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Pharmacological actions of nobiletin in the modulation of platelet function.

Vaiyapuri S, Roweth H, Ali MS, Unsworth AJ, Stainer AR, Flora GD, Crescente M, Jones CI, Moraes LA, Gibbins JM - Br. J. Pharmacol. (2015)

Bottom Line: The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice.Nobiletin extended bleeding time in mice and reduced the phosphorylation of PKB (Akt) and PLCγ2 within the collagen receptor (glycoprotein VI)-stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of vasodilator-stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling.Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK.

No MeSH data available.


Related in: MedlinePlus

Nobiletin inhibits agonist-induced platelet activation. Human-washed platelet aggregation performed in the presence or absence of various concentrations of nobiletin was monitored for 5 min by optical aggregometry following stimulation with 1 μg·mL−1 (A and B) and 5 μg·mL−1 collagen (C and D), 0.5 μg·mL−1 (E and F) and 1 μg·mL−1 CRP-XL (G and H) and 0.1 U·mL−1 thrombin (I and J). Similarly, the effects of nobiletin on PRP were analysed following stimulation with 1 μg·mL−1 collagen (K and L). Indomethacin (10 μM)-treated platelets were stimulated with 0.5 μg·mL−1 CRP-XL in the presence and absence of nobiletin (M and N). Whole blood aggregation was performed using 10 μg·mL−1 collagen in the presence and absence of nobiletin by impedance aggregometry (O and P). Cumulative data represent mean values ± SD (n = 3), the level of aggregation obtained at 5 min with vehicle-treated samples (0 μM) was taken as 100%. The P-values shown in the figure are calculated by non-parametric repeated measures anova (Friedman test; ****P < 0.0001).
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fig01: Nobiletin inhibits agonist-induced platelet activation. Human-washed platelet aggregation performed in the presence or absence of various concentrations of nobiletin was monitored for 5 min by optical aggregometry following stimulation with 1 μg·mL−1 (A and B) and 5 μg·mL−1 collagen (C and D), 0.5 μg·mL−1 (E and F) and 1 μg·mL−1 CRP-XL (G and H) and 0.1 U·mL−1 thrombin (I and J). Similarly, the effects of nobiletin on PRP were analysed following stimulation with 1 μg·mL−1 collagen (K and L). Indomethacin (10 μM)-treated platelets were stimulated with 0.5 μg·mL−1 CRP-XL in the presence and absence of nobiletin (M and N). Whole blood aggregation was performed using 10 μg·mL−1 collagen in the presence and absence of nobiletin by impedance aggregometry (O and P). Cumulative data represent mean values ± SD (n = 3), the level of aggregation obtained at 5 min with vehicle-treated samples (0 μM) was taken as 100%. The P-values shown in the figure are calculated by non-parametric repeated measures anova (Friedman test; ****P < 0.0001).

Mentions: Optical aggregometry was used to analyse the effects of nobiletin on platelet function upon activation with collagen, CRP-XL and thrombin. Washed human platelets were incubated with vehicle (0.01% DMSO) or nobiletin (6.25, 12.5, 25, 50 and 100 μM) for 5 min before stimulation with collagen (1 μg·mL−1) for up to 5 min (Figure 1A) in an optical aggregometer. Nobiletin reduced platelet aggregation in a concentration-dependent manner at 5 min (Figure 1A and B) with 95% inhibition achieved at 100 μM. The inhibitory effects of nobiletin were reduced modestly when a higher concentration of collagen (5 μg·mL−1) was used (Figure 1C and D). Since collagen activates platelets by binding both glycoprotein VI (GPVI) and integrin α2β1, a GPVI-selective agonist, CRP-XL, was used in aggregation assays to determine whether the inhibition of platelet aggregation occurred through the blockade of GPVI signalling. Platelet aggregation stimulated with CRP-XL (0.5 μg·mL−1) was also inhibited by nobiletin in a concentration-dependent manner (Figure 1E and F). Similar to collagen, a higher concentration of CRP-XL (1 μg·mL−1) reduced the level of inhibition induced by nobiletin (Figure 1G and H). In order to determine whether nobiletin is able to affect the signalling cascades activated by G-protein coupled receptors, aggregation assays were performed using thrombin as an agonist. A concentration of 0.1 U·mL−1 of thrombin was chosen to obtain similar levels of aggregation as obtained with other agonists such as collagen (1 μg·mL−1) and CRP-XL (0.5 μg·mL−1). Thrombin (0.1 U·mL−1)-stimulated platelet activation was inhibited by nobiletin only at higher concentrations (50, 100, 150 and 200 μM; Figure 1I and J). Lower concentrations of nobiletin did not inhibit thrombin-induced platelet aggregation (data not shown).


Pharmacological actions of nobiletin in the modulation of platelet function.

Vaiyapuri S, Roweth H, Ali MS, Unsworth AJ, Stainer AR, Flora GD, Crescente M, Jones CI, Moraes LA, Gibbins JM - Br. J. Pharmacol. (2015)

Nobiletin inhibits agonist-induced platelet activation. Human-washed platelet aggregation performed in the presence or absence of various concentrations of nobiletin was monitored for 5 min by optical aggregometry following stimulation with 1 μg·mL−1 (A and B) and 5 μg·mL−1 collagen (C and D), 0.5 μg·mL−1 (E and F) and 1 μg·mL−1 CRP-XL (G and H) and 0.1 U·mL−1 thrombin (I and J). Similarly, the effects of nobiletin on PRP were analysed following stimulation with 1 μg·mL−1 collagen (K and L). Indomethacin (10 μM)-treated platelets were stimulated with 0.5 μg·mL−1 CRP-XL in the presence and absence of nobiletin (M and N). Whole blood aggregation was performed using 10 μg·mL−1 collagen in the presence and absence of nobiletin by impedance aggregometry (O and P). Cumulative data represent mean values ± SD (n = 3), the level of aggregation obtained at 5 min with vehicle-treated samples (0 μM) was taken as 100%. The P-values shown in the figure are calculated by non-parametric repeated measures anova (Friedman test; ****P < 0.0001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543618&req=5

fig01: Nobiletin inhibits agonist-induced platelet activation. Human-washed platelet aggregation performed in the presence or absence of various concentrations of nobiletin was monitored for 5 min by optical aggregometry following stimulation with 1 μg·mL−1 (A and B) and 5 μg·mL−1 collagen (C and D), 0.5 μg·mL−1 (E and F) and 1 μg·mL−1 CRP-XL (G and H) and 0.1 U·mL−1 thrombin (I and J). Similarly, the effects of nobiletin on PRP were analysed following stimulation with 1 μg·mL−1 collagen (K and L). Indomethacin (10 μM)-treated platelets were stimulated with 0.5 μg·mL−1 CRP-XL in the presence and absence of nobiletin (M and N). Whole blood aggregation was performed using 10 μg·mL−1 collagen in the presence and absence of nobiletin by impedance aggregometry (O and P). Cumulative data represent mean values ± SD (n = 3), the level of aggregation obtained at 5 min with vehicle-treated samples (0 μM) was taken as 100%. The P-values shown in the figure are calculated by non-parametric repeated measures anova (Friedman test; ****P < 0.0001).
Mentions: Optical aggregometry was used to analyse the effects of nobiletin on platelet function upon activation with collagen, CRP-XL and thrombin. Washed human platelets were incubated with vehicle (0.01% DMSO) or nobiletin (6.25, 12.5, 25, 50 and 100 μM) for 5 min before stimulation with collagen (1 μg·mL−1) for up to 5 min (Figure 1A) in an optical aggregometer. Nobiletin reduced platelet aggregation in a concentration-dependent manner at 5 min (Figure 1A and B) with 95% inhibition achieved at 100 μM. The inhibitory effects of nobiletin were reduced modestly when a higher concentration of collagen (5 μg·mL−1) was used (Figure 1C and D). Since collagen activates platelets by binding both glycoprotein VI (GPVI) and integrin α2β1, a GPVI-selective agonist, CRP-XL, was used in aggregation assays to determine whether the inhibition of platelet aggregation occurred through the blockade of GPVI signalling. Platelet aggregation stimulated with CRP-XL (0.5 μg·mL−1) was also inhibited by nobiletin in a concentration-dependent manner (Figure 1E and F). Similar to collagen, a higher concentration of CRP-XL (1 μg·mL−1) reduced the level of inhibition induced by nobiletin (Figure 1G and H). In order to determine whether nobiletin is able to affect the signalling cascades activated by G-protein coupled receptors, aggregation assays were performed using thrombin as an agonist. A concentration of 0.1 U·mL−1 of thrombin was chosen to obtain similar levels of aggregation as obtained with other agonists such as collagen (1 μg·mL−1) and CRP-XL (0.5 μg·mL−1). Thrombin (0.1 U·mL−1)-stimulated platelet activation was inhibited by nobiletin only at higher concentrations (50, 100, 150 and 200 μM; Figure 1I and J). Lower concentrations of nobiletin did not inhibit thrombin-induced platelet aggregation (data not shown).

Bottom Line: The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice.Nobiletin extended bleeding time in mice and reduced the phosphorylation of PKB (Akt) and PLCγ2 within the collagen receptor (glycoprotein VI)-stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of vasodilator-stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling.Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, UK.

No MeSH data available.


Related in: MedlinePlus