RESUMEN
Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 µM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca2+ ]i) levels and thromboxane A2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoproteinSer157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs.
Asunto(s)
Biflavonoides , Nucleótidos Cíclicos , Fosfolipasas , Humanos , Animales , Ratones , Nucleótidos Cíclicos/metabolismo , Nucleótidos Cíclicos/farmacología , Fosfolipasa C gamma/metabolismo , Ácido Araquidónico/farmacología , Ácido Araquidónico/metabolismo , Fosfolipasas/metabolismo , Fosfolipasas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Activación Plaquetaria , Plaquetas/metabolismo , Agregación Plaquetaria , Proteína Quinasa C/metabolismo , Fosforilación , Colágeno/metabolismoRESUMEN
Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.
Asunto(s)
Eugenol , Embolia Pulmonar , Humanos , Ratones , Animales , Eugenol/farmacología , Eugenol/uso terapéutico , Eugenol/metabolismo , Fosfolipasa C gamma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Modelos Animales de Enfermedad , Activación Plaquetaria , Agregación Plaquetaria , Plaquetas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Tromboxano A2/metabolismo , Colágeno/metabolismo , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/metabolismo , Fosfolipasas A2 Citosólicas/metabolismoRESUMEN
Platelets are crucial for hemostasis and arterial thrombosis, which may lead to severe cardiovascular diseases (CVDs). Thus, therapeutic agents must be developed to prevent pathological platelet activation. Glabridin, a major bioalkaloid extracted from licorice root, improves metabolic abnormalities (i.e., obesity and diabetes) and protects against CVDs and neuronal disorders. To the best of our knowledge, no studies have focused on glabridin's effects on platelet activation. Therefore, we investigated these effects in humans and mice. Glabridin exhibited the highest inhibitory effects on collagen-stimulated platelet aggregation and moderate effects on arachidonic-acid-stimulated activation; however, no effects were observed for any other agonists (e.g., thrombin or U46619). Glabridin evidently reduced P-selectin expression, ATP release, and intracellular Ca2+ ([Ca2+]i) mobilization and thromboxane A2 formation; it further reduced the activation of phospholipase C (PLC)γ2/protein kinase C (PKC), phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3ß (GSK3ß), mitogen-activated protein kinase (MAPK), and NF-κB. In mice, glabridin reduced the mortality rate caused by acute pulmonary thromboembolism without altering bleeding time. Thus, glabridin effectively inhibits the PLCγ2/PKC cascade and prevents the activation of the PI3K/Akt/GSK3ß and MAPK pathways; this leads to a reduction in [Ca2+]i mobilization, which eventually inhibits platelet aggregation. Therefore, glabridin may be a promising therapeutic agent for thromboembolic disorders.
Asunto(s)
Glycyrrhiza , Selectina-P , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Adenosina Trifosfato/metabolismo , Animales , Plaquetas/metabolismo , Colágeno/metabolismo , Flavonoides/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Isoflavonas , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Selectina-P/metabolismo , Fenoles , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C gamma/metabolismo , Fosforilación , Activación Plaquetaria , Agregación Plaquetaria , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Trombina/metabolismo , Tromboxanos/metabolismoRESUMEN
Lipoteichoic acid (LTA) is a key cell wall component and virulence factor of Gram-positive bacteria. LTA contributes a major role in infection and it mediates inflammatory responses in the host. Rutaecarpine, an indolopyridoquinazolinone alkaloid isolated from Evodia rutaecarpa, has shown a variety of fascinating biological properties such as anti-thrombotic, anticancer, anti-obesity and thermoregulatory, vasorelaxing activity. It has also potent effects on the cardiovascular and endocrine systems. Herein, we investigated rutaecarpine's (Rut) anti-inflammatory effects in LTA-stimulated RAW macrophage cells. The Western blot and spectrophotometric results revealed that Rut inhibited the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and interleukin (IL)-1ß in the LTA-induced macrophage cells. Successively, our mechanistic studies publicized that Rut inhibited LTA-induced phosphorylation of mitogen-activated protein kinase (MAPK) including the extracellular signal-regulated kinase (ERK), and p38, but not c-Jun NH2-terminal kinase (JNK). In addition, the respective Western blot and confocal image analyses exhibited that Rut reserved nuclear transcription factor kappa-B (NF-κB) by hindering inhibitor of nuclear factor κB-α (IκBα) and NF-κB p65 phosphorylation and p65 nuclear translocation. These results indicate that Rut exhibits its anti-inflammatory effects mainly through attenuating NF-κB and ERK/p38 signaling pathways. Overall, this result suggests that Rut could be a potential therapeutic agent for the treatment of Gram-positive bacteria induced inflammatory diseases.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular , FN-kappa B , Animales , Antiinflamatorios/farmacología , Ciclooxigenasa 2/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Alcaloides Indólicos/farmacología , Lipopolisacáridos/farmacología , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Quinazolinas , Células RAW 264.7 , Ácidos Teicoicos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Platelets play a critical role in arterial thrombosis. Rutaecarpine (RUT) was purified from Tetradium ruticarpum, a well-known Chinese medicine. This study examined the relative activity of RUT with NF-κB inhibitors in human platelets. BAY11-7082 (an inhibitor of IκB kinase [IKK]), Ro106-9920 (an inhibitor of proteasomes), and RUT concentration-dependently (1-6 µM) inhibited platelet aggregation and P-selectin expression. RUT was found to have a similar effect to that of BAY11-7082; however, it exhibits more effectiveness than Ro106-9920. RUT suppresses the NF-κB pathway as it inhibits IKK, IκBα, and p65 phosphorylation and reverses IκBα degradation in activated platelets. This study also investigated the role of p38 and NF-κB in cell signaling events and found that SB203580 (an inhibitor of p38) markedly reduced p38, IKK, and p65 phosphorylation and reversed IκBα degradation as well as p65 activation in a confocal microscope, whereas BAY11-7082 had no effects in p38 phosphorylation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay shows that RUT and BAY11-7082 did not exhibit free radical scavenging activity. In the in vivo study, compared with BAY11-7082, RUT more effectively reduced mortality in adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism without affecting the bleeding time. In conclusion, a distinctive pathway of p38-mediated NF-κB activation may involve RUT-mediated antiplatelet activation, and RUT could act as a strong prophylactic or therapeutic drug for cardiovascular diseases.
Asunto(s)
Fibrinolíticos/farmacología , Alcaloides Indólicos/farmacología , FN-kappa B/metabolismo , Nitrilos/farmacología , Quinazolinas/farmacología , Sulfonas/farmacología , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Modelos Animales de Enfermedad , Fibrinolíticos/uso terapéutico , Depuradores de Radicales Libres/farmacología , Depuradores de Radicales Libres/uso terapéutico , Radicales Libres/antagonistas & inhibidores , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Imidazoles/farmacología , Imidazoles/uso terapéutico , Alcaloides Indólicos/uso terapéutico , Masculino , Ratones Endogámicos ICR , FN-kappa B/antagonistas & inhibidores , Nitrilos/uso terapéutico , Selectina-P/metabolismo , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Quinazolinas/uso terapéutico , Sulfonas/uso terapéutico , Factor de Transcripción ReIA/metabolismoRESUMEN
Inflammation is a major root of several diseases such as allergy, cancer, Alzheimer's, and several others, and the present state of existing drugs provoked researchers to search for new treatment strategies. Plants are regarded to be unique sources of active compounds holding pharmacological properties, and they offer novel designs in the development of therapeutic agents. Therefore, this study aimed to explore the anti-inflammatory mechanism of esculetin in lipoteichoic acid (LTA)-induced macrophage cells (RAW 264.7). The relative expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO) production and COX-2 expression were intensified in LTA-induced RAW cells. The phosphorylation status of mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and c-Jun N-terminal kinase (JNK)) and nuclear factor kappa B (NF-κB) p65 were detected by using Western blot assay. The nuclear translocation of p65 was assessed by confocal microscopic image analysis. Esculetin significantly and concentration-dependently inhibited LTA-induced NO production and iNOS expression, but not COX-2 expression, in RAW cells. Esculetin was not effective in LTA-induced MAPK molecules (ERK, p38 and JNK). However, esculetin recovered LTA-induced IκBα degradation and NF-κB p65 phosphorylation. Moreover, esculetin at a higher concentration of 20 µM evidently inhibited the nuclear translocation of NF-κB p65. At the same high concentration, esculetin augmented Nrf2 expression and decreased DPPH radical generation in RAW 264.7 cells. This study exhibits the value of esculetin for the treatment of LTA-induced inflammation by targeting NF-κB signaling pathways via its antioxidant properties.
Asunto(s)
Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B , Animales , Ciclooxigenasa 2/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Células RAW 264.7 , Transducción de SeñalRESUMEN
Studies have discovered that different extracts of Evodia rutaecarpa and its phytochemicals show a variety of biological activities associated with inflammation. Although rutaecarpine, an alkaloid isolated from the unripe fruit of E. rutaecarpa, has been exposed to have anti-inflammatory properties, the mechanism of action has not been well studied. Thus, this study investigated the molecular mechanisms of rutaecarpine (RUT) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. RUT reserved the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukin (IL)-1ß in the LPS-induced macrophages. RUT showed an inhibitory effect on the mitogen-activated protein kinases (MAPKs), and it also inhibited nuclear transcription factor kappa-B (NF-κB) by hindering IκBα and NF-κB p65 phosphorylation and p65 nuclear translocation. The phospho-PI3K and Akt was concentration-dependently suppressed by RUT. However, RUT not only suggestively reduced the migratory ability of macrophages and their numbers induced by LPS but also inhibited the phospho-Src, and FAK. Taken together, these results indicate that RUT participates a vital role in the inhibition of LPS-induced inflammatory processes in RAW 264.7 macrophages and that the mechanisms involve PI3K/Akt and MAPK-mediated downregulation of NF-κB signaling pathways. Notably, reducing the migration and number of cells induced by LPS via inhibiting of Src/FAK pathway was also included to the anti-inflammatory mechanism of RUT. Therefore, RUT may have potential benefits as a therapeutic agent against chronic inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Quinasa 1 de Adhesión Focal/metabolismo , Alcaloides Indólicos/farmacología , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Quinazolinas/farmacología , Animales , Línea Celular , Ciclooxigenasa 2/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Inhibidor NF-kappaB alfa/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The role of activated platelets in acute and chronic cardiovascular diseases (CVDs) is well established. Therefore, antiplatelet drugs significantly reduce the risk of severe CVDs. Evodia rutaecarpa (Wu-Chu-Yu) is a well-known Chinese medicine, and rutaecarpine (Rut) is a main bioactive component with substantial beneficial properties including vasodilation. To address a research gap, we investigated the inhibitory mechanisms of Rut in washed human platelets and experimental mice. At low concentrations (1-5 µM), Rut strongly inhibited collagen-induced platelet aggregation, whereas it exerted only a slight or no effect on platelets stimulated with other agonists (e.g., thrombin). Rut markedly inhibited P-selectin expression; adenosine triphosphate release; [Ca2+]i mobilization; hydroxyl radical formation; and phospholipase C (PLC)γ2/protein kinase C (PKC), mitogen-activated protein kinase, and phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3ß (GSK3ß) phosphorylation stimulated by collagen. SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor) did not reverse Rut-mediated antiplatelet aggregation. Rut was not directly responding to vasodilator-stimulated phosphoprotein phosphorylation. Rut significantly increased the occlusion time of fluorescence irradiated thrombotic platelet plug formation. The findings demonstrated that Rut exerts a strong effect against platelet activation through the PLCγ2/PKC and PI3K/Akt/GSK3ß pathways. Thus, Rut can be a potential therapeutic agent for thromboembolic disorders.
Asunto(s)
Alcaloides Indólicos/farmacología , Activación Plaquetaria/efectos de los fármacos , Quinazolinas/farmacología , Trombosis/prevención & control , Alcaloides/química , Alcaloides/farmacología , Animales , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Evodia/química , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Alcaloides Indólicos/aislamiento & purificación , Alcaloides Indólicos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos ICR , Proteínas de Microfilamentos/metabolismo , Nucleótidos Cíclicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Agregación Plaquetaria/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinazolinas/aislamiento & purificación , Quinazolinas/uso terapéutico , Quinolinas/química , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2-8 µM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbß3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbß3-mediated outside-in signaling, such as integrin ß3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbß3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.
Asunto(s)
Plaquetas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/efectos de los fármacos , Estilbenos/metabolismo , Animales , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Retracción del Coagulo/efectos de los fármacos , Colágeno , Fibrinógeno/metabolismo , Hemostasis/efectos de los fármacos , Humanos , Integrina alfa2/efectos de los fármacos , Integrina alfa2/metabolismo , Integrina beta3/efectos de los fármacos , Integrina beta3/metabolismo , Integrinas/efectos de los fármacos , Integrinas/metabolismo , Ratones , Selectina-P/metabolismo , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal/efectos de los fármacos , Estilbenos/farmacología , Trombosis/metabolismoRESUMEN
BACKGROUND: Columbianadin (CBN) is one of the main coumarin constituents isolated from Angelica pubescens. The pharmacological value of CBN is well demonstrated, especially in the prevention of several cancers and analgesic activity. A striking therapeutic target for arterial thrombosis is inhibition of platelet activation because platelet activation significantly contributes to these diseases. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo. METHODS: Aggregometry, immunoblotting, immunoprecipitation, confocal microscopic analysis, fibrin clot retraction, and thrombogenic animals were used in this study. RESULTS: CBN markedly inhibited platelet aggregation in washed human platelets stimulated only by collagen, but was not effective in platelets stimulated by other agonists such as thrombin, arachidonic acid, and U46619. CBN evidently inhibited ATP release, intracellular ([Ca2+]i) mobilization, and P-selectin expression. It also inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] 1/2 and c-Jun N-terminal kinase [JNK] 1/2, but not p38 MAPK) in collagen-activated platelets. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the CBN-mediated inhibition of platelet aggregation. CBN had no significant effect in triggering vasodilator-stimulated phosphoprotein phosphorylation. Moreover, it markedly hindered integrin αIIbß3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin αIIbß3-mediated outside-in signaling such as adhesion number and spreading area of platelets on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Additionally, CBN did not attenuate FITC-triflavin binding or phosphorylation of proteins, such as integrin ß3, Src, and focal adhesion kinase, in platelets spreading on immobilized fibrinogen. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation. CONCLUSION: This study demonstrated that CBN exhibits an exceptional activity against platelet activation through inhibition of the PLCγ2-PKC cascade, subsequently suppressing the activation of Akt and ERKs/JNKs and influencing platelet aggregation. Consequently, this work provides solid evidence and considers that CBN has the potential to serve as a therapeutic agent for the treatment of thromboembolic disorders.
Asunto(s)
Cumarinas/farmacología , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Sustancias Protectoras/farmacología , Transducción de Señal/efectos de los fármacos , Trombosis/tratamiento farmacológico , Animales , Humanos , RatonesRESUMEN
Ruthenium metal complex has been shown to exert several chemical and biological activities. A series of three novel ruthenium derivatives (TQ 1, 2 and 4) were synthesized to evaluate the anti-inflammatory and hepatoprotective activities in lipopolysaccharide (LPS)-stimulated macrophages and mice liver injury. The hydroxyl radical (OH°) scavenging activity of these derivatives has also been evaluated. The results revealed that among the tested compounds, TQ-4 effectively attenuated LPS-induced abnormal alteration in liver histoarchistructure via reducing alanine transaminase (ALT) and aspartate transaminase (AST). This compound exhibited significant inhibition of inflammatory cytokines (TNF-α and IL-1ß), inflammatory enzyme (iNOS), the component of NF-κB signaling pathway (p65) and JNK phosphorylation in LPS-induced mice liver tissues. In vitro results showed that TQ-4 had the best inhibition of NO production and iNOS expression in LPS-induced RAW 264.7 cells. Mechanistic approach indicated that TQ-4 inhibited the LPS-induced JNK phosphorylation, IκBα degradation, NF-κB p65 phosphorylation and its nuclear translocation, and hydroxyl radical (OH°) productions in RAW 264.7 cells. However, the compounds TQ-1 and 2 had no effects in this study. TQ-4 also inhibited LPS-induced OH° production. This study reveals the protective effect of TQ-4 against LPS-induced acute liver injury, inflammation, and oxidative reaction by destructing JNK/NF-κB signaling pathways. The result of this study may infer that TQ-4 might be a promising ruthenium metal derivative and/or therapeutic agent for treating liver injury.
Asunto(s)
Antiinflamatorios/farmacología , Complejos de Coordinación/farmacología , Inflamación/tratamiento farmacológico , Hígado/efectos de los fármacos , Sustancias Protectoras/farmacología , Rutenio/farmacología , Animales , Radicales Libres/metabolismo , Inflamación/metabolismo , Lipopolisacáridos/efectos adversos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Células RAW 264.7 , Transducción de Señal/efectos de los fármacosRESUMEN
Platelets are major players in the occurrence of cardiovascular diseases. Auraptene is the most abundant coumarin derivative from plants, and it has been demonstrated to possess a potent capacity to inhibit platelet activation. Although platelets are anucleated cells, they also express the transcription factor, nuclear factor-κB (NF-κB), that may exert non-genomic functions in platelet activation. In the current study, we further investigated the inhibitory roles of auraptene in NF-κB-mediated signal events in platelets. MG-132 (an inhibitor of proteasome) and BAY11-7082 (an inhibitor of IκB kinase; IKK), obviously inhibited platelet aggregation; however, BAY11-7082 exhibited more potent activity than MG-132 in this reaction. The existence of NF-κB (p65) in platelets was observed by confocal microscopy, and auraptene attenuated NF-κB activation such as IκBα and p65 phosphorylation and reversed IκBα degradation in collagen-activated platelets. To investigate cellular signaling events between PLCγ2-PKC and NF-κB, we found that BAY11-7082 abolished PLCγ2-PKC activation; nevertheless, neither U73122 nor Ro31-8220 had effect on NF-κB activation. Furthermore, both auraptene and BAY11-7082 significantly diminished HO⢠formation in activated platelets. For in vivo study, auraptene prolonged the occlusion time of platelet plug in mice. In conclusion, we propose a novel inhibitory pathway of NF-κB-mediated PLCγ2-PKC activation by auraptene in human platelets, and further supported that auraptene possesses potent activity for thromboembolic diseases.
Asunto(s)
Arterias/efectos de los fármacos , Plaquetas/efectos de los fármacos , Cumarinas/farmacología , FN-kappa B/antagonistas & inhibidores , Activación Plaquetaria/efectos de los fármacos , Trombosis/prevención & control , Animales , Arterias/metabolismo , Plaquetas/metabolismo , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Proteína Quinasa C/metabolismo , Transducción de Señal/efectos de los fármacos , Trombosis/metabolismoRESUMEN
Esculetin, a bioactive 6,7-dihydroxy derivative of coumarin, possesses pharmacological activities against obesity, diabetes, renal failure, and cardiovascular disorders (CVDs). Platelet activation plays a major role in CVDs. Thus, disrupting platelet activation represents an attractive therapeutic target. We examined the effect of esculetin in human platelet activation and experimental mouse models. At 10-80 µM, esculetin inhibited collagen- and arachidonic acid-induced platelet aggregation in washed human platelets. However, it had no effects on other agonists such as thrombin and U46619. Esculetin inhibited adenosine triphosphate release, P-selectin expression, hydroxyl radical (OH·) formation, Akt activation, and phospholipase C (PLC)γ2/protein kinase C (PKC) phosphorylation, but did not diminish mitogen-activated protein kinase phosphorylation in collagen-activated human platelets. Platelet function analysis indicated that esculetin substantially prolonged the closure time of whole blood. In experimental mice, esculetin significantly increased the occlusion time in thrombotic platelet plug formation and reduced mortality associated with acute pulmonary thromboembolism. However, it did not prolong the bleeding time. This study demonstrates that esculetin inhibits human platelet activation via hindering the PLCγ2-PKC cascade, hydroxyl radical formation, Akt activation, and ultimately suppressing platelet activation. Therefore, esculetin may act as an essential therapeutic agent for preventing thromboembolic diseases.
Asunto(s)
Plaquetas/metabolismo , Trombosis/etiología , Trombosis/prevención & control , Umbeliferonas/uso terapéutico , Biomarcadores , Plaquetas/efectos de los fármacos , Humanos , Fosfolipasa C gamma/metabolismo , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Umbeliferonas/química , Umbeliferonas/farmacologíaRESUMEN
Auraptene is the most abundant coumarin derivative from plants. The pharmacological value of this compound has been well demonstrated, especially in the prevention of cancer and neurodegenerative diseases. Platelet activation is a major factor contributing to arterial thrombosis. Thus, this study evaluated the influence of auraptene in platelet aggregation and thrombotic formation. Auraptene inhibited platelet aggregation in human platelets stimulated with collagen only. However, auraptene was not effective in inhibiting platelet aggregation stimulated with thrombin, arachidonic acid, and U46619. Auraptene also repressed ATP release, [Ca2+]i mobilization, and P-selectin expression. Moreover, it markedly blocked PAC-1 binding to integrin αIIbß3. However, it had no influence on properties related to integrin αIIbß3-mediated outside-in signaling, such as the adhesion number, spreading area of platelets, and fibrin clot retraction. Auraptene inhibited the phosphorylation of Lyn-Fyn-Syk, phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), Akt, and mitogen-activated protein kinases (MAPKs; extracellular-signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK1/2), but not p38 MAPK). Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the auraptene-mediated inhibition of platelet aggregation. Auraptene reduced mortality caused by adenosine diphosphate (ADP)-induced pulmonary thromboembolism. In conclusion, this study provides definite evidence that auraptene signifies a potential therapeutic agent for preventing thromboembolic disorders.
Asunto(s)
Cumarinas/uso terapéutico , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/mortalidad , Transducción de Señal , Adenosina Trifosfato/metabolismo , Animales , Plaquetas/efectos de los fármacos , Calcio/metabolismo , Cumarinas/química , Cumarinas/farmacología , Humanos , Ratones , Nucleótidos Cíclicos/metabolismo , Selectina-P/metabolismo , Fosforilación/efectos de los fármacos , Embolia Pulmonar/sangre , Transducción de Señal/efectos de los fármacosRESUMEN
In oncotherapy, ruthenium (Ru) complexes are reflected as potential alternatives for platinum compounds and have been proved as encouraging anticancer drugs with high efficacy and low side effects. Cardiovascular diseases (CVDs) are mutually considered as the number one killer globally, and thrombosis is liable for the majority of CVD-related deaths. Platelets, an anuclear and small circulating blood cell, play key roles in hemostasis by inhibiting unnecessary blood loss of vascular damage by making blood clot. Platelet activation also plays a role in cancer metastasis and progression. Nevertheless, abnormal activation of platelets results in thrombosis under pathological settings such as the rupture of atherosclerotic plaques. Thrombosis diminishes the blood supply to the heart and brain resulting in heart attacks and strokes, respectively. While currently used anti-platelet drugs such as aspirin and clopidogrel demonstrate efficacy in many patients, they exert undesirable side effects. Therefore, the development of effective therapeutic strategies for the prevention and treatment of thrombotic diseases is a demanding priority. Recently, precious metal drugs have conquered the subject of metal-based drugs, and several investigators have motivated their attention on the synthesis of various ruthenium (Ru) complexes due to their prospective therapeutic values. Similarly, our recent studies established that novel ruthenium-based compounds suppressed platelet aggregation via inhibiting several signaling cascades. Our study also described the structure antiplatelet-activity relationship (SAR) of three newly synthesized ruthenium-based compounds. This review summarizes the antiplatelet activity of newly synthesized ruthenium-based compounds with their potential molecular mechanisms.
Asunto(s)
Plaquetas/efectos de los fármacos , Terapia Molecular Dirigida , Compuestos Organometálicos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Rutenio/farmacología , Animales , Humanos , Compuestos Organometálicos/química , Compuestos Organometálicos/uso terapéutico , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/uso terapéutico , Rutenio/química , Rutenio/uso terapéutico , Trombosis/tratamiento farmacológico , Trombosis/prevención & controlRESUMEN
Morin hydrate, a bioactive flavonoid, has been proven to prevent inflammation and apoptosis of cells. Flavonoids can reduce the risk of cardiovascular diseases, in which platelet activation plays a major role. This study investigated the effect of morin hydrate on platelet activation in vitro and in vivo. Morin hydrate markedly inhibited platelet aggregation stimulated by collagen in human platelets but not that stimulated by other agonists. In collagen-activated platelets, morin hydrate inhibited adenosine triphosphate (ATP) release; intracellular Ca2+ mobilization; P-selectin expression; and phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), and Akt. In mitogen-activated protein kinase (MAPK) activation, morin hydrate evidently diminished ERK2 or JNK1 activation, except for p38 MAPK. Additionally, morin hydrate markedly reduced the OH· signals in platelet suspensions but not in the cell-free system (Fenton reaction solution). Moreover, morin hydrate substantially increased the occlusion time of thrombotic platelet plug formation but had no effect on bleeding time in mice. In conclusion, morin hydrate crucially inhibits platelet activation through inhibition of the PLCγ2â»PKC cascade and subsequent suppression of Akt and MAPK activation, thereby ultimately inhibiting platelet aggregation. Therefore, this paper suggests that morin hydrate constitutes a novel and potential natural therapeutic product for preventing or treating thromboembolic disorders.
Asunto(s)
Plaquetas/metabolismo , Flavonoides/farmacología , Agregación Plaquetaria/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Calcio/metabolismo , Flavonoides/química , Flavonoides/uso terapéutico , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Selectina-P/metabolismo , Activación Plaquetaria/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Trombosis/tratamiento farmacológicoRESUMEN
Platelets play a major role in hemostatic events and are associated with various pathological events, such as arterial thrombosis and atherosclerosis. Iridium (Ir) compounds are potential alternatives to platinum compounds, since they exert promising anticancer effects without cellular toxicity. Our recent studies found that Ir compounds show potent antiplatelet properties. In this study, we evaluated the in vitro antiplatelet, in vivo antithrombotic and structureâ»activity relationship (SAR) of newly synthesized Ir complexes, Ir-1, Ir-2 and Ir-4, in agonists-induced human platelets. Among the tested compounds, Ir-1 was active in inhibiting platelet aggregation induced by collagen; however, Ir-2 and Ir-4 had no effects even at their maximum concentrations of 50 µM against collagen and 500 µM against U46619-induced aggregation. Similarly, Ir-1 was potently inhibiting of adenosine triphosphate (ATP) release, calcium mobilization ([Ca2+]i) and P-selectin expression induced by collagen-induced without cytotoxicity. Likewise, Ir-1 expressively suppressed collagen-induced Akt, PKC, p38MAPKs and JNK phosphorylation. Interestingly, Ir-2 and Ir-4 had no effect on platelet function analyzer (PFA-100) collagen-adenosine diphosphate (C-ADP) and collagen-epinephrine (C-EPI) induced closure times in mice, but Ir-1 caused a significant increase when using C-ADP stimulation. Other in vivo studies revealed that Ir-1 significantly prolonged the platelet plug formation, increased tail bleeding times and reduced the mortality of adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism in mice. Ir-1 has no substitution on its phenyl group, a water molecule (like cisplatin) can replace its chloride ion and, hence, the rate of hydrolysis might be tuned by the substituent on the ligand system. These features might have played a role for the observed effects of Ir-1. These results indicate that Ir-1 may be a lead compound to design new antiplatelet drugs for the treatment of thromboembolic diseases.
Asunto(s)
Complejos de Coordinación/química , Complejos de Coordinación/uso terapéutico , Iridio/uso terapéutico , Trombosis/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Adulto , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Calcio/metabolismo , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Colágeno/farmacología , Complejos de Coordinación/farmacología , Femenino , Hemorragia/patología , Humanos , Proteínas Inmovilizadas/farmacología , Iridio/química , Iridio/farmacología , Ligandos , Masculino , Ratones Endogámicos ICR , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/patología , Relación Estructura-Actividad , Trombosis/patología , Factores de Tiempo , Adulto JovenRESUMEN
The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven to be a positive approach to the prevention of thrombosis. Ruthenium complexes are fascinating for the development of new drugs, as they possess numerous chemical and biological properties. The present study aims to evaluate the structure-activity relationship (SAR) of newly synthesized ruthenium (II) complexes, TQ-1, TQ-2 and TQ-3 in agonists-induced washed human platelets. Silica gel column chromatography, aggregometry, immunoblotting, NMR, and X-ray analyses were performed in this study. Of the three tested compounds, TQ-3 showed a concentration (1-5 µM) dependent inhibitory effect on platelet aggregation induced by collagen (1 µg/mL) and thrombin (0.01 U/mL) in washed human platelets; however, TQ-1 and TQ-2 had no response even at 250 µM of collagen and thrombin-induced aggregation. TQ-3 was effective with inhibiting collagen-induced ATP release, calcium mobilization ([Ca2+]i) and P-selectin expression without cytotoxicity. Moreover, TQ-3 significantly abolished collagen-induced Lyn-Fyn-Syk, Akt-JNK and p38 mitogen-activated protein kinases (p38 MAPKs) phosphorylation. The compound TQ-3 containing an electron donating amino group with two phenyl groups of the quinoline core could be accounted for by its hydrophobicity and this nature might be the reason for the noted antiplatelet effects of TQ-3. The present results provide a molecular basis for the inhibition by TQ-3 in collagen-induced platelet aggregation, through the suppression of multiple machineries of the signaling pathway. These results may suggest that TQ-3 can be considered a potential agent for the treatment of vascular diseases.
Asunto(s)
Plaquetas/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Agregación Plaquetaria/efectos de los fármacos , Compuestos de Rutenio/química , Colágeno/química , Humanos , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/síntesis química , Inhibidores de Agregación Plaquetaria/uso terapéutico , Rutenio/química , Compuestos de Rutenio/síntesis química , Compuestos de Rutenio/uso terapéutico , Relación Estructura-Actividad , Trombosis/tratamiento farmacológicoRESUMEN
Platelet activation plays a major role in cardio and cerebrovascular diseases, and cancer progression. Disruption of platelet activation represents an attractive therapeutic target for reducing the bidirectional cross talk between platelets and tumor cells. Platinum (Pt) compounds have been used for treating cancer. Hence, replacing Pt with iridium (Ir) is considered a potential alternative. We recently developed an Ir(III)-derived complex, [Ir(Cp*)1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine Cl]BF4 (Ir-11), which exhibited strong antiplatelet activity; hence, we assessed the therapeutic potential of Ir-11 against arterial thrombosis. In collagen-activated platelets, Ir-11 inhibited platelet aggregation, adenosine triphosphate (ATP) release, intracellular Ca2+ mobilization, P-selectin expression, and OH· formation, as well as the phosphorylation of phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Akt. Neither the adenylate cyclase inhibitor nor the guanylate cyclase inhibitor reversed the Ir-11-mediated antiplatelet effects. In experimental mice, Ir-11 prolonged the bleeding time and reduced mortality associated with acute pulmonary thromboembolism. Ir-11 plays a crucial role by inhibiting platelet activation through the inhibition of the PLCγ2-PKC cascade, and the subsequent suppression of Akt and MAPK activation, ultimately inhibiting platelet aggregation. Therefore, Ir-11 can be considered a new therapeutic agent against either arterial thrombosis or the bidirectional cross talk between platelets and tumor cells.
Asunto(s)
Iridio/química , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/prevención & control , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfolipasa C gamma/metabolismo , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Proteína Quinasa C/metabolismoRESUMEN
BACKGROUND: Platelets, a type of anucleated cell, play a crucial role in cardiovascular diseases (CVDs). Therefore, targeting platelet activation is essential for mitigating CVDs. Endogenous agonists, such as collagen, activate platelets by initiating signal transduction through specific platelet receptors, leading to platelet aggregation. Eugenol, primarily sourced from clove oil, is known for its antibacterial, anticancer, and anti-inflammatory properties, making it a valuable medicinal agent. In our previous study, eugenol was shown to inhibit platelet aggregation induced by collagen and arachidonic acid. We concluded that eugenol exerts a potent inhibitory effect on platelet activation by targeting the PLCγ2-PKC and cPLA2-TxA2 pathways, thereby suppressing platelet aggregation. In our current study, we found that eugenol significantly inhibits NF-κB activation. This led us to investigate the relationship between the NF-κB and cPLA2 pathways to elucidate how eugenol suppresses platelet activation. METHODS: In this study, we prepared platelet suspensions from the blood of healthy human donors to evaluate the inhibitory mechanisms of eugenol on platelet activation. We utilized immunoblotting and confocal microscopy to analyze these mechanisms in detail. Additionally, we assessed the anti-thrombotic effect of eugenol by observing fluorescein-induced platelet plug formation in the mesenteric microvessels of mice. RESULTS: For immunoblotting and confocal microscopy studies, eugenol significantly inhibited NF-κB-mediated signaling events stimulated by collagen in human platelets. Specifically, it reduced the phosphorylation of IKK and p65 and prevented the degradation of IκBα. Additionally, CAY10502, a cPLA2 inhibitor, significantly reduced NF-κB-mediated signaling events. In contrast, BAY11-7082, an IKK inhibitor, did not affect collagen-stimulated cPLA2 phosphorylation. These findings suggest that cPLA2 acts as an upstream regulator of NF-κB activation during platelet activation. Furthermore, both BAY11-7082 and CAY10502 significantly reduced the collagen-induced rise in intracellular calcium levels. In the animal study, eugenol demonstrated potential as an anti-thrombotic agent by significantly reducing platelet plug formation in fluorescein-irradiated mouse mesenteric microvessels. CONCLUSION: Our study uncovered a novel pathway in platelet activation involving the cPLA2-NF-κB axis, which plays a key role in the antiplatelet effects of eugenol. These findings suggest that eugenol could serve as a valuable and potent prophylactic or therapeutic option for arterial thrombosis.