International Journal of
Molecular Sciences Article
Licochalcone A Prevents Platelet Activation and Thrombus Formation through the Inhibition of PLCγ2-PKC, Akt, and MAPK Pathways Li-Ming Lien 1,2,† , Kuan-Hung Lin 3,4,† , Li-Ting Huang 5 , Mei-Fang Tseng 5 , Hou-Chang Chiu 2,6 , Ray-Jade Chen 1,5, * and Wan-Jung Lu 3,5,7, * 1 2 3 4 5 6 7
* †
School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; [email protected] Department of Neurology, Shin Kong Wu Ho Su Memorial Hospital, Taipei 111, Taiwan; [email protected] Department of Pharmacology and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 110, Taiwan; [email protected] Central Laboratory, Shin Kong Wu Ho Su Memorial Hospital, Taipei 111, Taiwan Department of Medical Research and Division of General Surgery, Department of Surgery, Taipei Medical University Hospital, Taipei 110, Taiwan; [email protected] (L.-T.H.); [email protected] (M.-F.T.) College of Medicine, Fu-Jen Catholic University, Taipei 242, Taiwan Graduate Institute of Metabolism and Obesity Sciences, College of Public Health and Nutrition, Taipei Medical University, Taipei 110, Taiwan Correspondence: [email protected] (R.-J.C.); [email protected] (W.-J.L.); Tel.: +886-2-2737-2181 (ext. 3310) (R.-J.C.); +886-2-2736-1661 (ext. 3201) (W.-J.L.) These authors contributed equally to this work.
Received: 5 June 2017; Accepted: 9 July 2017; Published: 12 July 2017
Abstract: Platelet activation is involved in cardiovascular diseases, such as atherosclerosis and ischemic stroke. Licochalcone A (LA), an active ingredient of licorice, exhibits multiple biological activities such as anti-oxidation and anti-inflammation. However, its role in platelet activation remains unclear. Therefore, the study investigated the antiplatelet mechanism of LA. Our data revealed that LA (2–10 µM) concentration dependently inhibited platelet aggregation induced by collagen, but not thrombin and U46619. LA markedly attenuated collagen-stimulated ATP release, P-selectin secretion, calcium mobilization, and GPIIbIIIa activation, but did not interfere with the collagen binding to platelets. Moreover, LA significantly reduced the activation of PLCγ2, PKC, Akt and MAPKs. Thus, LA attenuates platelet activation, possibly by inhibiting collagen receptor downstream signaling but not by blocking the collagen receptors. In addition, LA prevented adenosine diphosphate (ADP)-induced acute pulmonary thrombosis, fluorescein sodium-induced platelet thrombus formation, and middle cerebral artery occlusion/reperfusion-induced brain injury in mice, but did not affect normal hemostasis. This study demonstrated that LA effectively reduced platelet activation and thrombus formation, in part, through the inhibition of PLCγ2–PKC, Akt, and MAPK pathways, without the side effect of bleeding. These findings also indicate that LA may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders such as stroke. Keywords: Licochalcone A; middle cerebral artery occlusion; platelet activation; PLCγ2–PKC; thrombus formation
1. Introduction Platelet activation is involved in normal hemostasis and in pathological processes such as atherosclerosis and stroke [1,2]. When blood vessels are injured, exposed extracellular matrix proteins Int. J. Mol. Sci. 2017, 18, 1500; doi:10.3390/ijms18071500
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 1500
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(e.g., collagen and von Willebrand factor) activate platelets and further recruit additional platelets from the bloodstream. These platelets form a firm platelet plug at the injury site to stop blood loss; however, under pathological conditions, the platelets are prone to uncontrolled activation and aggregation and may cause vessel occlusion. Collagen signaling is mediated through the interaction of collagen and its receptor glycoprotein VI (GPVI) and integrin α2β1 [3], which are located on the plasma membrane of platelets; these receptors transmit the activation signals, including those of phospholipase Cγ2 (PLCγ2) and protein kinase C (PKC) activation, and mediate platelet granule release and calcium mobilization [3], all of which finally lead to glycoprotein IIbIIIa (GPIIbIIIa) activation and subsequent platelet aggregation. These processes are crucial for platelet activation and thrombus formation [4]. Moreover, GPVI serves as a promising pharmacological target for the effective and safe treatment of thrombotic and possibly inflammatory diseases [4]. Aspirin and clopidogrel are commonly used to prevent stroke, but account for only a 20% reduction in all recurrent stroke events [5]. Thus, targeting collagen signaling may provide an alternative therapeutic approach to mitigate the recurrence of secondary stroke. Licochalcone A (LA), a natural chalcone derived from the roots and rhizomes of Glycyrrhiza spp., exhibits multiple biological activities such as antibacterial, antioxidant, anti-inflammatory, antimalarial, antiviral, and antitumor effects [6–11]. LA inhibits lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) production and cytokine release in the RAW 264.7 mouse macrophage cell line [12]. LA also alleviates LPS-induced acute lung and kidney injury through NF-κB and p38/ERK mitogen-activated protein kinase (MAPK) signaling and attenuates pertussis toxin-induced autoimmune encephalomyelitis by reducing the production of tumor necrosis factor-α and interferon-γ in vivo [12–14]. In addition, LA induces tumor cell cycle arrest, apoptosis, and autophagy in a various cancer cell lines [10,11]. These observations reveals that LA protects against several pathological processes. Although, recently, LA was also reported to reduce rabbit and rat platelet activation through the inhibition of cyclooxygenase-1 (COX-1) activity [15,16], its role in platelet activation and thrombosis remains unclear. In the present study, our preliminary data revealed that LA significantly inhibited collagen-induced platelet aggregation through PLCγ2–PKC pathway, suggesting that, in addition to inhibiting COX-1 activity, LA may attenuate platelet activation through other mechanisms. Therefore, we further systemically investigated the mechanism of LA in platelet activation and for the first time determined whether LA has antithrombotic effect in in vivo studies. 2. Results 2.1. Licochalcone A (LA) Inhibited Collagen-Induced Platelet Aggregation As shown in Figure 1A, LA (2–10 µM) was used to determine its effect on the platelet aggregation induced by collagen (1 µg/mL). The data indicated that LA (at concentrations of 2, 5 and 10 µM) inhibited collagen-induced platelet aggregation by 16.6%, 45.3% and 90.8%, respectively. The IC50 was approximately 5.6 µM. In addition, only at a higher concentration of 80 µM did LA affect thrombin (0.01 U/mL)- or U46619 (1 µM)-mediated platelet aggregation (Figure 1B). These results are consistent with that Okuda-Tanino et al. reported [15] and indicate that LA is more sensitive to the inhibition of collagen-mediated platelet activation. Accordingly, in the following experiments, we mainly evaluated the mechanism of LA at concentrations of 2–10 µM in collagen-mediated platelet activation events. 2.2. LA Inhibited Collagen-Mediated ATP Release and P-Selectin Secretion Platelet granule release plays a crucial role in the amplification of platelet activation and aggregation. Here, two experiments on ATP release and P-selectin secretion, which were assessed by the microplate reader and through flow cytometry, respectively, were performed to determine whether LA interferes with the granule release induced by collagen. As shown in Figure 1C, luciferase/luciferin was used to detect ATP. The data revealed that collagen markedly induced ATP release, which
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by the microplate reader and through flow cytometry, respectively, were performed to determine whether LA interferes with the granule release induced by collagen. As shown in Figure 1C, luciferase/luciferin usedµM) to detect The data revealed that manner. collagen markedly induced ATP was reversed by LAwas (2–10 in a ATP. concentration-dependent Moreover, LA (5 and release, which was reversed by LA (2–10 μM) in a concentration-dependent manner. Moreover, LAby 10 µM) significantly reduced collagen-induced P-selectin secretion (Figure 1D), as determined and 10 ofμM) significantly reduced (FITC)–P-selectin. collagen-induced These P-selectin secretion (Figure 1D), as the(5intensity fluorescein isothiocyanate findings suggest that LA inhibits determined by the intensity of fluorescein isothiocyanate (FITC)–P-selectin. These findings suggest collagen-mediated platelet activation through the blockade of granule release. that LA inhibits collagen-mediated platelet activation through the blockade of granule release.
Figure 1. Effects of Licochalcone A (LA) on the collagen-induced platelet aggregation, ATP release, Figure 1. Effects of Licochalcone A (LA) on the collagen-induced platelet aggregation, ATP release, and P-selectin secretion. (A,B) Washed platelets (3.6 × 108 cells/mL) were preincubated with DMSO and P-selectin secretion. (A,B) Washed platelets (3.6 × 108 cells/mL) were preincubated with DMSO (solvent control) and LA (2–80 μM), and were then stimulated using collagen (1 μg/mL), thrombin (solvent control) and LA (2–80 µM), and were then stimulated using collagen (1 µg/mL), thrombin (0.01 U/mL), or U46619 (1 μM) to trigger platelet aggregation, as measured by a transmission (0.01 U/mL), or U46619 (1 µM) to trigger platelet aggregation, as measured by a transmission aggregometer; (C) The effect of LA on collagen-induced ATP release was characterized by the aggregometer; (C) The effect of LA on collagen-induced ATP release was characterized by the detection detection of chemiluminescent emission from the luciferin–luciferase reaction, which was of chemiluminescent emission from the luciferin–luciferase reaction, which was continually recorded continually recorded using a microplate reader; (D) The effect of LA on collagen-induced P-selectin using a microplate reader; (D) The effect of LA on collagen-induced P-selectin secretion was detected secretion was detected using FITC–P-selectin antibody. The fluorescence was immediately detected using FITC–P-selectin antibody. The fluorescence was immediately detected through flow cytometry. through flow cytometry. The profiles (B) are representative examples of five similar experiments. The profiles (B) are representative examples of five similar experiments. Data (A,C) are presented as Data (A,C) are presented as the mean ± SEM. (A, n = 5; C, n = 3). * p < 0.05 and *** p < 0.001, compared thewith mean ± SEM. (A, n = 5; C, n = 3). * p < 0.05 and *** p < 0.001, compared with the DMSO group; Data the DMSO group; Data (D) are presented as the mean ± SEM. (n = 3). ** p < 0.01, compared with (D) are presented as# the ± SEM. (nwith = 3). **collagen p < 0.01,(positive) comparedgroup. with the resting group; # p < 0.05, the resting group; p < mean 0.05, compared the compared with the collagen (positive) group.
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2.3. LA LA Inhibited Inhibited Collagen-Mediated Collagen-Mediated Calcium Calcium Mobilization Mobilizationand andGPIIbIIIa GPIIbIIIaActivation Activationwithout withoutInterfering Interferingwith with 2.3. CollagenReceptors Receptors Collagen Calcium signaling signaling isis the the common commonplatelet plateletactivation activationsignaling signalingpathway. pathway. Receptor-stimulated Receptor-stimulated Calcium PLC catalyzes catalyzes the the hydrolysis hydrolysis of ofphosphatidylinositol phosphatidylinositol biphosphate biphosphate to to release releaseinositol inositoltrisphosphate trisphosphate PLC and diacyglycerol, diacyglycerol, which which activate activatecalcium calcium mobilization mobilization and andPKC, PKC,respectively respectively[17]. [17]. The Theelevation elevation of of and 2+ 2+ intracellular Ca Ca contributes to several events of platelet platelet activation, activation, such such as as shape shape change, change, granule granule intracellular release,and andGPIIbIIIa GPIIbIIIaactivaiton activaiton[18]. [18]. release, Asshown shownininFigure Figure2A, 2A,fura-2 fura-2was was used measure change in calcium according to As used to to measure thethe change in calcium levellevel according to the the ratio of F340/F380, which is directly correlated amount intracellularcalcium. calcium.The The data data ratio of F340/F380, which is directly correlated to to thethe amount ofofintracellular revealed that that LA LA (2–10 (2–10 µM) μM) markedly markedly inhibited inhibited calcium calcium mobilization, mobilization, as as detected detected using using F-4500 F-4500 revealed FluorescenceSpectrophotometer. Spectrophotometer.Moreover, Moreover, FITC–PAC-1 antibody to demonstrate Fluorescence thethe FITC–PAC-1 antibody waswas usedused to demonstrate that that(5–10 LA µM) (5–10markedly μM) markedly inhibited (Figure stepaggregation, in platelet LA inhibited GPIIbIIIaGPIIbIIIa activationactivation (Figure 2B), a final2B), stepainfinal platelet aggregation, as detected through flow cytometry. These findings that LA blocks calcium as detected through flow cytometry. These findings indicate that LA indicate blocks calcium mobilization and mobilizationGPIIbIIIa and subsequent activation, thereby inhibitingIn platelet In subsequent activation, GPIIbIIIa thereby inhibiting platelet aggregation. addition,aggregation. FITC–collagen addition, FITC–collagen was used to determine directlyleading blocks to collagen receptors, was used to determine whether LA directly blockswhether collagenLA receptors, the inhibition of leading to the inhibition downstream As obtained shown infrom Figure 2C,cytometry the data revealed obtained its downstream signaling. of Asits shown in Figuresignaling. 2C, the data flow fromFITC–collagen flow cytometry revealedbinds that FITC–collagen markedlypretreated binds to platelets. Moreover, that markedly to platelets. Moreover, LA did not interferepretreated with the LA did of notFITC–collagen interfere withtothe binding of finding FITC–collagen platelets. This collagen-induced finding suggests that LA binding platelets. This suggeststothat LA inhibits platelet inhibits collagen-induced plateletcollagen activation, possibly by inhibiting collagen downstream activation, possibly by inhibiting receptor downstream signaling butreceptor not by blocking the signalingreceptors. but not In byaddition, blockingLA the(10–80 collagen In addition, (10–80 μM) did as notdetected exhibit collagen µM)receptors. did not exhibit cytotoxicLA effects on platelets, cytotoxic on platelets, as detected by the LDH (Figure 2D), activation indicatingis that by the LDHeffects assay (Figure 2D), indicating that LA-mediated the assay inhibition of platelet not LA-mediated the inhibition of platelet activation is not due to the cytotoxicity. due to the cytotoxicity.
Figure 2. Cont.
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Figure Figure2.2.Effects EffectsofofLA LAon oncalcium calciummobilization, mobilization,GPIIbIIIa GPIIbIIIaactivation, activation,collagen collagenreceptors receptorsand andlactate lactate dehydrogenase (LDH) release. Washed platelets were preincubated with DMSO and LA (2–10 dehydrogenase (LDH) release. Washed platelets were preincubated with DMSO and LA (2–10µM), μM), and andwere werethen thenstimulated stimulatedusing using1 1µg/mL μg/mLcollagen collagen(A,B) (A,B)or orFITC–collagen FITC–collagen(C). (C).(A) (A)The Theratio ratioofoffura-2 fura-2 fluorescence fluorescence(F340/F380) (F340/F380) was was used used to to determine determine calcium calcium mobilization, mobilization, as as measured measured by byaaHitachi Hitachi F4500 fluorescence spectrophotometer; (B,C) GPIIbIIIa activation and the competition with collagen F4500 fluorescence spectrophotometer; (B,C) GPIIbIIIa activation and the competition with collagen were weredetermined determinedusing usingthe theFITC–PAC-1 FITC–PAC-1antibody antibodyand andFITC–collagen, FITC–collagen,respectively. respectively.The Thefluorescence fluorescence intensity was measured through flow cytometry; (D) The platelets were preincubated intensity was measured through flow cytometry; (D) The platelets were preincubatedwith withTyrode’s Tyrode’s solution, control) or or various variousconcentrations concentrationsofofLA LA(10–80 (10–80 µM) min at °C, 37 ◦and C, solution,DMSO DMSO (solvent (solvent control) μM) forfor 10 10 min at 37 and the supernatant was collected to measure LDH release by the LDH assay kit. LDH activity was the supernatant was collected to measure LDH release by the LDH assay kit. LDH activity was expressed expressedas asthe the%%ofoftotal totalenzyme enzymeactivity, activity,which whichwas wasmeasured measuredininplatelets plateletslysed lysedwith with0.5% 0.5%Triton Triton X-100. Data (A) are presented as the means ± SEM (n = 3). ** p < 0.01 and *** p < 0.001, compared X-100. Data (A) are presented as the means ± SEM (n = 3). ** p < 0.01 and *** p < 0.001, comparedwith with the theDMSO DMSOgroup; group;Data Data(B) (B)are arepresented presentedasasthe themeans means±±SEM SEM(n(n==3). 3).****pp 0.05, n = 8). In = 8). In addition, the LA-treated groups did not prolong the tail-bleeding time, compared addition, with the the LA-treated groups did not the tail-bleeding agent. time, compared with the DMSO group, DMSO group, indicating that LAprolong is a safer antithrombotic indicating that LA is a safer antithrombotic agent.
Figure 5.5. Influence Influence of ofLA LAon onmiddle middlecerebral cerebralartery arteryocclusion occlusion(MCAO)/reperfusion-induced (MCAO)/reperfusion-induced brain brain Figure injuryand andtailtail bleeding in Mice mice.(male, Mice5–6(male, old) were intraperitoneally injury bleeding time time in mice. weeks 5–6 old) weeks were intraperitoneally administrated administrated with control) DMSO (solvent control) LA (1.8 3.6 mg/kg) forwere 30 min. (A) Mice were with DMSO (solvent or LA (1.8 and 3.6or mg/kg) forand 30 min. (A) Mice subjected to MCAO subjected MCAOby for24-h 30 min followedImmediately by 24-h reperfusion. Immediately sacrifice, coronal for 30 min to followed reperfusion. after sacrifice, coronal after sections were cut and sectionsusing were cut and stained usingchloride; 2,3,5-triphenyltetrazolium chloride; and white indicate stained 2,3,5-triphenyltetrazolium white areas indicate infarction, redareas areas indicate infarction, and (left red areas indicate tissuesratios (left panel). Edemawere and calculated infarct ratios (right image panel) normal tissues panel). Edemanormal and infarct (right panel) through were calculated analysis and are reportedhemisphere. as a ratio ofInfarct the non-ischemic hemisphere. analysis and are through reportedimage as a ratio of the non-ischemic ratio was corrected for Infarct Data ratio are waspresented correctedasfor are the mean ± SEM. (nthe = 8). *** p < 0.001, edema. theedema. mean ±Data SEM. (n presented = 8). *** p
Molecular Sciences Article
Licochalcone A Prevents Platelet Activation and Thrombus Formation through the Inhibition of PLCγ2-PKC, Akt, and MAPK Pathways Li-Ming Lien 1,2,† , Kuan-Hung Lin 3,4,† , Li-Ting Huang 5 , Mei-Fang Tseng 5 , Hou-Chang Chiu 2,6 , Ray-Jade Chen 1,5, * and Wan-Jung Lu 3,5,7, * 1 2 3 4 5 6 7
* †
School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan; [email protected] Department of Neurology, Shin Kong Wu Ho Su Memorial Hospital, Taipei 111, Taiwan; [email protected] Department of Pharmacology and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 110, Taiwan; [email protected] Central Laboratory, Shin Kong Wu Ho Su Memorial Hospital, Taipei 111, Taiwan Department of Medical Research and Division of General Surgery, Department of Surgery, Taipei Medical University Hospital, Taipei 110, Taiwan; [email protected] (L.-T.H.); [email protected] (M.-F.T.) College of Medicine, Fu-Jen Catholic University, Taipei 242, Taiwan Graduate Institute of Metabolism and Obesity Sciences, College of Public Health and Nutrition, Taipei Medical University, Taipei 110, Taiwan Correspondence: [email protected] (R.-J.C.); [email protected] (W.-J.L.); Tel.: +886-2-2737-2181 (ext. 3310) (R.-J.C.); +886-2-2736-1661 (ext. 3201) (W.-J.L.) These authors contributed equally to this work.
Received: 5 June 2017; Accepted: 9 July 2017; Published: 12 July 2017
Abstract: Platelet activation is involved in cardiovascular diseases, such as atherosclerosis and ischemic stroke. Licochalcone A (LA), an active ingredient of licorice, exhibits multiple biological activities such as anti-oxidation and anti-inflammation. However, its role in platelet activation remains unclear. Therefore, the study investigated the antiplatelet mechanism of LA. Our data revealed that LA (2–10 µM) concentration dependently inhibited platelet aggregation induced by collagen, but not thrombin and U46619. LA markedly attenuated collagen-stimulated ATP release, P-selectin secretion, calcium mobilization, and GPIIbIIIa activation, but did not interfere with the collagen binding to platelets. Moreover, LA significantly reduced the activation of PLCγ2, PKC, Akt and MAPKs. Thus, LA attenuates platelet activation, possibly by inhibiting collagen receptor downstream signaling but not by blocking the collagen receptors. In addition, LA prevented adenosine diphosphate (ADP)-induced acute pulmonary thrombosis, fluorescein sodium-induced platelet thrombus formation, and middle cerebral artery occlusion/reperfusion-induced brain injury in mice, but did not affect normal hemostasis. This study demonstrated that LA effectively reduced platelet activation and thrombus formation, in part, through the inhibition of PLCγ2–PKC, Akt, and MAPK pathways, without the side effect of bleeding. These findings also indicate that LA may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders such as stroke. Keywords: Licochalcone A; middle cerebral artery occlusion; platelet activation; PLCγ2–PKC; thrombus formation
1. Introduction Platelet activation is involved in normal hemostasis and in pathological processes such as atherosclerosis and stroke [1,2]. When blood vessels are injured, exposed extracellular matrix proteins Int. J. Mol. Sci. 2017, 18, 1500; doi:10.3390/ijms18071500
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 1500
2 of 16
(e.g., collagen and von Willebrand factor) activate platelets and further recruit additional platelets from the bloodstream. These platelets form a firm platelet plug at the injury site to stop blood loss; however, under pathological conditions, the platelets are prone to uncontrolled activation and aggregation and may cause vessel occlusion. Collagen signaling is mediated through the interaction of collagen and its receptor glycoprotein VI (GPVI) and integrin α2β1 [3], which are located on the plasma membrane of platelets; these receptors transmit the activation signals, including those of phospholipase Cγ2 (PLCγ2) and protein kinase C (PKC) activation, and mediate platelet granule release and calcium mobilization [3], all of which finally lead to glycoprotein IIbIIIa (GPIIbIIIa) activation and subsequent platelet aggregation. These processes are crucial for platelet activation and thrombus formation [4]. Moreover, GPVI serves as a promising pharmacological target for the effective and safe treatment of thrombotic and possibly inflammatory diseases [4]. Aspirin and clopidogrel are commonly used to prevent stroke, but account for only a 20% reduction in all recurrent stroke events [5]. Thus, targeting collagen signaling may provide an alternative therapeutic approach to mitigate the recurrence of secondary stroke. Licochalcone A (LA), a natural chalcone derived from the roots and rhizomes of Glycyrrhiza spp., exhibits multiple biological activities such as antibacterial, antioxidant, anti-inflammatory, antimalarial, antiviral, and antitumor effects [6–11]. LA inhibits lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) production and cytokine release in the RAW 264.7 mouse macrophage cell line [12]. LA also alleviates LPS-induced acute lung and kidney injury through NF-κB and p38/ERK mitogen-activated protein kinase (MAPK) signaling and attenuates pertussis toxin-induced autoimmune encephalomyelitis by reducing the production of tumor necrosis factor-α and interferon-γ in vivo [12–14]. In addition, LA induces tumor cell cycle arrest, apoptosis, and autophagy in a various cancer cell lines [10,11]. These observations reveals that LA protects against several pathological processes. Although, recently, LA was also reported to reduce rabbit and rat platelet activation through the inhibition of cyclooxygenase-1 (COX-1) activity [15,16], its role in platelet activation and thrombosis remains unclear. In the present study, our preliminary data revealed that LA significantly inhibited collagen-induced platelet aggregation through PLCγ2–PKC pathway, suggesting that, in addition to inhibiting COX-1 activity, LA may attenuate platelet activation through other mechanisms. Therefore, we further systemically investigated the mechanism of LA in platelet activation and for the first time determined whether LA has antithrombotic effect in in vivo studies. 2. Results 2.1. Licochalcone A (LA) Inhibited Collagen-Induced Platelet Aggregation As shown in Figure 1A, LA (2–10 µM) was used to determine its effect on the platelet aggregation induced by collagen (1 µg/mL). The data indicated that LA (at concentrations of 2, 5 and 10 µM) inhibited collagen-induced platelet aggregation by 16.6%, 45.3% and 90.8%, respectively. The IC50 was approximately 5.6 µM. In addition, only at a higher concentration of 80 µM did LA affect thrombin (0.01 U/mL)- or U46619 (1 µM)-mediated platelet aggregation (Figure 1B). These results are consistent with that Okuda-Tanino et al. reported [15] and indicate that LA is more sensitive to the inhibition of collagen-mediated platelet activation. Accordingly, in the following experiments, we mainly evaluated the mechanism of LA at concentrations of 2–10 µM in collagen-mediated platelet activation events. 2.2. LA Inhibited Collagen-Mediated ATP Release and P-Selectin Secretion Platelet granule release plays a crucial role in the amplification of platelet activation and aggregation. Here, two experiments on ATP release and P-selectin secretion, which were assessed by the microplate reader and through flow cytometry, respectively, were performed to determine whether LA interferes with the granule release induced by collagen. As shown in Figure 1C, luciferase/luciferin was used to detect ATP. The data revealed that collagen markedly induced ATP release, which
Int. J. Mol. Sci. 2017, 18, 1500
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3 of 16
by the microplate reader and through flow cytometry, respectively, were performed to determine whether LA interferes with the granule release induced by collagen. As shown in Figure 1C, luciferase/luciferin usedµM) to detect The data revealed that manner. collagen markedly induced ATP was reversed by LAwas (2–10 in a ATP. concentration-dependent Moreover, LA (5 and release, which was reversed by LA (2–10 μM) in a concentration-dependent manner. Moreover, LAby 10 µM) significantly reduced collagen-induced P-selectin secretion (Figure 1D), as determined and 10 ofμM) significantly reduced (FITC)–P-selectin. collagen-induced These P-selectin secretion (Figure 1D), as the(5intensity fluorescein isothiocyanate findings suggest that LA inhibits determined by the intensity of fluorescein isothiocyanate (FITC)–P-selectin. These findings suggest collagen-mediated platelet activation through the blockade of granule release. that LA inhibits collagen-mediated platelet activation through the blockade of granule release.
Figure 1. Effects of Licochalcone A (LA) on the collagen-induced platelet aggregation, ATP release, Figure 1. Effects of Licochalcone A (LA) on the collagen-induced platelet aggregation, ATP release, and P-selectin secretion. (A,B) Washed platelets (3.6 × 108 cells/mL) were preincubated with DMSO and P-selectin secretion. (A,B) Washed platelets (3.6 × 108 cells/mL) were preincubated with DMSO (solvent control) and LA (2–80 μM), and were then stimulated using collagen (1 μg/mL), thrombin (solvent control) and LA (2–80 µM), and were then stimulated using collagen (1 µg/mL), thrombin (0.01 U/mL), or U46619 (1 μM) to trigger platelet aggregation, as measured by a transmission (0.01 U/mL), or U46619 (1 µM) to trigger platelet aggregation, as measured by a transmission aggregometer; (C) The effect of LA on collagen-induced ATP release was characterized by the aggregometer; (C) The effect of LA on collagen-induced ATP release was characterized by the detection detection of chemiluminescent emission from the luciferin–luciferase reaction, which was of chemiluminescent emission from the luciferin–luciferase reaction, which was continually recorded continually recorded using a microplate reader; (D) The effect of LA on collagen-induced P-selectin using a microplate reader; (D) The effect of LA on collagen-induced P-selectin secretion was detected secretion was detected using FITC–P-selectin antibody. The fluorescence was immediately detected using FITC–P-selectin antibody. The fluorescence was immediately detected through flow cytometry. through flow cytometry. The profiles (B) are representative examples of five similar experiments. The profiles (B) are representative examples of five similar experiments. Data (A,C) are presented as Data (A,C) are presented as the mean ± SEM. (A, n = 5; C, n = 3). * p < 0.05 and *** p < 0.001, compared thewith mean ± SEM. (A, n = 5; C, n = 3). * p < 0.05 and *** p < 0.001, compared with the DMSO group; Data the DMSO group; Data (D) are presented as the mean ± SEM. (n = 3). ** p < 0.01, compared with (D) are presented as# the ± SEM. (nwith = 3). **collagen p < 0.01,(positive) comparedgroup. with the resting group; # p < 0.05, the resting group; p < mean 0.05, compared the compared with the collagen (positive) group.
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2.3. LA LA Inhibited Inhibited Collagen-Mediated Collagen-Mediated Calcium Calcium Mobilization Mobilizationand andGPIIbIIIa GPIIbIIIaActivation Activationwithout withoutInterfering Interferingwith with 2.3. CollagenReceptors Receptors Collagen Calcium signaling signaling isis the the common commonplatelet plateletactivation activationsignaling signalingpathway. pathway. Receptor-stimulated Receptor-stimulated Calcium PLC catalyzes catalyzes the the hydrolysis hydrolysis of ofphosphatidylinositol phosphatidylinositol biphosphate biphosphate to to release releaseinositol inositoltrisphosphate trisphosphate PLC and diacyglycerol, diacyglycerol, which which activate activatecalcium calcium mobilization mobilization and andPKC, PKC,respectively respectively[17]. [17]. The Theelevation elevation of of and 2+ 2+ intracellular Ca Ca contributes to several events of platelet platelet activation, activation, such such as as shape shape change, change, granule granule intracellular release,and andGPIIbIIIa GPIIbIIIaactivaiton activaiton[18]. [18]. release, Asshown shownininFigure Figure2A, 2A,fura-2 fura-2was was used measure change in calcium according to As used to to measure thethe change in calcium levellevel according to the the ratio of F340/F380, which is directly correlated amount intracellularcalcium. calcium.The The data data ratio of F340/F380, which is directly correlated to to thethe amount ofofintracellular revealed that that LA LA (2–10 (2–10 µM) μM) markedly markedly inhibited inhibited calcium calcium mobilization, mobilization, as as detected detected using using F-4500 F-4500 revealed FluorescenceSpectrophotometer. Spectrophotometer.Moreover, Moreover, FITC–PAC-1 antibody to demonstrate Fluorescence thethe FITC–PAC-1 antibody waswas usedused to demonstrate that that(5–10 LA µM) (5–10markedly μM) markedly inhibited (Figure stepaggregation, in platelet LA inhibited GPIIbIIIaGPIIbIIIa activationactivation (Figure 2B), a final2B), stepainfinal platelet aggregation, as detected through flow cytometry. These findings that LA blocks calcium as detected through flow cytometry. These findings indicate that LA indicate blocks calcium mobilization and mobilizationGPIIbIIIa and subsequent activation, thereby inhibitingIn platelet In subsequent activation, GPIIbIIIa thereby inhibiting platelet aggregation. addition,aggregation. FITC–collagen addition, FITC–collagen was used to determine directlyleading blocks to collagen receptors, was used to determine whether LA directly blockswhether collagenLA receptors, the inhibition of leading to the inhibition downstream As obtained shown infrom Figure 2C,cytometry the data revealed obtained its downstream signaling. of Asits shown in Figuresignaling. 2C, the data flow fromFITC–collagen flow cytometry revealedbinds that FITC–collagen markedlypretreated binds to platelets. Moreover, that markedly to platelets. Moreover, LA did not interferepretreated with the LA did of notFITC–collagen interfere withtothe binding of finding FITC–collagen platelets. This collagen-induced finding suggests that LA binding platelets. This suggeststothat LA inhibits platelet inhibits collagen-induced plateletcollagen activation, possibly by inhibiting collagen downstream activation, possibly by inhibiting receptor downstream signaling butreceptor not by blocking the signalingreceptors. but not In byaddition, blockingLA the(10–80 collagen In addition, (10–80 μM) did as notdetected exhibit collagen µM)receptors. did not exhibit cytotoxicLA effects on platelets, cytotoxic on platelets, as detected by the LDH (Figure 2D), activation indicatingis that by the LDHeffects assay (Figure 2D), indicating that LA-mediated the assay inhibition of platelet not LA-mediated the inhibition of platelet activation is not due to the cytotoxicity. due to the cytotoxicity.
Figure 2. Cont.
Int.J.J.Mol. Mol.Sci. Sci.2017, 2017,18, 18,1500 1500 Int.
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Figure Figure2.2.Effects EffectsofofLA LAon oncalcium calciummobilization, mobilization,GPIIbIIIa GPIIbIIIaactivation, activation,collagen collagenreceptors receptorsand andlactate lactate dehydrogenase (LDH) release. Washed platelets were preincubated with DMSO and LA (2–10 dehydrogenase (LDH) release. Washed platelets were preincubated with DMSO and LA (2–10µM), μM), and andwere werethen thenstimulated stimulatedusing using1 1µg/mL μg/mLcollagen collagen(A,B) (A,B)or orFITC–collagen FITC–collagen(C). (C).(A) (A)The Theratio ratioofoffura-2 fura-2 fluorescence fluorescence(F340/F380) (F340/F380) was was used used to to determine determine calcium calcium mobilization, mobilization, as as measured measured by byaaHitachi Hitachi F4500 fluorescence spectrophotometer; (B,C) GPIIbIIIa activation and the competition with collagen F4500 fluorescence spectrophotometer; (B,C) GPIIbIIIa activation and the competition with collagen were weredetermined determinedusing usingthe theFITC–PAC-1 FITC–PAC-1antibody antibodyand andFITC–collagen, FITC–collagen,respectively. respectively.The Thefluorescence fluorescence intensity was measured through flow cytometry; (D) The platelets were preincubated intensity was measured through flow cytometry; (D) The platelets were preincubatedwith withTyrode’s Tyrode’s solution, control) or or various variousconcentrations concentrationsofofLA LA(10–80 (10–80 µM) min at °C, 37 ◦and C, solution,DMSO DMSO (solvent (solvent control) μM) forfor 10 10 min at 37 and the supernatant was collected to measure LDH release by the LDH assay kit. LDH activity was the supernatant was collected to measure LDH release by the LDH assay kit. LDH activity was expressed expressedas asthe the%%ofoftotal totalenzyme enzymeactivity, activity,which whichwas wasmeasured measuredininplatelets plateletslysed lysedwith with0.5% 0.5%Triton Triton X-100. Data (A) are presented as the means ± SEM (n = 3). ** p < 0.01 and *** p < 0.001, compared X-100. Data (A) are presented as the means ± SEM (n = 3). ** p < 0.01 and *** p < 0.001, comparedwith with the theDMSO DMSOgroup; group;Data Data(B) (B)are arepresented presentedasasthe themeans means±±SEM SEM(n(n==3). 3).****pp 0.05, n = 8). In = 8). In addition, the LA-treated groups did not prolong the tail-bleeding time, compared addition, with the the LA-treated groups did not the tail-bleeding agent. time, compared with the DMSO group, DMSO group, indicating that LAprolong is a safer antithrombotic indicating that LA is a safer antithrombotic agent.
Figure 5.5. Influence Influence of ofLA LAon onmiddle middlecerebral cerebralartery arteryocclusion occlusion(MCAO)/reperfusion-induced (MCAO)/reperfusion-induced brain brain Figure injuryand andtailtail bleeding in Mice mice.(male, Mice5–6(male, old) were intraperitoneally injury bleeding time time in mice. weeks 5–6 old) weeks were intraperitoneally administrated administrated with control) DMSO (solvent control) LA (1.8 3.6 mg/kg) forwere 30 min. (A) Mice were with DMSO (solvent or LA (1.8 and 3.6or mg/kg) forand 30 min. (A) Mice subjected to MCAO subjected MCAOby for24-h 30 min followedImmediately by 24-h reperfusion. Immediately sacrifice, coronal for 30 min to followed reperfusion. after sacrifice, coronal after sections were cut and sectionsusing were cut and stained usingchloride; 2,3,5-triphenyltetrazolium chloride; and white indicate stained 2,3,5-triphenyltetrazolium white areas indicate infarction, redareas areas indicate infarction, and (left red areas indicate tissuesratios (left panel). Edemawere and calculated infarct ratios (right image panel) normal tissues panel). Edemanormal and infarct (right panel) through were calculated analysis and are reportedhemisphere. as a ratio ofInfarct the non-ischemic hemisphere. analysis and are through reportedimage as a ratio of the non-ischemic ratio was corrected for Infarct Data ratio are waspresented correctedasfor are the mean ± SEM. (nthe = 8). *** p < 0.001, edema. theedema. mean ±Data SEM. (n presented = 8). *** p
