綜 合 本 研 究 實 驗 結 果 ， 啤 酒 花(Humulus lupulus) 成 分 中 的 xanthohumol 抑制血小板凝集反應的作用主要涉及下列路徑：
(一) xanthohumol 抑制PLCγ2 pathway 與 ptotein kinase C 的活化 以 及 47 kDa protein 磷 酸 化 反 應 ； xanthohumol 抑 制 mitogen-activated protein kinases pathway 包含 p38 MAPK、
ERK、JNK 的磷酸化；xanthohumol 抑制Akt 的磷酸化反應。
(二) xanthohumol 可 有 效 抑 制 血 小 板 活 化 時 產 生 的 hydroxyl radical。
(三) 綜合以上的作用，xanthohumol 最終抑制了血小板細胞內鈣離 子的移動和濃度的增加，阻斷由鈣離子引起的一連串訊息傳 遞反應和最終的血小板凝集反應。
Table 1. Effect of xanthohumol and collagen on nitrate formation in washed human platelets
Washed human platelets suspensions (3.5 × 108/ml) were preincubated with xanthohumol (1.5 and 3 μM) for 3 min at 37℃.
Addition of collagen (1 μg/ml) to platelet suspensions served as positive control. Data are presented as the means ± S.E.M (n=4). **P < 0.01 as compared with the resting group.
OH HO
O
OH
O H
3C CH
3H
3C
Figure 1. Chemical structure of xanthohumol (C21H22O5, MW.354.40).
DMSO
Figure 2. Effect of xanthohumol on various agonists-induces platelet aggregation in washed platelets. Platelets were preincubated with various concentrations of xanthohumol (1.5-10 μM) and stirred for 3 min, then collagen (1 μg/ml), U46619 (1 μM),thrombin (0.05 IU/ml), or AA (60 μM) to trigger platelet aggregation. Data are presented as percent inhibition of the control (means ± S.E.M., n=4).
T (%)
ΔT (%)min
Δmin
Figure 3. Concentration-inhibition curves of xanthohumol on collagen (1 μg/ml, ○)-, U46619 (1 μM, ▽)-, thrombin (0.05 U/ml,
◇)-, arachidonic acid (60 μM, □)-induced platelet aggregation in human platelet suspensions. Human platelet suspensions were preincubated with various concentrations of xanthohumol (0.5-10 μM) at 37℃ for 3 min, and agonists were then added to trigger platelet aggregation. Data are presented as percent inhibition of the control (means ± S.E.M., n = 6).
ATP release
collagen DMSO
xanthohumol
1.5 3
T (%)
Δmin
Figure 4. Effects of xanthohumol on collagen-induced aggregation in human platelet suspensions. Platelets were preincubated with xanthohumol (1.5 and 3 μM) and stirred for 3 min, then collagen (1 μg/ml) was added to trigger platelet aggregation (lower tracing) and ATP release (upper tracing). Luciferin-luciferase (10 μg/ml) was added 1 min before the agonist in order to measure the ATP release reaction. The profiles are representative examples of four similar experiments.
T (%)
Δmin
collagen DMSO
xanthohumol
3 35 70
Figure 5. Effects of xanthohumol on collagen-induced aggregation in human platelet-rich plasma. Platelets were preincubated with xanthohumol (3, 35 and 70 μM) and stirred for 3 min, and then collagen (1 μg/ml) was added to trigger platelet aggregation. The profiles are representative examples of four similar experiments.
Figure 6. Effect of xanthohumol on collagen-induced intracellular Ca2+
mobilization of Fura 2-AM loaded human platelets. Platelet suspensions were incubated with Fura 2-AM (5 μM) at 37℃ for 60 min, followed by the addition of collagen (1 μg/ml) in the absence or presence of xanthohumol (1.5 and 3 μM), which was added 3 min prior to the addition of collagen. The profiles are representative examples of three similar experiments.
xanthohumol
1.5 3
DMSO
Fluorescence intensity
collagen
Figure 7. Effect of xanthohumol on collagen-induced intracellular Ca2+
mobilization of Fura 2-AM loaded human platelets. Platelet suspensions were incubated with Fura 2-AM (5 μM) at 37℃ for 60 min, followed by the addition of collagen (1 μg/ml) in the absence or presence of xanthohumol (1.5 and 3 μM), which was added 3 min prior to the addition of collagen. Data are presented as means ± S.E.M. (n = 3). ** P <
0.01 as compared with the resting group; ##P < 0.01 as compared with the collagen group.
C D
A B
Fluorescence intensity
Number of cells
A B C D
*** *** ***
Figure 8. Flow cytometric analysis of FITC-triflavin binding to human platelets in the absence or presence of xanthohumol (1.5 and 3 μM). (A) The solid line represents the fluorescence profiles of only FITC-triflavin (2 μg/ml) the absence of xanthohumol as a positive control;
(B) in the presence of EDTA (5 mM) as the negative control; or in the presence of xanthohumol (C) 1.5 μM and (D) 3 μM. Data are presented as means ± S.E.M. (n = 5). *** P < 0.001 as compared with the negative group.
*
PLCγ2 phosphorylation (folds/basal)
0.0
Figure 9. Effect of xanthohumol on protein phosphorylation of PLCγ2 in human platelets challenged with collagen. Platelets were preincubated with Tyrode’s solution only (lane 1), or platelets were preincubated with isovolumetric solvent control (0.5 % DMSO, lane 2) or xanthohumol (1.5 μM, lane 3; 3 μM, lane 4) followed by the addition of collagen (1 μg/ml) to trigger PLCγ2 activation. The bar graph depicts the ratios of quantitative results obtained by scanning the anti-phospho- PLCγ2 and anti-PLCγ2 reactive bands and quantify optical density using Bio-1D version 99 image software. Data are presented as means ± S.E.M.
(n = 6). *P < 0.05 as compared with the resting group (lane 1), #P < 0.05 as compared with the resting group (lane 2).
p-p47
Figure 10. Effect of xanthohumol on protein phosphorylation of Mw 47,000 (p47) in human platelets challenged with collagen. Platelets were preincubated with Tyrode’s solution only (lane 1), or platelets were preincubated with isovolumetric solvent control (0.5 % DMSO, lane 2) or xanthohumol (1.5 μM, lane 3; 3 μM, lane 4) followed by the addition of collagen (1 μg/ml) to trigger protein kinase C activation. The bar graph depicts the ratios of quantitative results obtained by scanning the anti-phospho-47 kDa and anti-α-tubulin reactive bands and quantify optical density using Bio-1D version 99 image software. Data are presented as means ± S.E.M. (n = 5). ***P < 0.001 as compared with the resting group (lane 1); #P < 0.05 and ##P < 0.01 as compared with the collagen group (lane 2)
**
Figure 11. Effect of xanthohumol on protein phosphorylation of Mw 47,000 (p47) in human platelets challenged with PDBu. Platelets were preincubated with Tyrode’s solution only (lane 1), or platelets were preincubated with isovolumetric solvent control (0.5 % DMSO, lane 2) or xanthohumol (1.5 μM, lane 3; 3 μM, lane 4) followed by the addition of PDBu (150 nM) to trigger protein kinase C activation. The bar graph depicts the ratios of quantitative results obtained by scanning the anti-phospho-47 kDa and anti-α-tubulin reactive bands and quantify optical density using Bio-1D version 99 image software. Data are presented as means ± S.E.M. (n = 5). **P < 0.01 as compared with the resting group (lane 1).
PDBu
DMSO xanthohumol
1.5 3
T (%)
Δmin
Figure 12. Effect of xanthohumol on PDBu-induced platelet aggregation of human platelet suspensions. Platelets were incubated with xanthohumol (1.5 and 3 μM) for 3 min, the PDBu (150 nM) was added to trigger platelet aggregation. The profiles are representative examples of four similar experiments.
phospho-p38 MAPK
p38 phosphorylation (folds/basal)
Figure 13. Effect of xanthohumol on protein phosphorylation of p38 MAPK in human platelets challenged with collagen. Platelets were preincubated with Tyrode’s solution only (lane 1), or platelets were preincubated with isovolumetric solvent control (0.5 % DMSO, lane 2) or xanthohumol (1.5 μM, lane 3; 3 μM, lane 4) followed by the addition of collagen (1 μg/ml) to trigger p38 MAPK activation. The bar graph depicts the ratios of quantitative results obtained by scanning the anti-phospho-p38 and anti-p38 reactive bands and quantify optical density using Bio-1D version 99 image software. Data are presented as means ± S.E.M. (n = 3). **P < 0.01 as compared with the resting group (lane 1), #P < 0.05 as compared with the collagen group (lane 2).
***
ERKs phosphorylation (folds/basal)
0
Figure 14. Effect of xanthohumol on protein phosphorylation of ERKs in human platelets challenged with collagen. Platelets were preincubated with Tyrode’s solution only (lane 1), or platelets were preincubated with isovolumetric solvent control (0.5 % DMSO, lane 2) or xanthohumol (1.5 μM, lane 3; 3 μM, lane 4) followed by the addition of collagen (1 μg/ml) to trigger ERKs activation. The bar graph depicts the ratios of quantitative results obtained by scanning the anti-phospho-ERK and anti-ERK reactive bands and quantify optical density using Bio-1D version 99 image software. Data are presented as means ± S.E.M. (n = 6).
***P < 0.001 as compared with the resting group (lane 1); #P < 0.05 and
###P < 0.001 as compared with the collagen group (lane 2).
*
JNK1 phosphorylation (folds/basal)
0.0
Figure 15. Effect of xanthohumol on protein phosphorylation of JNK1 in human platelets challenged with collagen. Platelets were preincubated with Tyrode’s solution only (lane 1), or platelets were preincubated with isovolumetric solvent control (0.5 % DMSO, lane 2) or xanthohumol (1.5 μM, lane 3; 3 μM, lane 4) followed by the addition of collagen (1 μg/ml) to trigger JNK1 activation. The bar graph depicts the ratios of quantitative results obtained by scanning the anti-phospho-JNK and anti-JNK reactive bands and quantify optical density using Bio-1D version 99 image software. Data are presented as means ± S.E.M. (n=5).
*P < 0.05 as compared with the resting group (lane 1), #P < 0.05 as compared with the collagen group (lane 2) .
Figure 16. Effect of xanthohumol on protein phosphorylation of Akt in human platelets challenged with collagen. Platelets were preincubated with Tyrode’s solution only (lane 1), or platelets were preincubated with isovolumetric solvent control (0.5 % DMSO, lane 2) or xanthohumol (1.5 μM, lane 3; 3 μM, lane 4) followed by the addition of collagen (1 μg/ml) to trigger Akt activation. The bar graph depicts the ratios of quantitative results obtained by scanning the anti-phospho-Akt and anti-α-tubulin reactive bands and quantify optical density using Bio-1D version 99 image software. Data are presented as means ± S.E.M.
(n = 8). *P < 0.05 as compared with the resting group (lane 1), #P < 0.05 as compared with the collagen group (lane 2).
collagen - + + +
xanthohumol - - 1.5 3
phospho-Akt
α-tubulin
collagen
xanthohumol
3 ODQ
T (%)
Δmin
SQ
collagen
xanthohumol 3
T (%)
Δmin
Figure 17. Effects of xanthohumol on collagen-induced aggregation in human platelet suspension. Platelets were preincubated with xanthohumol (3 μM) and stirred for 3 min, then collagen (1 μg/ml) was added to trigger platelet aggregation; Platelets were preincubated with ODQ (10 μM) and SQ (100 μM) and stirred for 3 min, then preincubated with xanthohumol (3 μM) and stirred for 3 min, then collagen (1 μg/ml) was added to trigger platelet aggregation.
Figure 18. Effect of xanthohumol on collagen-induced free radical-scavenging activity of human platelet suspensions. ESR conditions are described in “material and methods”. Scanning was begun 1 min after the mixing of all reagents. ESR spectra of free radical-scavenging activity of platelet suspensions were follows by the addition of DMPO (100 mM) (A) and the addition of collagen (1 μg/ml) in the absence (B) or presence of xanthohumol 1.5 μM (C) and 3 μM (D), which was added 3 min prior to collagen (1 μg/ml). The spectra are representative examples of three similar experiments.
(B)
(C)
(D) (A)
##
Figure 19. Effects of SB203580 (10 μM), Ro318220 (2 μM) or Ly294002 (10 μM) on p47 phosphorylation stimulated by collagen in human platelets. Platelets were pretreated with SB203580 (10 μM), Ro318220 (2 μM) or Ly294002 (10 μM) followed by the addition of collagen (1mg/ml)；then the p47 phosphorylation was mwasured as described in “materials and methods”. Results are expressed as the mean
± S.E.M (n = 3).*P < 0.05 as compared with the resting group (lane 1), #P
< 0.05 as compared with the collagen group (lane 2).
*
p38 MAPK phosphorylation (folds/basal)
0.0
Figure 20. Effects of SB203580 (10 μM), Ro318220 (2 μM) or Ly294002 (10 μM) on p47 phosphorylation stimulated by collagen in human platelets. Platelets were pretreated with SB203580 (10 μM), Ro318220 (2 μM) or Ly294002 (10 μM) followed by the addition of collagen (1mg/ml)；then the p38 MAPK phosphorylation was mwasured as described in “materials and methods”. Results are expressed as the mean ± S.E.M (n = 3).*P < 0.05 as compared with the resting group (lane 1), #P < 0.05 as compared with the collagen group (lane 2).
phospho-Akt
Akt
collagen - + + + +
SB203580 - - + -
-Ro318220 - - - +
-Ly294002 - - - - +
Figure 21. Effects of SB203580 (10 μM), Ro318220 (2 μM) or Ly294002 (10 μM) on p47 phosphorylation stimulated by collagen in human platelets. Platelets were pretreated with SB203580 (10 μM), Ro318220 (2 μM) or Ly294002 (10 μM) followed by the addition of collagen (1mg/ml)；then the Akt phosphorylation was mwasured as described in “materials and methods”. Results are expressed as the mean
± S.E.M (n = 6).*P < 0.05 as compared with the resting group (lane 1), #P
< 0.05 as compared with the collagen group (lane 2).
Ca2+
Figure 22. Signal transduction of platelet aggregation. Agonists can activate several phospholipase, including phospholipase C (PLC) and phospholipase A2 (PLA2). The products of the action of phospholipase C on phosphatidylinositol 4,5-bisphosphate (PIP2) include 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG stimulates protein kinase C (PKC), followed by phosphorylation of a 47 kDa protein. IP3 induces the release of Ca2+ from dense tubular systems (DTS). The major metabolite of arachidonic acid (AA) in platelet is thromboxane A2 (TxA2).
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