-induced platelet aggregation, the release of dense granules and α-granules, and the activation of integrin αIIbß3 in a dose-dependent manner. In addition, ILTG suppressed the spreading of platelets on immobilized fibrinogen. In collagen-activated platelets, ILTG markedly inhibited the expression of phosphorylation of phospholipase C gamma-2 (PLCγ2) and protein kinase B (Akt). Conclusions: These results indicated that ILTG could inhibit the collagen- and thrombin-induced platelet aggregation and granule release via the glycoprotein VI-mediated signal pathway in vitro.

Pharmacological actions of neferine in the modulation of human platelet function.

Neferine has long been recognized as a medicinal herbal ingredient with various physiological and pharmacological activities. Although previous studies have reported its antithrombotic effect, the underlying mechanisms have not been thoroughly investigated. Since platelets play a key role in thrombosis, we investigated the effects of neferine on human platelet function and the potential mechanisms. Platelet aggregation, adhesion and spreading were performed to investigate the effect of neferine on inhibition of platelet function. Flow cytometry was used to determine platelet alpha granule secretion and integrin IIb/IIIa activation, as detected by CD62P (P-selectin) expression, PAC-1 and fibrinogen binding. Western blotting was utilized to investigate the effect of neferine on intracellular signaling of activated platelet. We found that neferine significantly suppressed platelet aggregation and remarkably promoted the dissociation of platelet aggregates induced by collagen, thrombin, U46619, ADP and adrenaline in a dose-dependent manner. Flow cytometry analysis showed that neferine inhibited thrombin-induced platelet P-selectin expression, PAC-1 and fibrinogen binding. In addition, neferine reduced the adhesion of human platelets on coated collagen under both static and shearing condition at an arterial shear rate of 40 dyne/cm2. Neferine also inhibited the spreading of human platelets on immobilized fibrinogen. Western blot analysis showed that neferine inhibited PI3K activation, and decreased the levels of phosphorylation of Akt, GSK3ß and p38 MAPK in platelets. In summary, neferine has the potential to be an antiplatelet and antithrombotic agent by inhibiting the PI3K-Akt-GSK3ß/p38 MAPK signaling pathway.

Resolvin D1 attenuates imiquimod-induced mice psoriasiform dermatitis through MAPKs and NF-κB pathways.

BACKGROUND: Resolvin D1 (RvD1), a pro-resolution lipid mediator derived from docosahexaenoic acid (DHA), has been described to promote several kinds of inflammatory resolution. However, the effects and anti-inflammatory mechanisms of RvD1 on psoriasis have not been previously reported. OBJECTIVE: The present study aimed to determine the protective effects and the underlying mechanisms of RvD1 on imiquimod (IMQ)-induced psoriasiform dermatitis. METHODS: Mice were topically treated with IMQ to develop psoriasiform dermatitis on their shaved back, pretreated intraperitoneally (i.p.) with or without RvD1 or tert-butoxycarbonyl Met-Leu-Phe peptide (Boc), a lipoxin A4 (ALX) receptor antagonist. The severity was monitored and graded using a modified human scoring system, the Psoriasis Area and Severity Index (PASI), histopathology, and the signature cytokines of psoriasis (IL-23, IL-17, IL-22 and TNF-α). The mRNA and protein levels of inflammatory cytokines were quantified by quantitative real-time PCR (QRT-PCR) and ELISA. The expressions of signaling proteins MAPKs and NF-κB p65 were analyzed using western blotting. Electrophoretic mobility shift assay (EMSA) was used to check NF-κB p65 DNA binding activity. RESULTS: Our study showed that RvD1 alleviated IMQ-induced psoriasiform dermatitis and improved skin pathological changes. RvD1 markedly inhibited IMQ-induced activation of ERK1/2, p38, JNK (c-Jun N-terminal protein kinase, a subfamily of MAPKs), and NF-κB. Furthermore, pretreatment with Boc, would not exacerbate skin inflammation of IMQ-induced mice, but significantly reversed the beneficial effects of RvD1 on IMQ-induced psoriasiform inflammation. CONCLUSION: RvD1 can obviously improve skin inflammation in IMQ-induced mice psoriasiform dermatitis. The protective mechanisms might be related to its selective reaction with lipoxin A4 receptor/Formyl-peptide receptor 2 (ALX/FPR2), by downregulating relevant cytokines of the IL-23/IL-17 axis expression, the inhibition of MAPKs and NF-κB signaling transduction pathways. Thus, these results show that RvD1 could be a possible candidate for psoriasis therapy.

Antiplatelet activity of loureirin A by attenuating Akt phosphorylation: In vitro studies.

Loureirin A is a flavonoid extracted from Dragon׳s Blood that has been used to promote blood circulation and remove stasis in Chinese traditional medicine. However, the mechanisms of these effects are not fully understood. We explored the anti-platelet activity and underlying mechanism of loureirin A in vitro. Our results indicated that loureirin A negatively affected agonist-induced platelet aggregation such as collagen, collagen-related peptide (CRP), ADP and thrombin. Loureirin A inhibited collagen-induced platelet ATP secretion and thrombin-stimulated P-selectin expression in a dose-dependent manner. Platelet spreading on immobilized fibrinogen was significantly impaired in the presence of loureirin A. Immunoblotting analysis indicated that 100µM of loureirin A almost completely eliminated collagen-induced Akt phosphorylation at Ser473. Interestingly, a submaximal dose (50µM) of loureirin A had an additive inhibitory effect with the phosphoinositide 3-kinase (PI3K) inhibitor Ly294002 on collage-induced Akt phosphorylation in platelets. Taken together, loureirin A had an inhibitory effect on platelet activation, perhaps through an impairment of PI3K/Akt signaling.

Neferine exerts its antithrombotic effect by inhibiting platelet aggregation and promoting dissociation of platelet aggregates.

INTRODUCTION: Neferine, a kind of isoquinoline alkaloid, extracted from the seed embryo of Nelumbo nucifera Gaertn, has long been recognized in traditional medicine as a medicinal plant with various usages. Neferine has many biological activities, including anti-hypertensive, anti-arrhythmic, negative inotropic effect and relaxation on vascular smooth muscle. Although previous studies have reported its antithrombotic effect, the mechanisms by which it exerts antithrombotic effect have not been thoroughly studied. METHOD: Washed mice platelets and mice platelet-rich-plasma (PRP) were used to investigate the effects of neferine on platelet aggregation, secretion induced by various agonists and dissociation of agonist-formed platelet aggregates. Bioflux plates coated with collagen were used to investigate the effect of neferine on platelet adhesion and thrombosis in vitro. With collagen-epinephrine-induced acute pulmonary thrombus formation mouse model, the effect of neferine on thrombosis in vivo was also examined. RESULTS: Neferine, significantly and dose-dependently, inhibited collagen-, thrombin-, U46619-induced platelet aggregation in mice washed platelets, or ADP-induced platelet aggregation in PRP. Neferine treatment decreased platelet dense granule secretion initiated by collagen, thrombin and U46619. Also, Neferine dramatically and dose-dependently promoted the dissociation of platelet aggregates pre-formed by various agonists including collagen, thrombin, U46619 or ADP. Neferine can significantly reduce the area of mice platelets adhesion to the collagen and inhibit thrombosis in vitro. In collagen-epinephrine-induced acute pulmonary thrombus mouse model, neferine, at 6 mg/kg, significantly attenuated thrombus formation. CONCLUSIONS: Neferine remarkably prevents thrombus formation by inhibiting platelet activation, adhesion and aggregation, as well as promoting disassembly of pre-formed platelet aggregates. The inhibitory effects of neferine on platelet activation might be relevant in cases involving aberrant platelet activation where neferine could be used as an anti-platelet and antithrombotic agent.