- Email: [email protected]
Antiplatelet activity of hesperetin, a bioflavonoid, is mainly mediated by inhibition of PLC-γ2 phosphorylation and cyclooxygenase-1 activity
Atherosclerosis 194 (2007) 144–152
Antiplatelet activity of hesperetin, a bioflavonoid, is mainly mediated by inhibition of PLC-␥2 phosphorylation and cyclooxygenase-1 activity Yong-Ri Jin a , Xiang-Hua Han b , Yong-He Zhang c , Jung-Jin Lee b , Yong Lim b , Jin-Ho Chung d , Yeo-Pyo Yun b,∗ a
Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea b College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea c Department of Pharmacology, School of Basic Medical Science, Peking University, Beijing 100083, China d College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea Received 21 June 2006; received in revised form 27 September 2006; accepted 6 October 2006 Available online 7 November 2006
Abstract Diet can be one of the most important factors that influence risks for atherothrombotic diseases. Hesperetin included in grapefruits and oranges is one candidate that may benefit the cardiovascular system. Here, we investigated antiplatelet activity of hesperetin in vitro. In addition, possible antiplatelet mechanism was also investigated. Hesperetin concentration-dependently inhibited washed rabbit platelet aggregation induced by collagen and arachidonic acid, with IC50 of 20.5 ± 3.5 and 69.2 ± 5.1 M, respectively, while has little effect on thrombin- or U46619-, a thromboxane (TX) A2 mimic, mediated platelet aggregation, suggesting that hesperetin may selectively inhibit collagen- and arachidonic acid-mediated signal transduction. In accordance with these findings, hesperetin revealed blocking of the collagen-mediated phospholipase (PL) C-␥2 phosphorylation, and caused concentration-dependent decreases of cytosolic calcium mobilization, arachidonic acid liberation and serotonin secretion. In addition, hesperetin inhibited arachidonic acid-mediated platelet aggregation by interfering with cyclooxygenase-1 activity as established by the measurement of arachidonic acid-mediated TXA2 and prostaglandin D2 formations as well as cyclooxygenase-1 and TXA2 synthase activity assays. Taken together, the present results provide a cellular mechanism for the antiplatelet activity of hesperetin through inhibition of PLC-␥2 phosphorylation and cyclooxygenase-1 activity, which may contribute to the beneficial effects of grapefruits and oranges on cardiovascular system. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Hesperetin; Platelet aggregation; Phospholipase C; Cyclooxygenase-1
1. Introduction The platelet activation and subsequent platelet aggregation play an essential role in the pathogenesis of cardiovascular, cerebrovascular, and peripheral vascular diseases. When blood vessels become damaged, subendothelial macromolecules such as collagen and thrombin, which are exposed or generated at the site of damage, stimulate platelet activa∗
Corresponding author. Tel.: +82 43 261 2821; fax: +82 43 268 2732. E-mail address: [email protected] (Y.-P. Yun).
0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2006.10.011
tion and the initiation of hemostasis [1–4]. Collagen, which supports the adhesion of platelets to the site of injury via von Willebrand factor (VWF) as well as glycoprotein VI (GPVI) and integrin ␣21, induces platelet activation through a tyrosine kinase-based signaling pathway that involves the kinase Syk and phospholipase (PL) C-␥2, which results cytoplasmic calcium increase, shape change and granule release; adhesion is partly and aggregation is largely dependent on ADP and prostaglandin (PG) H2 /thromboxane (TX) A2 release [5–8]. This results in platelet shape change and spreading, and the release or secretion of proactivatory substances that
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
activate and recruit platelets to the developing thrombus [9]. The formation and release of thromboxane (TX) A2 is a central component in the platelet in response to a variety of agonists. TXA2 is an eicosanoid, a metabolite of arachidonic acid formed via the cyclooxygenase-TXA2 synthase pathway, and it is considered to be one of the most powerful agonists for platelet activation and a major contributor to the thrombus formation [10]. TXA2 binds to a G protein-coupled receptor to induce phospholipase C activation which results an increase of [Ca2+ ]i and protein kinase C activation, and causes platelets to change shape, extend pseudopods and adhere to platelets on the damaged surface. The clinical evidences have proven that inhibition of the synthesis or the action of TXA2 was effective for the patients with acute coronary syndromes and myocardial infarction [11]. Platelet activation may be inhibited by a number of dietary components including some dietary fats and antioxidants [12–15]. Flavonoids are a large group of low molecular weight polyphenolic compounds, and have been shown to be one of the most important classes in free states and as glycosides. Large amounts of bioflavonoids are ingested because of their abundance and wide distribution in foods and beverages. A dietary antioxidant that has received attention with regard to antioxidant effect is the polyphenolic compound, hesperidin (hesperetin 7-rhammnoglucoside) and its aglycone hesperetin (Fig. 1; 3 ,5,7-trihydroxy-4 -methoxy flavanone). Both flavonoids present extensively in the plant kingdom especially in many citrus fruits such as grapefruits and oranges, which are commonly used in traditional medicines [16]. It has been reported that hesperetin shows a wide spectrum of pharmacological effects such as anti-inflammatory, anticarcinogenic, antihypertensive and anti-atherogenic effects [16,17]. Hesperetin has been reported to inhibit low-density lipoprotein oxidation in vitro [18]. It has also been reported that hesperetin inhibited HMG-CoA reductase and lowers the plasma cholesterol level in rats [19]. The role of hesperetin and the structurally related naringenin citrus flavanone in the prevention and treatment of atherogenic disease has recently received considerable attention, with particular interest in the use of these flavanones as anticancer and anti-atherogenic compounds [20,21]. However, the anti-platelet activity as
Fig. 1. Chemical structure of hesperetin.
145
well as possible mechanism of hesperetin has not yet been elucidated. In this study, we have investigated the anti-platelet activity of hesperetin by measurement of platelet aggregation in vitro. The possible cellular mechanisms involved in PLC␥2 activation, arachidonic acid cascade, calcium mobilization and granule secretion were also investigated.
2. Materials and methods 2.1. Materials Hesperetin, indomethacin, imipramine, imidazole, serotonin creatinine sulfate, o-phthalaldehyde (OPT) and BSA were from Sigma Chemical Co. (St. Louis, MO). [3 H]Arachidonic acid (220 Ci/mmol) was obtained from Perkin-Elmer Life and Analytical Sciences (Boston, MA). Collagen, arachidonic acid and thrombin were purchased from Chrono-Log Co. (Havertown, PA). U46619, TXB2 , prostaglandin (PG) D2 , PGH2 and cyclooxygenase-1 inhibitor assay kit were purchased from Cayman Chemical Co. (Ann Arbor, MI). Anti-PLC-␥2 and anti-phospho-PLC␥2 antibody (Q-20) were obtained from Upstate Biotechnology Incorporate (Lake Placid, NY). U73122 was obtained from New England Biolabs, Inc. (Bevely, MA). The other chemicals were of analytical grade. 2.2. Animals New Zealand white rabbits were purchased from SamTako Animal Co. (Osan, Kyungi-do) and acclimatized for 1 week at 24 ◦ C and 55% humidity, with free access to a commercial pellet diet obtained from Samyang Co. (Wonju, Kangwon-do) and drinking water before experiments. The animal studies have been carried out in accordance with the Guide for the Care and Use of Laboratory Animals, Chungbuk National University, Korea. 2.3. Preparation of washed rabbit platelet Fresh blood was obtained from ear artery of male rabbits (New Zealand white rabbits weighing about 2–3 kg), collected into plastic tubes containing acid citrate dextrose (ACD) solution (1/6 volume of blood) composed of citric acid (65 mM), trisodium citrate (85 mM), and dextrose (2%) at pH 4.5, subsequently centrifuged at 250 × g for 10 min to obtain platelet-rich plasma. Platelet-rich plasma was centrifuged at 650 × g for 10 min at room temperature (20–25 ◦ C). The pellet was washed twice with Tyrode/HEPES solution (pH 6.35). The resultant pellet was resuspended in the second Tyrode/HEPES solution (pH 7.35) with a final density of approximately 4 × 108 platelets/mL. The Tyrode/HEPES solution was composed of NaCl 138.3 mM, KCl 2.68 mM, MgCl2 ·6H2 O 1.0 mM, NaHCO3 4.0 mM, HEPES 10 mM, glucose 0.1% (w/v) and albumin 0.35% (w/v) at pH 6.35 or 7.35.
146
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
2.4. Measurement of platelet aggregation Platelet aggregation was determined by a standard turbidometric method [22] using an aggregometer (Chrono-Log Co., Havertown, PA). Platelet aggregation was expressed as an increase in light transmission. The levels of light transmission were calibrated as 0% for a platelet suspension and 100% for the Tyrode/HEPES solution. Platelet suspension (0.25 mL) in the aggregometer cuvette was preincubated at 37 ◦ C for 2 min under continuous stirring at 1000 rpm. And then CaCl2 was added at the final concentration of 1 mM. After 2 min, various concentrations of hesperetin (1–100 M), which was in line with its use on other cells [23–25], was added. The incubation time was set as 3 min, which is long enough to reveal its effect, and platelet aggregation was induced by various agonists and aggregation percent was monitored for 5 min. 2.5. Measurement of arachidonic acid metabolites The arachidonic acid metabolites, TXB2 and PGD2 generations, were measured as previously described [26]. In brief, washed rabbit platelets (4 × 108 cells/mL) were preincubated with various concentrations of hesperetin at 37 ◦ C for 3 min, and further incubated with a mixture of [3 H]arachidonic acid and unlabeled arachidonic acid (2 M, 1 Ci/mL) for 5 min. The reaction was terminated by addition of stop solution (2.6 mM EGTA, 130 M BW755C). Lipids were extracted and separated by TLC on silica gel G plates with the following development system: ethyl acetate/isooctane/acetic acid/H2 O (11/5/2/10, v/v/v/v). The area corresponding to each lipid was scraped off and the radioactivity was determined by liquid scintillation counting. 2.6. TXA2 synthase activity assay The TXA2 synthase activity was assayed as previously described [26]. In brief, aliquots of PGH2 in anhydrous acetone, were pipetted into glass tubes, and then the acetone was evaporated under a gentle stream of nitrogen, and PGH2 was re-dissolved immediately in ethanol. Platelet suspensions were incubated with the test compounds at 37 ◦ C for 3 min prior to the addition of 5 M PGH2 . The final concentration of ethanol was 0.1% (v/v). At 5 min after addition of PGH2 , the incubations were terminated by addition of cooling EGTA (2 mM) and centrifuged at 12,000 × g at 4 ◦ C for 4 min. Because TXA2 is very unstable and rapidly converted to more stable metabolite TXB2 , the amount of TXB2 in the supernatants was assayed by a commercial enzyme immunoassay kit according to the manufacturers’ instructions (Amersham Biosciences, Ltd., Bucking Hamshire). TXA2 synthase activity is reflected by the production of TXB2 . 2.7. Cytosolic calcium mobilization assay Cytosolic Ca2+ mobilization was measured by using the fluorescent dye fura-2/AM as previously described [26],
which involved incubating the platelets with cell permeant acetoxymethyl ester. Rabbit platelets (isolated as described above) were incubated with 2 M fura-2/AM at room temperature for 1 h (on a rocking platform) in the loading buffer (137 mM NaCl, 27 mM KCl, 0.4 mM NaH2 PO4 , 10 mM HEPES, 12 mM NaHCO3 , 5.5 mM dextrose, 0.35% BSA, pH 7.4). Excess fura-2/AM was removed by centrifugation (500 × g for 10 min) and the platelets were suspended in fresh buffer, without added EGTA. Aliquots of platelet suspension (2.5 mL) were added to 4 mL cuvettes containing a teflon coated stirrer bar (Chrono-Log, Havertown, PA). Just before [Ca2+ ]i measurement was performed, Ca2+ was added back to the buffer to a final concentration of 1 mM, and then hesperetin (various concentrations in 25 L) and collagen were added. The measurement of [Ca2+ ]i was performed at room temperature in a MSIII fluorimeter (Photon Technology International, S. Brunswick, NJ) using excitation wavelengths of 340 and 380 as well as an emission wavelength of 505 nm. [Ca2+ ]i was calculated by using the SPEX dM3000 software package. 2.8. Immunoblotting For analysis of total platelet proteins, the reaction was terminated by addition of LaemmLi sample buffer, and the mixture was then boiled for 5 min and analyzed on a 7.5% SDS-PAGE. Immunoblot assays were performed as previously described [27], with slight modifications. For immunoblotting, proteins were electrically transferred to the polyvinylidene difluoride membrane for 80 min at 120 mA. Blots were incubated for 4 h with 5% (w/v) BSA in TBS to block residual protein binding sites. Immunodetection of phospho-PLC-␥2 was detected by rabbit anti-phosphoPLC-␥2 antibody (Q-20, 1 g/mL) in TBS containing 5% BSA for 4 h. The primary antibody was removed, and blots were washed in TBS with 0.05% Tween-20 three times. To detect the primary antibody, blots were incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit antibody (New England Biolabs, MA) diluted to 1:5000 in TBS containing 5% BSA for 5 h, and then washed five times in TBS with 0.05% Tween-20. After the blots were exposed to enhanced chemiluminescence reagents (Amersham Biosciences, Ltd., Bucking Hamshire) for 5 min, they were then exposed to hyper film-enhanced chemiluminescence (Amersham) for 5 min. The intensities of phospho-PLC-␥2 and total PLC-␥2 bands were quantified by Scion-Image for Windows Program (Scion Corporation, MA). 2.9. Measurement of serotonin secretion Serotonin release was measured by the fluorimetric method of Holmsen and Dangelmaier with a little modification [28]. In brief, to prevent the reuptake of sereted serotonin, imipramine (a serotonin re-uptake inhibitor; 5 M) was added to platelet suspension. Washed rabbit platelets were treated with hesperetin at 37 ◦ C for 3 min prior to addi-
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
147
tion of collagen. A 0.35 mL aliquot of the washed rabbit platelet was mixed with 5 mM EDTA in ice, and centrifuged at 12,000 × g for 2 min. The supernatant was mixed with 6 M trichloroacetic acid (TCA) and centrifuged at 12,000 × g for 2 min. A 0.3 mL aliquot of TCA supernatant was mixed with 2.4 mL of the solution (0.5% o-phthalaldehyde in ethanol diluted 1:10 with 8N HCl), placed in a boiling water bath for 10 min, and then cooled in ice. The excess TCA was extracted with chloroform, and fluorophore was measured at the wavelength of excitation (360 nm) and emission (475 nm). Serotonin creatinine sulfate was used as a standard solution to calculate the extent of serotonin release. 2.10. Measurement of arachidonic acid liberation The arachidonic acid liberation was measured as previously described [26]. In brief, PRP was preincubated with [3 H]arachidonic acid (1 Ci/mL) at 37 ◦ C for 1.5 h, and then washed as described above. The [3 H]arachidonic acidlabeled platelets (4 × 108 cells/mL) were pretreated with 100 M BW755C, a COX and LOX inhibitor, and various concentrations of hesperetin at 37 ◦ C for 3 min in the presence of 1 mM CaCl2 , and then stimulated with collagen (50 g/mL). The reaction was terminated by addition of chloroform/methanol/HCl (200/200/1, v/v/v). Lipids were extracted and separated by TLC on silica gel G plates with the following development system: petroleum ether/diethyl ether/acetic acid (40/40/1, v/v/v). The area corresponding to each lipid was scraped off, and the radioactivity was determined by liquid scintillation counting. 2.11. Statistical analysis The experimental results are expressed as mean ± SEM. A one-way analysis of variance (ANOVA) was used for multiple comparison (Sigma Stat® . SPSS Inc., CA). If there was a significant variation between treated-groups, Dunnett’s test was applied. The data were considered significant with a probability less than 0.05.
3. Results 3.1. Effect of hesperetin on washed rabbit platelet aggregation in vitro To compare the selectivity of hesperetin on various agonists-induced platelet aggregation, the agonists concentration were determined by a preliminary study and set as 10 g/mL for collagen, 100 M for arachidonic acid, 1 M for U46619 and 0.05 U/mL for thrombin. Under these concentrations, these agonists almost cause equal platelet aggregation (70 ± 5.5%). As shown in Fig. 2, hesperetin concentration-dependently inhibited collagen (10 g/mL)and arachidonic acid (100 M)-challenged washed rabbit platelet aggregation, with IC50 values of 20.5 ± 3.5
Fig. 2. Effect of hesperetin on washed rabbit platelet aggregation in vitro. Washed rabbit platelet suspension was incubated at 37 ◦ C in an aggregometer with stirring at 1000 rpm, and then hesperetin was added. After 3 min preincubation, the platelet aggregation was induced by addition of arachidonic acid (100 M), collagen (10 g/mL) in the absence or presence of indomethacin (20 M), U46619 (1 M), or thrombin (0.05 U/mL). The aggregation percents were expressed as percentage of maximum aggregation induced by respective inducers. Data are expressed as mean ± S.E.M. (n = 4).
and 69.2 ± 5.1 M, respectively. However, hesperetin only slightly inhibited U46619 (1 M)- and had no effect on thrombin (0.05 U/mL)-induced platelet aggregation. These results indicate that hesperetin may selectively affect collagen- and arachidonic acid-mediated signal transductions in platelets. To clarify whether the inhibition of hesperetin on collagen-induced platelet aggregation was mediated by arachidonic cascade, effect of hesperetin on collagen-induced indomethacin (20 M)-pretreated platelet aggregation was also determined. The maximum platelet aggregation induced by collagen was reduced to 50 ± 3% after treatment of indomethacin, however, the inhibition-curve of hesperetin on collagen-induced platelet aggregation was not changed, which indicates that in addition to the inhibition of arachidonic cascade, other pathways may also be involved in the inhibition of hesperetin on collagen-mediated platelet aggregation. 3.2. Effect of hesperetin on conversions of arachidonic acid to TXB2 and PGD2 Hesperetin concentration-dependently suppressed TXB2 and PGD2 generations induced by addition of [3 H]arachidonic acid in intact rabbit platelets (Fig. 3A and B). The TXB2 formations were inhibited by 8.5, 25.4 and 75.3% at the concentrations of 20, 50 and 100 M, respectively. And PGD2 formations were also inhibited by 0.5, 10.4 and 45.3% at the concentrations of 20, 50 and 100 M, respectively. These results suggest that hesperetin may inhibit cyclooxygenase activity rather than that of TXA2
148
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
As shown in Fig. 3C, hesperetin had no effect on the conversion of PGH2 into TXB2 in all concentrations of hesperetin used. However, imidazole, a typical TXA2 synthase inhibitor, markedly inhibited the conversion of PGH2 into TXB2 . 3.4. Effect of hesperetin on collagen-induced [Ca2+ ]i The representative traces in which collagen were added to induce [Ca2+ ]i mobilization were shown in Fig. 4A. After 3 min incubation of platelets with various concentrations of hesperetin, collagen (10 g/mL) was added to induce [Ca2+ ]i mobilization. Collagen induced a slow but stable increase of [Ca2+ ]i which reaches the peak level of 300 nM after 5–7 min. Treatment of the platelet suspension with hesperetin inhibited the elevation of the [Ca2+ ]i in response to collagen by 20.2, 50.8 and 77.2% at concentrations of 10, 20 and 50 M, respectively. The right panel (Fig. 4B) is the average of three times separated experiments, similar to that shown in left panel. 3.5. Effect of hesperetin on serotonin secretion
Fig. 3. Effects of hesperetin on arachidonic acid-induced metabolite and TXA2 synthase activity. The arachidonic acid-mediated [3 H]TXB2 and [3 H]PGD2 formations were assayed by using [3 H]arachidonic acid. The [3 H]TXB2 (A) and [3 H]PGD2 (B) were extracted and separated by TLC on silica gel G plate. The area corresponding to each lipid was scraped off and the radioactivity was determined by liquid scintillation counting. TXA2 synthase activity (C) was assayed by using PGH2 . TXA2 synthase activity is reflected by the production of TXB2 , which was determined by an enzymeimmunoassay kit. Data are expressed as mean ± S.E.M. (n = 3). * P < 0.05 and ** P < 0.01 vs. stimulus control.
synthase, because TXA2 and PGD2 are simultaneously produced from arachidonic acid through COX pathway. Indomethacin, a COX inhibitor used as a positive control, almost completely inhibited TXA2 and PGD2 formations at a concentration of 20 M. It was also demonstrated by a COX-1 activity assay that hesperetin inhibited COX-1 activity in a concentration-dependent manner (data not shown). 3.3. Effect of hesperetin on TXA2 synthase activity The conversion of arachidonic acid to TXA2 in platelets requires the action of two enzymes, COX and TXA2 synthase. TXA2 synthase catalyzes the conversion of PGH2 to TXA2 in platelets. By utilizing PGH2 , it is possible to circumvent the COX step during arachidonic acid metabolism.
Serotonin is secreted from activated platelets during platelet aggregation. Since hesperetin inhibited platelet aggregation induced by collagen and arachidonic acid, it also reduced the release of serotonin in a concentration-dependent manner, with inhibition percentages of 25.2 and 83.5% at concentrations of 50 and 100 M for arachidonic acid (Fig. 5A), respectively, and 10.5, 42.6 and 83.3% at concentrations of 10, 20 and 50 M for collagen (Fig. 5B), respectively. 3.6. Effect of hesperetin on collagen-induced PLC-γ2 phosphorylation To further examine the underlying antiplatelet mechanism of hesperetin, washed rabbit platelets were stimulated with 10 g/mL of collagen in the presence or absence of hesperetin, and the phosphorylation of PLC-␥2 was assayed. As shown in Fig. 6, pretreatment with hesperetin at concentrations of 10, 20, 50 and 100 M significantly inhibited collagen-induced PLC-␥2 phosphorylation with the inhibition percentages of 10.1, 45.4, 60.3 and 87.5%, respectively. Suppressive effect of hesperetin on collagen-induced PLC␥2 phosphorylation was also confirmed after 1, 3 and 5 min incubations, in which pretreatment of hesperetin at a concentration of 200 M completely abrogated this induction (data not shown). 3.7. Effect of hesperetin on collagen-induced arachidonic acid liberation As shown in Fig. 7, pretreatment of hesperetin concentration-dependently inhibited collagen-induced arachidonic acid liberation in [3 H]arachidonic acid prelabeled rabbit platelets by 4.5, 22.4, 44.7 and 64.3% at the
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
149
Fig. 4. Effect of hesperetin on collagen-stimulated [Ca2+ ]i . Calcium (1 mM) was added to the platelet suspension 10 s before data collection started (zero time). Hesperetin solution was added to yield final concentrations of 10, 20 and 50 M in the platelet suspension. Collagen (10 g/mL) was added 3 min later. The traces (A) are from a representative experiment; similar results were obtained from three separate experiments, and average data were shown in right panel (B) as mean ± S.E.M. * P < 0.05 and ** P < 0.01 vs. stimulus control.
concentrations of 5, 10, 20 and 50 M, respectively. U73122, a phospholipase C inhibitor which was used as a positive control, completely blocked arachidonic acid liberation at a concentration of 20 M.
Fig. 6. Effect of hesperetin on collagen-induced PLC-␥2 phosphorylation. Washed rabbit platelets in the presence of EGTA (1 mM) and indomethacin (20 M) were incubated with various concentrations of hesperetin (10, 20, 50 and 100 M) or DMSO for 3 min, and then stimulated with collagen (10 g/mL) for 5 min. Cells were lysed and 20 g/mL protein was analyzed with SDS-PAGE. Relative densities were quantified by scanning densitometry and showed the levels of each density as relative value of total PLC-␥2, respectively. Data are expressed as mean ± S.E.M. (n = 3). * P < 0.05 and ** P < 0.01 vs. stimulus control.
4. Discussion Fig. 5. Effect of hesperetin on serotonin secretion. Washed rabbit platelet suspension was incubated with imipramine (5 M) and various concentrations of hesperetin at 37 ◦ C for 3 min prior to addition of arachidonic acid (A) or collagen (B). The serotonin concentration was determined by a fluorimetric method as described in Section 2. Data are expressed as mean ± S.E.M. (n = 3). * P < 0.05 and ** P < 0.01 vs. stimulus control.
A group of antioxidants of the flavonoid family has been reported to inhibit platelet aggregation in vitro and ex vivo, and human studies have also suggested that a diet high in flavonoids may inhibit platelet aggregation in vivo
150
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
Fig. 7. Effect of hesperetin on collagen-induced arachidonic acid liberation. [3 H]Arachidonic acid-prelabeled platelets were incubated with various concentrations of hesperetin at 37 ◦ C for 3 min in the presence of 100 M BW755C, and then stimulated with 50 g/mL of collagen for 2 min. Liberated [3 H]arachidonic acid was determined as described in Section 2. Data are expressed as mean ± S.E.M. (n = 3). * P < 0.05 and ** P < 0.01 vs. stimulus control.
[12,13,29,30]. Hesperetin included in grapefruits and oranges is one candidate that may benefit the cardiovascular system [21]. In the present study, we demonstrated that hesperetin was a potent inhibitor of platelet aggregation in response to collagen and arachidonic acid, and the cellular mechanism for the antiplatelet activity of hesperetin might be mediated by the inhibition of PLC-␥2 phosphorylation and cyclooxygenase-1 activity. It is well known that U46619, a TXA2 mimic, acts directly on the TXA2 receptor to induce G protein-coupled phospholipase C activation, resulting an increase of [Ca2+ ]i and protein kinase C activation [3]. Similarly, arachidonic acid, which acts directly on membrane COX-TXA2 synthase pathway to produce TXA2 , mediates platelet activation in the same way as U46619 [31]. From the platelet aggregation study (Fig. 2), hesperetin inhibited arachidonic acidmediated platelet aggregation in a concentration-dependent manner, while even at a highest concentration of 100 M slightly suppressed U46619-induced platelet aggregation. These results suggest that hesperetin may be more potent in inhibition of arachidonic acid-mediated TXA2 production than a direct inhibition of TXA2 receptor. Therefore the effect of hesperetin on the generation of TXA2 was firstly determined by using [3 H]arachidonic acid in intact rabbit platelet. As shown in Fig. 3A and B, hesperetin inhibited both [3 H]TXA2 and [3 H]PGD2 in the same concentrationdependent manner. Considering that TXA2 and PGD2 are simultaneously produced from arachidonic acid through COX-1 pathway in platelets [32], it seems that hesperetin may selectively inhibit activity of COX-1 rather than that of TXA2 , because indomethacin, a potent COX inhibitor, also completely inhibited TXA2 and PGD2 productions. It was also confirmed by a COX-1 activity as well as a TXA2 synthase activity results that hesperetin concentrationdependently inhibited COX-1 activity (data not shown) but had no effect on PGH2 —a precursor of the PGs and TXA2 , mediated TXB2 formation (Fig. 3C) in all concentrationranges. Present results were consistent with other reports that some flavonoids could inhibit platelet by the inhibition
of COX-1 activity [33,34]. Moreover, AA-induced serotonin secretion was also inhibited by hesperetin in a concentrationdependent manner (Fig. 5A). The reason that hesperetin inhibited U46619-induced platelet aggregation by 30% at a concentration of 100 M may be due to a direct inhibition of TXA2 receptor, although it is very weak, which may be due to its lack of a double bond in C2–C3. It has been reported that flavonoids (such as apigenin, luteolin, genistein and quercetin, etc.) share a characteristic conjugation between A, C and B rings with the presence of a lactone structure, which is also present within the TXA2 molecule and may account for competition between these compounds for the same receptors. The structural features such as the presence of the double bond in C2–C3 and/or keto group in C4 are important for the binding to the TXA2 receptor [35]. In addition, hesperetin also inhibited collagen-mediated platelet aggregation in a concentration-dependent manner, and the IC50 value (20.5 M) for collagen was more potent than that (69.2 M) for arachidonic acid. To exclude the possibility that the inhibition of collagen-induced platelet aggregation was mediated by the inhibition of COX-1 activity, the effect of hesperetin on collagen-induced indomethacinpretreated platelet aggregation was also determined. In the presence of indomethacin (20 M), which is sufficient enough to block COX-1 activity completely, the maximum platelet aggregation induced by collagen (10 g/mL) was reduced about 25% compared with the control (data not shown). As shown in Fig. 2, however, pretreatment of indomethacin did not change the inhibition curve of hesperetin on collagen-induced platelet aggregation. It seems that, in addition to the inhibition of COX-1 activity, hesperetin may specifically affect some components involved in collagen-mediated signal transduction. It has been reported that collagen induces platelet activation through a tyrosine kinase-based signaling pathway that involves the kinase Syk and PLC-␥2, which results in [Ca2+ ]i increase, shape change and granule release [9]; adhesion is partly and aggregation is largely dependent on ADP and TXA2 /PGH2 release [5,6]. Considering that calcium mobilization is a major cellular process in collagen-mediated platelet activation, the cytosolic Ca2+ was determined in the platelets loaded with fura-2/AM. Concomitant with the inhibition of platelet aggregation data, hesperetin inhibited cytosolic Ca2+ mobilization in a concentration-dependent manner (Fig. 4). Accordingly the granule secretion, which was determined as the level of serotonin, was also inhibited by hesperetin in the same pattern as inhibition of platelet aggregation and cytosolic Ca2+ mobilization (Fig. 5B). Because the collagen-mediated cytosolic Ca2+ mobilization are believed to be caused by activation of PLC-␥2 to give inositol-1,4,5-trisphosphate (IP3) [36], we further examined the effect of hesperetin on PLC-␥2 activation. Hesperetin inhibited levels of collageninduced phosphorylated PLC-␥2 in the same concentration range that inhibited platelet aggregation (Fig. 6). In addition, collagen-induced time-dependent increase of PLC-␥2 phosphorylation was completely abrogated after treatment
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
of hesperetin at a highest concentration of 100 M (data not shown). These results suggest that inhibition of PLC-␥2 phosphorylation may be important in the inhibition of hesperetin on collagen-mediated platelet aggregation. The precise target of hesperetin on collagen-mediated signaling transduction remains unknown, however, it is not unreasonable to speculate that this target may be involved in a prior inhibition of the upstream signal(s) of PLC-␥2 rather than a direct inhibition of PLC, as hesperetin has no effect on thrombininduced platelet aggregation even at a highest concentration of 100 M. In the case of thrombin-mediated platelet aggregation, PLC activation plays a central role [37], and U73122, a well-known PLC inhibitor, can inhibit both collagen and thrombin-induced platelet aggregation (data not shown). Fc receptor gamma chain of collagen receptor may be a potential target for the inhibition of hesperetin on collagen-induced platelet aggregation; however, for lacking such a commercial antibody against Fc receptor gamma chain, we cannot confirm such a hypothesis. On the other hand, the inhibition of tyrosine kinase by hesperetin maybe also contributes to the inhibition of collagen-induced platelet aggregation, as flavonoids have been reported to exhibit such inhibitions on tyrosine kinases [35]. Further study in this direction is still needed. In addition, collagen-induced arachidonic acid release from [3 H]arachidonic acid-prelabeled rabbit platelets was also inhibited by hesperetin in a concentration-dependent manner (Fig. 7), which is in agreement with a previous study that inhibition of phosphoinositide breakdown was able to block the arachidonic acid liberation completely [38]. It has been reported that volunteers given a commercial orange juice reached maximum plasma concentration of 1.28–2.72 M hesperetin [39,40]. Concerning the strong inhibitory effect of hesperetin on platelet aggregation, this effect is related to the high concentrations of hesperetin treatment (>10 M). This raises the question of whether the observed antiplatelet effect of hesperetin at higher concentrations is exact and of value in vivo. Because hesperetin can bind to membrane proteins and partition within cells, however, the effective cellular concentration achieved remains to be determined. In fact, we have observed a significant inhibition of collagen-induced platelet aggregation ex vivo after oral administration of hesperetin in rats with inhibition values of 66 and 16.5% when stimulated with 1.0 and 2.0 g/mL collagen, respectively. In addition, it has also been reported that micronized purified flavonoid fraction (MPFF, which was commercialized as Daflon® 500 mg, or Arvenum® 500, Capiven, Detralex, Variton, Ardium, and Venitol), consisting of 90% diosmin and 10% hesperidin, significantly inhibited in vivo platelet functions [15]. Further study on human subjects may help to elucidate this question. In summary, our study showed that hesperetin was able to inhibit platelet aggregation induced by collagen and arachidonic acid, and the cellular mechanism for the antiplatelet activity of hesperetin was mainly mediated by the inhibition of PLC-␥2 phosphorylation and cyclooxygenase-1 activity. This beneficial property of hesperetin may be of importance
151
in thrombosis in which platelet aggregation is an important determinant of thrombus initiation and development, and may contribute to the beneficial effects of grapefruits and oranges on cardiovascular system. Acknowledgement This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (No. R05-2004-000-12436-0). References [1] Lange RA, Hillis LD. Antiplatelet therapy for ischemic heart disease. N Engl J Med 2004;350:277–80. [2] Corti R, Fuster V, Badimon JJ. Pathogenetic concepts of acute coronary syndromes. J Am Coll Cardiol 2003;41:7S–14S. [3] Jackson SP, Nesbitt WS, Kulkarni S. Signaling events underlying thrombus formation. J Thromb Haemost 2003;1:1602–12. [4] Corti R, Farkouh ME, Badimon JJ. The vulnerable plaque and acute coronary syndromes. Am J Med 2002;113:668–80. [5] Cowan DH, Robertson AL, Shook P, Giroski P. Platelet adherence to collagen: role of plasma, ADP, and divalent cations. Br J Haematol 1981;47:257–67. [6] Cowan DH. Platelet adherence to collagen: role of prostaglandin– thromboxane synthesis. Br J Haematol 1981;49:425–34. [7] Sixma JJ, van Zanten GH, Huizinga EG, et al. Platelet adhesion to collagen: an update. Thromb Haemost 1997;78:434–8. [8] Nieswandt B, Watson SP. Platelet–collagen interaction: is GPVI the central receptor? Blood 2003;102:449–61. [9] Gibbins JM. Platelet adhesion signalling and the regulation of thrombus formation. J Cell Sci 2004;117:3415–25. [10] Woulfe DS. Platelet G protein-coupled receptors in hemostasis and thrombosis. J Thromb Haemost 2005. [11] Awtry EH, Loscalzo J. Aspirin. Circulation 2000;101:1206–18. [12] Stangl V, Lorenz M, Ludwig A, et al. The flavonoid phloretin suppresses stimulated expression of endothelial adhesion molecules and reduces activation of human platelets. J Nutr 2005;135:172–8. [13] Hubbard GP, Wolffram S, Lovegrove JA, Gibbins JM. Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans. J Thromb Haemost 2004;2:2138–45. [14] Cheng J, Kondo K, Suzuki Y, et al. Inhibitory effects of total flavones of Hippophae Rhamnoides L on thrombosis in mouse femoral artery and in vitro platelet aggregation. Life Sci 2003;72:2263–71. [15] McGregor L, Bellangeon M, Chignier E, et al. Effect of a micronized purified flavonoid fraction on in vivo platelet functions in the rat. Thromb Res 1999;94:235–40. [16] Garg A, Garg S, Zaneveld LJ, Singla AK. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother Res 2001;15: 655–69. [17] Galati EM, Trovato A, Kirjavainen S, et al. Biological effects of hesperidin, a citrus flavonoid. (Note III). Antihypertensive and diuretic activity in rat. Farmaco 1996;51:219–21. [18] Shin YW, Bok SH, Jeong TS, et al. Hypocholesterolemic effect of naringin associated with hepatic cholesterol regulating enzyme changes in rats. Int J Vitam Nutr Res 1999;69:341–7. [19] Bok SH, Lee SH, Park YB, et al. Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA: cholesterol transferase are lower in rats fed citrus peel extract or a mixture of citrus bioflavonoids. J Nutr 1999;129:1182–5. [20] Wilcox LJ, Borradaile NM, de Dreu LE, Huff MW. Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin,
152
[21] [22] [23]
[24] [25]
[26]
[27]
[28] [29]
[30]
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152 via reduced activity and expression of ACAT2 and MTP. J Lipid Res 2001;42:725–34. Wilcox LJ, Borradaile NM, Huff MW. Antiatherogenic properties of naringenin, a citrus flavonoid. Cardiovasc Drug Rev 1999;17:160–78. Born GV, Cross MJ. The aggregation of blood platelets. J Physiol 1963;168:178–95. Orallo F, Alvarez E, Basaran H, Lugnier C. Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin. Naunyn-Schmiedeberg’s Arch Pharmacol 2004;370:452–63. Kim JY, Jung KJ, Choi JS, Chung HY. Hesperetin: a potent antioxidant against peroxynitrite. Free Radic Res 2004;38:761–9. Proteggente AR, Basu-Modak S, Kuhnle G, et al. Hesperetin glucuronide, a photoprotective agent arising from flavonoid metabolism in human skin fibroblasts. Photochem Photobiol 2003;78:256–61. Jin YR, Cho MR, Ryu CK, et al. Antiplatelet activity of J78 (2-Chloro-3-[2 -bromo, 4 -fluoro-phenyl]-amino-8-hydroxy-1,4naphthoquinone), an antithrombotic agent, is mediated by thromboxane (TX) A2 receptor blockade with TXA2 synthase inhibition and suppression of cytosolic Ca2+ mobilization. J Pharmacol Exp Ther 2005;312:214–9. Kim JH, Jin YR, Park BS, et al. Luteolin prevents PDGF-BBinduced proliferation of vascular smooth muscle cells by inhibition of PDGF beta-receptor phosphorylation. Biochem Pharmacol 2005;69: 1715–21. Holmsen H, Dangelmarier CA. Measurement of sesretion of serotonin. Meth Enzymol 1989;169:205–10. Kang WS, Lim IH, Yuk DY, et al. Antithrombotic activities of green tea catechins and (−)-epigallocatechin gallate. Thromb Res 1999;96:229–37. Tzeng SH, Ko WC, Ko FN, Teng CM. Inhibition of platelet aggregation by some flavonoids. Thromb Res 1991;64:91–100.
[31] Parise LV, Venton DL, Le Breton GC. Arachidonic acid-induced platelet aggregation is mediated by a thromboxane A2 /prostaglandin H2 receptor interaction. J Pharmacol Exp Ther 1984;228:240–4. [32] Coleman RA, Smith WL, Narumiya S. International union of pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 1994;46:205–29. [33] Mower RL, Landolfi R, Steiner M. Inhibition in vitro of platelet aggregation and arachidonic acid metabolism by flavone. Biochem Pharmacol 1984;33:357–63. [34] Landolfi R, Mower RL, Steiner M. Modification of platelet function and arachidonic acid metabolism by bioflavonoids. Structure–activity relations. Biochem Pharmacol 1984;33:1525–30. [35] Guerrero JA, Lozano ML, Castillo J, et al. Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor. J Thromb Haemost 2005;3:369–76. [36] Farndale RW, Sixma JJ, Barnes MJ, De Groot PG. The role of collagen in thrombosis and hemostasis. J Thromb Haemost 2004;2:561–73. [37] Molino M, Bainton DF, Hoxie JA, Coughlin SR, Brass LF. Thrombin receptors on human platelets. Initial localization and subsequent redistribution during platelet activation. J Biol Chem 1997;272:6011–7. [38] Lockhart LK, Pampolina C, Nickolaychuk BR, McNicol A. Evidence for a role for phospholipase C, but not phospholipase A2 , in platelet activation in response to low concentrations of collagen. Thromb Haemost 2001;85:882–9. [39] Nielsen IL, Chee WS, Poulsen L, et al. Bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover trial. J Nutr 2006;136:404–8. [40] Manach C, Morand C, Gil-Izquierdo A, Bouteloup-Demange C, Remesy C. Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. Eur J Clin Nutr 2003;57:235–42.
Antiplatelet activity of hesperetin, a bioflavonoid, is mainly mediated by inhibition of PLC-␥2 phosphorylation and cyclooxygenase-1 activity Yong-Ri Jin a , Xiang-Hua Han b , Yong-He Zhang c , Jung-Jin Lee b , Yong Lim b , Jin-Ho Chung d , Yeo-Pyo Yun b,∗ a
Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea b College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea c Department of Pharmacology, School of Basic Medical Science, Peking University, Beijing 100083, China d College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea Received 21 June 2006; received in revised form 27 September 2006; accepted 6 October 2006 Available online 7 November 2006
Abstract Diet can be one of the most important factors that influence risks for atherothrombotic diseases. Hesperetin included in grapefruits and oranges is one candidate that may benefit the cardiovascular system. Here, we investigated antiplatelet activity of hesperetin in vitro. In addition, possible antiplatelet mechanism was also investigated. Hesperetin concentration-dependently inhibited washed rabbit platelet aggregation induced by collagen and arachidonic acid, with IC50 of 20.5 ± 3.5 and 69.2 ± 5.1 M, respectively, while has little effect on thrombin- or U46619-, a thromboxane (TX) A2 mimic, mediated platelet aggregation, suggesting that hesperetin may selectively inhibit collagen- and arachidonic acid-mediated signal transduction. In accordance with these findings, hesperetin revealed blocking of the collagen-mediated phospholipase (PL) C-␥2 phosphorylation, and caused concentration-dependent decreases of cytosolic calcium mobilization, arachidonic acid liberation and serotonin secretion. In addition, hesperetin inhibited arachidonic acid-mediated platelet aggregation by interfering with cyclooxygenase-1 activity as established by the measurement of arachidonic acid-mediated TXA2 and prostaglandin D2 formations as well as cyclooxygenase-1 and TXA2 synthase activity assays. Taken together, the present results provide a cellular mechanism for the antiplatelet activity of hesperetin through inhibition of PLC-␥2 phosphorylation and cyclooxygenase-1 activity, which may contribute to the beneficial effects of grapefruits and oranges on cardiovascular system. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Hesperetin; Platelet aggregation; Phospholipase C; Cyclooxygenase-1
1. Introduction The platelet activation and subsequent platelet aggregation play an essential role in the pathogenesis of cardiovascular, cerebrovascular, and peripheral vascular diseases. When blood vessels become damaged, subendothelial macromolecules such as collagen and thrombin, which are exposed or generated at the site of damage, stimulate platelet activa∗
Corresponding author. Tel.: +82 43 261 2821; fax: +82 43 268 2732. E-mail address: [email protected] (Y.-P. Yun).
0021-9150/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2006.10.011
tion and the initiation of hemostasis [1–4]. Collagen, which supports the adhesion of platelets to the site of injury via von Willebrand factor (VWF) as well as glycoprotein VI (GPVI) and integrin ␣21, induces platelet activation through a tyrosine kinase-based signaling pathway that involves the kinase Syk and phospholipase (PL) C-␥2, which results cytoplasmic calcium increase, shape change and granule release; adhesion is partly and aggregation is largely dependent on ADP and prostaglandin (PG) H2 /thromboxane (TX) A2 release [5–8]. This results in platelet shape change and spreading, and the release or secretion of proactivatory substances that
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
activate and recruit platelets to the developing thrombus [9]. The formation and release of thromboxane (TX) A2 is a central component in the platelet in response to a variety of agonists. TXA2 is an eicosanoid, a metabolite of arachidonic acid formed via the cyclooxygenase-TXA2 synthase pathway, and it is considered to be one of the most powerful agonists for platelet activation and a major contributor to the thrombus formation [10]. TXA2 binds to a G protein-coupled receptor to induce phospholipase C activation which results an increase of [Ca2+ ]i and protein kinase C activation, and causes platelets to change shape, extend pseudopods and adhere to platelets on the damaged surface. The clinical evidences have proven that inhibition of the synthesis or the action of TXA2 was effective for the patients with acute coronary syndromes and myocardial infarction [11]. Platelet activation may be inhibited by a number of dietary components including some dietary fats and antioxidants [12–15]. Flavonoids are a large group of low molecular weight polyphenolic compounds, and have been shown to be one of the most important classes in free states and as glycosides. Large amounts of bioflavonoids are ingested because of their abundance and wide distribution in foods and beverages. A dietary antioxidant that has received attention with regard to antioxidant effect is the polyphenolic compound, hesperidin (hesperetin 7-rhammnoglucoside) and its aglycone hesperetin (Fig. 1; 3 ,5,7-trihydroxy-4 -methoxy flavanone). Both flavonoids present extensively in the plant kingdom especially in many citrus fruits such as grapefruits and oranges, which are commonly used in traditional medicines [16]. It has been reported that hesperetin shows a wide spectrum of pharmacological effects such as anti-inflammatory, anticarcinogenic, antihypertensive and anti-atherogenic effects [16,17]. Hesperetin has been reported to inhibit low-density lipoprotein oxidation in vitro [18]. It has also been reported that hesperetin inhibited HMG-CoA reductase and lowers the plasma cholesterol level in rats [19]. The role of hesperetin and the structurally related naringenin citrus flavanone in the prevention and treatment of atherogenic disease has recently received considerable attention, with particular interest in the use of these flavanones as anticancer and anti-atherogenic compounds [20,21]. However, the anti-platelet activity as
Fig. 1. Chemical structure of hesperetin.
145
well as possible mechanism of hesperetin has not yet been elucidated. In this study, we have investigated the anti-platelet activity of hesperetin by measurement of platelet aggregation in vitro. The possible cellular mechanisms involved in PLC␥2 activation, arachidonic acid cascade, calcium mobilization and granule secretion were also investigated.
2. Materials and methods 2.1. Materials Hesperetin, indomethacin, imipramine, imidazole, serotonin creatinine sulfate, o-phthalaldehyde (OPT) and BSA were from Sigma Chemical Co. (St. Louis, MO). [3 H]Arachidonic acid (220 Ci/mmol) was obtained from Perkin-Elmer Life and Analytical Sciences (Boston, MA). Collagen, arachidonic acid and thrombin were purchased from Chrono-Log Co. (Havertown, PA). U46619, TXB2 , prostaglandin (PG) D2 , PGH2 and cyclooxygenase-1 inhibitor assay kit were purchased from Cayman Chemical Co. (Ann Arbor, MI). Anti-PLC-␥2 and anti-phospho-PLC␥2 antibody (Q-20) were obtained from Upstate Biotechnology Incorporate (Lake Placid, NY). U73122 was obtained from New England Biolabs, Inc. (Bevely, MA). The other chemicals were of analytical grade. 2.2. Animals New Zealand white rabbits were purchased from SamTako Animal Co. (Osan, Kyungi-do) and acclimatized for 1 week at 24 ◦ C and 55% humidity, with free access to a commercial pellet diet obtained from Samyang Co. (Wonju, Kangwon-do) and drinking water before experiments. The animal studies have been carried out in accordance with the Guide for the Care and Use of Laboratory Animals, Chungbuk National University, Korea. 2.3. Preparation of washed rabbit platelet Fresh blood was obtained from ear artery of male rabbits (New Zealand white rabbits weighing about 2–3 kg), collected into plastic tubes containing acid citrate dextrose (ACD) solution (1/6 volume of blood) composed of citric acid (65 mM), trisodium citrate (85 mM), and dextrose (2%) at pH 4.5, subsequently centrifuged at 250 × g for 10 min to obtain platelet-rich plasma. Platelet-rich plasma was centrifuged at 650 × g for 10 min at room temperature (20–25 ◦ C). The pellet was washed twice with Tyrode/HEPES solution (pH 6.35). The resultant pellet was resuspended in the second Tyrode/HEPES solution (pH 7.35) with a final density of approximately 4 × 108 platelets/mL. The Tyrode/HEPES solution was composed of NaCl 138.3 mM, KCl 2.68 mM, MgCl2 ·6H2 O 1.0 mM, NaHCO3 4.0 mM, HEPES 10 mM, glucose 0.1% (w/v) and albumin 0.35% (w/v) at pH 6.35 or 7.35.
146
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
2.4. Measurement of platelet aggregation Platelet aggregation was determined by a standard turbidometric method [22] using an aggregometer (Chrono-Log Co., Havertown, PA). Platelet aggregation was expressed as an increase in light transmission. The levels of light transmission were calibrated as 0% for a platelet suspension and 100% for the Tyrode/HEPES solution. Platelet suspension (0.25 mL) in the aggregometer cuvette was preincubated at 37 ◦ C for 2 min under continuous stirring at 1000 rpm. And then CaCl2 was added at the final concentration of 1 mM. After 2 min, various concentrations of hesperetin (1–100 M), which was in line with its use on other cells [23–25], was added. The incubation time was set as 3 min, which is long enough to reveal its effect, and platelet aggregation was induced by various agonists and aggregation percent was monitored for 5 min. 2.5. Measurement of arachidonic acid metabolites The arachidonic acid metabolites, TXB2 and PGD2 generations, were measured as previously described [26]. In brief, washed rabbit platelets (4 × 108 cells/mL) were preincubated with various concentrations of hesperetin at 37 ◦ C for 3 min, and further incubated with a mixture of [3 H]arachidonic acid and unlabeled arachidonic acid (2 M, 1 Ci/mL) for 5 min. The reaction was terminated by addition of stop solution (2.6 mM EGTA, 130 M BW755C). Lipids were extracted and separated by TLC on silica gel G plates with the following development system: ethyl acetate/isooctane/acetic acid/H2 O (11/5/2/10, v/v/v/v). The area corresponding to each lipid was scraped off and the radioactivity was determined by liquid scintillation counting. 2.6. TXA2 synthase activity assay The TXA2 synthase activity was assayed as previously described [26]. In brief, aliquots of PGH2 in anhydrous acetone, were pipetted into glass tubes, and then the acetone was evaporated under a gentle stream of nitrogen, and PGH2 was re-dissolved immediately in ethanol. Platelet suspensions were incubated with the test compounds at 37 ◦ C for 3 min prior to the addition of 5 M PGH2 . The final concentration of ethanol was 0.1% (v/v). At 5 min after addition of PGH2 , the incubations were terminated by addition of cooling EGTA (2 mM) and centrifuged at 12,000 × g at 4 ◦ C for 4 min. Because TXA2 is very unstable and rapidly converted to more stable metabolite TXB2 , the amount of TXB2 in the supernatants was assayed by a commercial enzyme immunoassay kit according to the manufacturers’ instructions (Amersham Biosciences, Ltd., Bucking Hamshire). TXA2 synthase activity is reflected by the production of TXB2 . 2.7. Cytosolic calcium mobilization assay Cytosolic Ca2+ mobilization was measured by using the fluorescent dye fura-2/AM as previously described [26],
which involved incubating the platelets with cell permeant acetoxymethyl ester. Rabbit platelets (isolated as described above) were incubated with 2 M fura-2/AM at room temperature for 1 h (on a rocking platform) in the loading buffer (137 mM NaCl, 27 mM KCl, 0.4 mM NaH2 PO4 , 10 mM HEPES, 12 mM NaHCO3 , 5.5 mM dextrose, 0.35% BSA, pH 7.4). Excess fura-2/AM was removed by centrifugation (500 × g for 10 min) and the platelets were suspended in fresh buffer, without added EGTA. Aliquots of platelet suspension (2.5 mL) were added to 4 mL cuvettes containing a teflon coated stirrer bar (Chrono-Log, Havertown, PA). Just before [Ca2+ ]i measurement was performed, Ca2+ was added back to the buffer to a final concentration of 1 mM, and then hesperetin (various concentrations in 25 L) and collagen were added. The measurement of [Ca2+ ]i was performed at room temperature in a MSIII fluorimeter (Photon Technology International, S. Brunswick, NJ) using excitation wavelengths of 340 and 380 as well as an emission wavelength of 505 nm. [Ca2+ ]i was calculated by using the SPEX dM3000 software package. 2.8. Immunoblotting For analysis of total platelet proteins, the reaction was terminated by addition of LaemmLi sample buffer, and the mixture was then boiled for 5 min and analyzed on a 7.5% SDS-PAGE. Immunoblot assays were performed as previously described [27], with slight modifications. For immunoblotting, proteins were electrically transferred to the polyvinylidene difluoride membrane for 80 min at 120 mA. Blots were incubated for 4 h with 5% (w/v) BSA in TBS to block residual protein binding sites. Immunodetection of phospho-PLC-␥2 was detected by rabbit anti-phosphoPLC-␥2 antibody (Q-20, 1 g/mL) in TBS containing 5% BSA for 4 h. The primary antibody was removed, and blots were washed in TBS with 0.05% Tween-20 three times. To detect the primary antibody, blots were incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit antibody (New England Biolabs, MA) diluted to 1:5000 in TBS containing 5% BSA for 5 h, and then washed five times in TBS with 0.05% Tween-20. After the blots were exposed to enhanced chemiluminescence reagents (Amersham Biosciences, Ltd., Bucking Hamshire) for 5 min, they were then exposed to hyper film-enhanced chemiluminescence (Amersham) for 5 min. The intensities of phospho-PLC-␥2 and total PLC-␥2 bands were quantified by Scion-Image for Windows Program (Scion Corporation, MA). 2.9. Measurement of serotonin secretion Serotonin release was measured by the fluorimetric method of Holmsen and Dangelmaier with a little modification [28]. In brief, to prevent the reuptake of sereted serotonin, imipramine (a serotonin re-uptake inhibitor; 5 M) was added to platelet suspension. Washed rabbit platelets were treated with hesperetin at 37 ◦ C for 3 min prior to addi-
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
147
tion of collagen. A 0.35 mL aliquot of the washed rabbit platelet was mixed with 5 mM EDTA in ice, and centrifuged at 12,000 × g for 2 min. The supernatant was mixed with 6 M trichloroacetic acid (TCA) and centrifuged at 12,000 × g for 2 min. A 0.3 mL aliquot of TCA supernatant was mixed with 2.4 mL of the solution (0.5% o-phthalaldehyde in ethanol diluted 1:10 with 8N HCl), placed in a boiling water bath for 10 min, and then cooled in ice. The excess TCA was extracted with chloroform, and fluorophore was measured at the wavelength of excitation (360 nm) and emission (475 nm). Serotonin creatinine sulfate was used as a standard solution to calculate the extent of serotonin release. 2.10. Measurement of arachidonic acid liberation The arachidonic acid liberation was measured as previously described [26]. In brief, PRP was preincubated with [3 H]arachidonic acid (1 Ci/mL) at 37 ◦ C for 1.5 h, and then washed as described above. The [3 H]arachidonic acidlabeled platelets (4 × 108 cells/mL) were pretreated with 100 M BW755C, a COX and LOX inhibitor, and various concentrations of hesperetin at 37 ◦ C for 3 min in the presence of 1 mM CaCl2 , and then stimulated with collagen (50 g/mL). The reaction was terminated by addition of chloroform/methanol/HCl (200/200/1, v/v/v). Lipids were extracted and separated by TLC on silica gel G plates with the following development system: petroleum ether/diethyl ether/acetic acid (40/40/1, v/v/v). The area corresponding to each lipid was scraped off, and the radioactivity was determined by liquid scintillation counting. 2.11. Statistical analysis The experimental results are expressed as mean ± SEM. A one-way analysis of variance (ANOVA) was used for multiple comparison (Sigma Stat® . SPSS Inc., CA). If there was a significant variation between treated-groups, Dunnett’s test was applied. The data were considered significant with a probability less than 0.05.
3. Results 3.1. Effect of hesperetin on washed rabbit platelet aggregation in vitro To compare the selectivity of hesperetin on various agonists-induced platelet aggregation, the agonists concentration were determined by a preliminary study and set as 10 g/mL for collagen, 100 M for arachidonic acid, 1 M for U46619 and 0.05 U/mL for thrombin. Under these concentrations, these agonists almost cause equal platelet aggregation (70 ± 5.5%). As shown in Fig. 2, hesperetin concentration-dependently inhibited collagen (10 g/mL)and arachidonic acid (100 M)-challenged washed rabbit platelet aggregation, with IC50 values of 20.5 ± 3.5
Fig. 2. Effect of hesperetin on washed rabbit platelet aggregation in vitro. Washed rabbit platelet suspension was incubated at 37 ◦ C in an aggregometer with stirring at 1000 rpm, and then hesperetin was added. After 3 min preincubation, the platelet aggregation was induced by addition of arachidonic acid (100 M), collagen (10 g/mL) in the absence or presence of indomethacin (20 M), U46619 (1 M), or thrombin (0.05 U/mL). The aggregation percents were expressed as percentage of maximum aggregation induced by respective inducers. Data are expressed as mean ± S.E.M. (n = 4).
and 69.2 ± 5.1 M, respectively. However, hesperetin only slightly inhibited U46619 (1 M)- and had no effect on thrombin (0.05 U/mL)-induced platelet aggregation. These results indicate that hesperetin may selectively affect collagen- and arachidonic acid-mediated signal transductions in platelets. To clarify whether the inhibition of hesperetin on collagen-induced platelet aggregation was mediated by arachidonic cascade, effect of hesperetin on collagen-induced indomethacin (20 M)-pretreated platelet aggregation was also determined. The maximum platelet aggregation induced by collagen was reduced to 50 ± 3% after treatment of indomethacin, however, the inhibition-curve of hesperetin on collagen-induced platelet aggregation was not changed, which indicates that in addition to the inhibition of arachidonic cascade, other pathways may also be involved in the inhibition of hesperetin on collagen-mediated platelet aggregation. 3.2. Effect of hesperetin on conversions of arachidonic acid to TXB2 and PGD2 Hesperetin concentration-dependently suppressed TXB2 and PGD2 generations induced by addition of [3 H]arachidonic acid in intact rabbit platelets (Fig. 3A and B). The TXB2 formations were inhibited by 8.5, 25.4 and 75.3% at the concentrations of 20, 50 and 100 M, respectively. And PGD2 formations were also inhibited by 0.5, 10.4 and 45.3% at the concentrations of 20, 50 and 100 M, respectively. These results suggest that hesperetin may inhibit cyclooxygenase activity rather than that of TXA2
148
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
As shown in Fig. 3C, hesperetin had no effect on the conversion of PGH2 into TXB2 in all concentrations of hesperetin used. However, imidazole, a typical TXA2 synthase inhibitor, markedly inhibited the conversion of PGH2 into TXB2 . 3.4. Effect of hesperetin on collagen-induced [Ca2+ ]i The representative traces in which collagen were added to induce [Ca2+ ]i mobilization were shown in Fig. 4A. After 3 min incubation of platelets with various concentrations of hesperetin, collagen (10 g/mL) was added to induce [Ca2+ ]i mobilization. Collagen induced a slow but stable increase of [Ca2+ ]i which reaches the peak level of 300 nM after 5–7 min. Treatment of the platelet suspension with hesperetin inhibited the elevation of the [Ca2+ ]i in response to collagen by 20.2, 50.8 and 77.2% at concentrations of 10, 20 and 50 M, respectively. The right panel (Fig. 4B) is the average of three times separated experiments, similar to that shown in left panel. 3.5. Effect of hesperetin on serotonin secretion
Fig. 3. Effects of hesperetin on arachidonic acid-induced metabolite and TXA2 synthase activity. The arachidonic acid-mediated [3 H]TXB2 and [3 H]PGD2 formations were assayed by using [3 H]arachidonic acid. The [3 H]TXB2 (A) and [3 H]PGD2 (B) were extracted and separated by TLC on silica gel G plate. The area corresponding to each lipid was scraped off and the radioactivity was determined by liquid scintillation counting. TXA2 synthase activity (C) was assayed by using PGH2 . TXA2 synthase activity is reflected by the production of TXB2 , which was determined by an enzymeimmunoassay kit. Data are expressed as mean ± S.E.M. (n = 3). * P < 0.05 and ** P < 0.01 vs. stimulus control.
synthase, because TXA2 and PGD2 are simultaneously produced from arachidonic acid through COX pathway. Indomethacin, a COX inhibitor used as a positive control, almost completely inhibited TXA2 and PGD2 formations at a concentration of 20 M. It was also demonstrated by a COX-1 activity assay that hesperetin inhibited COX-1 activity in a concentration-dependent manner (data not shown). 3.3. Effect of hesperetin on TXA2 synthase activity The conversion of arachidonic acid to TXA2 in platelets requires the action of two enzymes, COX and TXA2 synthase. TXA2 synthase catalyzes the conversion of PGH2 to TXA2 in platelets. By utilizing PGH2 , it is possible to circumvent the COX step during arachidonic acid metabolism.
Serotonin is secreted from activated platelets during platelet aggregation. Since hesperetin inhibited platelet aggregation induced by collagen and arachidonic acid, it also reduced the release of serotonin in a concentration-dependent manner, with inhibition percentages of 25.2 and 83.5% at concentrations of 50 and 100 M for arachidonic acid (Fig. 5A), respectively, and 10.5, 42.6 and 83.3% at concentrations of 10, 20 and 50 M for collagen (Fig. 5B), respectively. 3.6. Effect of hesperetin on collagen-induced PLC-γ2 phosphorylation To further examine the underlying antiplatelet mechanism of hesperetin, washed rabbit platelets were stimulated with 10 g/mL of collagen in the presence or absence of hesperetin, and the phosphorylation of PLC-␥2 was assayed. As shown in Fig. 6, pretreatment with hesperetin at concentrations of 10, 20, 50 and 100 M significantly inhibited collagen-induced PLC-␥2 phosphorylation with the inhibition percentages of 10.1, 45.4, 60.3 and 87.5%, respectively. Suppressive effect of hesperetin on collagen-induced PLC␥2 phosphorylation was also confirmed after 1, 3 and 5 min incubations, in which pretreatment of hesperetin at a concentration of 200 M completely abrogated this induction (data not shown). 3.7. Effect of hesperetin on collagen-induced arachidonic acid liberation As shown in Fig. 7, pretreatment of hesperetin concentration-dependently inhibited collagen-induced arachidonic acid liberation in [3 H]arachidonic acid prelabeled rabbit platelets by 4.5, 22.4, 44.7 and 64.3% at the
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
149
Fig. 4. Effect of hesperetin on collagen-stimulated [Ca2+ ]i . Calcium (1 mM) was added to the platelet suspension 10 s before data collection started (zero time). Hesperetin solution was added to yield final concentrations of 10, 20 and 50 M in the platelet suspension. Collagen (10 g/mL) was added 3 min later. The traces (A) are from a representative experiment; similar results were obtained from three separate experiments, and average data were shown in right panel (B) as mean ± S.E.M. * P < 0.05 and ** P < 0.01 vs. stimulus control.
concentrations of 5, 10, 20 and 50 M, respectively. U73122, a phospholipase C inhibitor which was used as a positive control, completely blocked arachidonic acid liberation at a concentration of 20 M.
Fig. 6. Effect of hesperetin on collagen-induced PLC-␥2 phosphorylation. Washed rabbit platelets in the presence of EGTA (1 mM) and indomethacin (20 M) were incubated with various concentrations of hesperetin (10, 20, 50 and 100 M) or DMSO for 3 min, and then stimulated with collagen (10 g/mL) for 5 min. Cells were lysed and 20 g/mL protein was analyzed with SDS-PAGE. Relative densities were quantified by scanning densitometry and showed the levels of each density as relative value of total PLC-␥2, respectively. Data are expressed as mean ± S.E.M. (n = 3). * P < 0.05 and ** P < 0.01 vs. stimulus control.
4. Discussion Fig. 5. Effect of hesperetin on serotonin secretion. Washed rabbit platelet suspension was incubated with imipramine (5 M) and various concentrations of hesperetin at 37 ◦ C for 3 min prior to addition of arachidonic acid (A) or collagen (B). The serotonin concentration was determined by a fluorimetric method as described in Section 2. Data are expressed as mean ± S.E.M. (n = 3). * P < 0.05 and ** P < 0.01 vs. stimulus control.
A group of antioxidants of the flavonoid family has been reported to inhibit platelet aggregation in vitro and ex vivo, and human studies have also suggested that a diet high in flavonoids may inhibit platelet aggregation in vivo
150
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
Fig. 7. Effect of hesperetin on collagen-induced arachidonic acid liberation. [3 H]Arachidonic acid-prelabeled platelets were incubated with various concentrations of hesperetin at 37 ◦ C for 3 min in the presence of 100 M BW755C, and then stimulated with 50 g/mL of collagen for 2 min. Liberated [3 H]arachidonic acid was determined as described in Section 2. Data are expressed as mean ± S.E.M. (n = 3). * P < 0.05 and ** P < 0.01 vs. stimulus control.
[12,13,29,30]. Hesperetin included in grapefruits and oranges is one candidate that may benefit the cardiovascular system [21]. In the present study, we demonstrated that hesperetin was a potent inhibitor of platelet aggregation in response to collagen and arachidonic acid, and the cellular mechanism for the antiplatelet activity of hesperetin might be mediated by the inhibition of PLC-␥2 phosphorylation and cyclooxygenase-1 activity. It is well known that U46619, a TXA2 mimic, acts directly on the TXA2 receptor to induce G protein-coupled phospholipase C activation, resulting an increase of [Ca2+ ]i and protein kinase C activation [3]. Similarly, arachidonic acid, which acts directly on membrane COX-TXA2 synthase pathway to produce TXA2 , mediates platelet activation in the same way as U46619 [31]. From the platelet aggregation study (Fig. 2), hesperetin inhibited arachidonic acidmediated platelet aggregation in a concentration-dependent manner, while even at a highest concentration of 100 M slightly suppressed U46619-induced platelet aggregation. These results suggest that hesperetin may be more potent in inhibition of arachidonic acid-mediated TXA2 production than a direct inhibition of TXA2 receptor. Therefore the effect of hesperetin on the generation of TXA2 was firstly determined by using [3 H]arachidonic acid in intact rabbit platelet. As shown in Fig. 3A and B, hesperetin inhibited both [3 H]TXA2 and [3 H]PGD2 in the same concentrationdependent manner. Considering that TXA2 and PGD2 are simultaneously produced from arachidonic acid through COX-1 pathway in platelets [32], it seems that hesperetin may selectively inhibit activity of COX-1 rather than that of TXA2 , because indomethacin, a potent COX inhibitor, also completely inhibited TXA2 and PGD2 productions. It was also confirmed by a COX-1 activity as well as a TXA2 synthase activity results that hesperetin concentrationdependently inhibited COX-1 activity (data not shown) but had no effect on PGH2 —a precursor of the PGs and TXA2 , mediated TXB2 formation (Fig. 3C) in all concentrationranges. Present results were consistent with other reports that some flavonoids could inhibit platelet by the inhibition
of COX-1 activity [33,34]. Moreover, AA-induced serotonin secretion was also inhibited by hesperetin in a concentrationdependent manner (Fig. 5A). The reason that hesperetin inhibited U46619-induced platelet aggregation by 30% at a concentration of 100 M may be due to a direct inhibition of TXA2 receptor, although it is very weak, which may be due to its lack of a double bond in C2–C3. It has been reported that flavonoids (such as apigenin, luteolin, genistein and quercetin, etc.) share a characteristic conjugation between A, C and B rings with the presence of a lactone structure, which is also present within the TXA2 molecule and may account for competition between these compounds for the same receptors. The structural features such as the presence of the double bond in C2–C3 and/or keto group in C4 are important for the binding to the TXA2 receptor [35]. In addition, hesperetin also inhibited collagen-mediated platelet aggregation in a concentration-dependent manner, and the IC50 value (20.5 M) for collagen was more potent than that (69.2 M) for arachidonic acid. To exclude the possibility that the inhibition of collagen-induced platelet aggregation was mediated by the inhibition of COX-1 activity, the effect of hesperetin on collagen-induced indomethacinpretreated platelet aggregation was also determined. In the presence of indomethacin (20 M), which is sufficient enough to block COX-1 activity completely, the maximum platelet aggregation induced by collagen (10 g/mL) was reduced about 25% compared with the control (data not shown). As shown in Fig. 2, however, pretreatment of indomethacin did not change the inhibition curve of hesperetin on collagen-induced platelet aggregation. It seems that, in addition to the inhibition of COX-1 activity, hesperetin may specifically affect some components involved in collagen-mediated signal transduction. It has been reported that collagen induces platelet activation through a tyrosine kinase-based signaling pathway that involves the kinase Syk and PLC-␥2, which results in [Ca2+ ]i increase, shape change and granule release [9]; adhesion is partly and aggregation is largely dependent on ADP and TXA2 /PGH2 release [5,6]. Considering that calcium mobilization is a major cellular process in collagen-mediated platelet activation, the cytosolic Ca2+ was determined in the platelets loaded with fura-2/AM. Concomitant with the inhibition of platelet aggregation data, hesperetin inhibited cytosolic Ca2+ mobilization in a concentration-dependent manner (Fig. 4). Accordingly the granule secretion, which was determined as the level of serotonin, was also inhibited by hesperetin in the same pattern as inhibition of platelet aggregation and cytosolic Ca2+ mobilization (Fig. 5B). Because the collagen-mediated cytosolic Ca2+ mobilization are believed to be caused by activation of PLC-␥2 to give inositol-1,4,5-trisphosphate (IP3) [36], we further examined the effect of hesperetin on PLC-␥2 activation. Hesperetin inhibited levels of collageninduced phosphorylated PLC-␥2 in the same concentration range that inhibited platelet aggregation (Fig. 6). In addition, collagen-induced time-dependent increase of PLC-␥2 phosphorylation was completely abrogated after treatment
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152
of hesperetin at a highest concentration of 100 M (data not shown). These results suggest that inhibition of PLC-␥2 phosphorylation may be important in the inhibition of hesperetin on collagen-mediated platelet aggregation. The precise target of hesperetin on collagen-mediated signaling transduction remains unknown, however, it is not unreasonable to speculate that this target may be involved in a prior inhibition of the upstream signal(s) of PLC-␥2 rather than a direct inhibition of PLC, as hesperetin has no effect on thrombininduced platelet aggregation even at a highest concentration of 100 M. In the case of thrombin-mediated platelet aggregation, PLC activation plays a central role [37], and U73122, a well-known PLC inhibitor, can inhibit both collagen and thrombin-induced platelet aggregation (data not shown). Fc receptor gamma chain of collagen receptor may be a potential target for the inhibition of hesperetin on collagen-induced platelet aggregation; however, for lacking such a commercial antibody against Fc receptor gamma chain, we cannot confirm such a hypothesis. On the other hand, the inhibition of tyrosine kinase by hesperetin maybe also contributes to the inhibition of collagen-induced platelet aggregation, as flavonoids have been reported to exhibit such inhibitions on tyrosine kinases [35]. Further study in this direction is still needed. In addition, collagen-induced arachidonic acid release from [3 H]arachidonic acid-prelabeled rabbit platelets was also inhibited by hesperetin in a concentration-dependent manner (Fig. 7), which is in agreement with a previous study that inhibition of phosphoinositide breakdown was able to block the arachidonic acid liberation completely [38]. It has been reported that volunteers given a commercial orange juice reached maximum plasma concentration of 1.28–2.72 M hesperetin [39,40]. Concerning the strong inhibitory effect of hesperetin on platelet aggregation, this effect is related to the high concentrations of hesperetin treatment (>10 M). This raises the question of whether the observed antiplatelet effect of hesperetin at higher concentrations is exact and of value in vivo. Because hesperetin can bind to membrane proteins and partition within cells, however, the effective cellular concentration achieved remains to be determined. In fact, we have observed a significant inhibition of collagen-induced platelet aggregation ex vivo after oral administration of hesperetin in rats with inhibition values of 66 and 16.5% when stimulated with 1.0 and 2.0 g/mL collagen, respectively. In addition, it has also been reported that micronized purified flavonoid fraction (MPFF, which was commercialized as Daflon® 500 mg, or Arvenum® 500, Capiven, Detralex, Variton, Ardium, and Venitol), consisting of 90% diosmin and 10% hesperidin, significantly inhibited in vivo platelet functions [15]. Further study on human subjects may help to elucidate this question. In summary, our study showed that hesperetin was able to inhibit platelet aggregation induced by collagen and arachidonic acid, and the cellular mechanism for the antiplatelet activity of hesperetin was mainly mediated by the inhibition of PLC-␥2 phosphorylation and cyclooxygenase-1 activity. This beneficial property of hesperetin may be of importance
151
in thrombosis in which platelet aggregation is an important determinant of thrombus initiation and development, and may contribute to the beneficial effects of grapefruits and oranges on cardiovascular system. Acknowledgement This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (No. R05-2004-000-12436-0). References [1] Lange RA, Hillis LD. Antiplatelet therapy for ischemic heart disease. N Engl J Med 2004;350:277–80. [2] Corti R, Fuster V, Badimon JJ. Pathogenetic concepts of acute coronary syndromes. J Am Coll Cardiol 2003;41:7S–14S. [3] Jackson SP, Nesbitt WS, Kulkarni S. Signaling events underlying thrombus formation. J Thromb Haemost 2003;1:1602–12. [4] Corti R, Farkouh ME, Badimon JJ. The vulnerable plaque and acute coronary syndromes. Am J Med 2002;113:668–80. [5] Cowan DH, Robertson AL, Shook P, Giroski P. Platelet adherence to collagen: role of plasma, ADP, and divalent cations. Br J Haematol 1981;47:257–67. [6] Cowan DH. Platelet adherence to collagen: role of prostaglandin– thromboxane synthesis. Br J Haematol 1981;49:425–34. [7] Sixma JJ, van Zanten GH, Huizinga EG, et al. Platelet adhesion to collagen: an update. Thromb Haemost 1997;78:434–8. [8] Nieswandt B, Watson SP. Platelet–collagen interaction: is GPVI the central receptor? Blood 2003;102:449–61. [9] Gibbins JM. Platelet adhesion signalling and the regulation of thrombus formation. J Cell Sci 2004;117:3415–25. [10] Woulfe DS. Platelet G protein-coupled receptors in hemostasis and thrombosis. J Thromb Haemost 2005. [11] Awtry EH, Loscalzo J. Aspirin. Circulation 2000;101:1206–18. [12] Stangl V, Lorenz M, Ludwig A, et al. The flavonoid phloretin suppresses stimulated expression of endothelial adhesion molecules and reduces activation of human platelets. J Nutr 2005;135:172–8. [13] Hubbard GP, Wolffram S, Lovegrove JA, Gibbins JM. Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans. J Thromb Haemost 2004;2:2138–45. [14] Cheng J, Kondo K, Suzuki Y, et al. Inhibitory effects of total flavones of Hippophae Rhamnoides L on thrombosis in mouse femoral artery and in vitro platelet aggregation. Life Sci 2003;72:2263–71. [15] McGregor L, Bellangeon M, Chignier E, et al. Effect of a micronized purified flavonoid fraction on in vivo platelet functions in the rat. Thromb Res 1999;94:235–40. [16] Garg A, Garg S, Zaneveld LJ, Singla AK. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother Res 2001;15: 655–69. [17] Galati EM, Trovato A, Kirjavainen S, et al. Biological effects of hesperidin, a citrus flavonoid. (Note III). Antihypertensive and diuretic activity in rat. Farmaco 1996;51:219–21. [18] Shin YW, Bok SH, Jeong TS, et al. Hypocholesterolemic effect of naringin associated with hepatic cholesterol regulating enzyme changes in rats. Int J Vitam Nutr Res 1999;69:341–7. [19] Bok SH, Lee SH, Park YB, et al. Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA: cholesterol transferase are lower in rats fed citrus peel extract or a mixture of citrus bioflavonoids. J Nutr 1999;129:1182–5. [20] Wilcox LJ, Borradaile NM, de Dreu LE, Huff MW. Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin,
152
[21] [22] [23]
[24] [25]
[26]
[27]
[28] [29]
[30]
Y.-R. Jin et al. / Atherosclerosis 194 (2007) 144–152 via reduced activity and expression of ACAT2 and MTP. J Lipid Res 2001;42:725–34. Wilcox LJ, Borradaile NM, Huff MW. Antiatherogenic properties of naringenin, a citrus flavonoid. Cardiovasc Drug Rev 1999;17:160–78. Born GV, Cross MJ. The aggregation of blood platelets. J Physiol 1963;168:178–95. Orallo F, Alvarez E, Basaran H, Lugnier C. Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin. Naunyn-Schmiedeberg’s Arch Pharmacol 2004;370:452–63. Kim JY, Jung KJ, Choi JS, Chung HY. Hesperetin: a potent antioxidant against peroxynitrite. Free Radic Res 2004;38:761–9. Proteggente AR, Basu-Modak S, Kuhnle G, et al. Hesperetin glucuronide, a photoprotective agent arising from flavonoid metabolism in human skin fibroblasts. Photochem Photobiol 2003;78:256–61. Jin YR, Cho MR, Ryu CK, et al. Antiplatelet activity of J78 (2-Chloro-3-[2 -bromo, 4 -fluoro-phenyl]-amino-8-hydroxy-1,4naphthoquinone), an antithrombotic agent, is mediated by thromboxane (TX) A2 receptor blockade with TXA2 synthase inhibition and suppression of cytosolic Ca2+ mobilization. J Pharmacol Exp Ther 2005;312:214–9. Kim JH, Jin YR, Park BS, et al. Luteolin prevents PDGF-BBinduced proliferation of vascular smooth muscle cells by inhibition of PDGF beta-receptor phosphorylation. Biochem Pharmacol 2005;69: 1715–21. Holmsen H, Dangelmarier CA. Measurement of sesretion of serotonin. Meth Enzymol 1989;169:205–10. Kang WS, Lim IH, Yuk DY, et al. Antithrombotic activities of green tea catechins and (−)-epigallocatechin gallate. Thromb Res 1999;96:229–37. Tzeng SH, Ko WC, Ko FN, Teng CM. Inhibition of platelet aggregation by some flavonoids. Thromb Res 1991;64:91–100.
[31] Parise LV, Venton DL, Le Breton GC. Arachidonic acid-induced platelet aggregation is mediated by a thromboxane A2 /prostaglandin H2 receptor interaction. J Pharmacol Exp Ther 1984;228:240–4. [32] Coleman RA, Smith WL, Narumiya S. International union of pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 1994;46:205–29. [33] Mower RL, Landolfi R, Steiner M. Inhibition in vitro of platelet aggregation and arachidonic acid metabolism by flavone. Biochem Pharmacol 1984;33:357–63. [34] Landolfi R, Mower RL, Steiner M. Modification of platelet function and arachidonic acid metabolism by bioflavonoids. Structure–activity relations. Biochem Pharmacol 1984;33:1525–30. [35] Guerrero JA, Lozano ML, Castillo J, et al. Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor. J Thromb Haemost 2005;3:369–76. [36] Farndale RW, Sixma JJ, Barnes MJ, De Groot PG. The role of collagen in thrombosis and hemostasis. J Thromb Haemost 2004;2:561–73. [37] Molino M, Bainton DF, Hoxie JA, Coughlin SR, Brass LF. Thrombin receptors on human platelets. Initial localization and subsequent redistribution during platelet activation. J Biol Chem 1997;272:6011–7. [38] Lockhart LK, Pampolina C, Nickolaychuk BR, McNicol A. Evidence for a role for phospholipase C, but not phospholipase A2 , in platelet activation in response to low concentrations of collagen. Thromb Haemost 2001;85:882–9. [39] Nielsen IL, Chee WS, Poulsen L, et al. Bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover trial. J Nutr 2006;136:404–8. [40] Manach C, Morand C, Gil-Izquierdo A, Bouteloup-Demange C, Remesy C. Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. Eur J Clin Nutr 2003;57:235–42.