RESUMO
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.
Assuntos
Eugenol , Embolia Pulmonar , Humanos , Camundongos , Animais , Eugenol/farmacologia , Eugenol/uso terapêutico , Eugenol/metabolismo , Fosfolipase C gama/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Ativação Plaquetária , Agregação Plaquetária , Plaquetas/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Tromboxano A2/metabolismo , Colágeno/metabolismo , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/metabolismo , Fosfolipases A2 Citosólicas/metabolismoRESUMO
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.
Assuntos
Biflavonoides , Nucleotídeos Cíclicos , Fosfolipases , Humanos , Animais , Camundongos , Nucleotídeos Cíclicos/metabolismo , Nucleotídeos Cíclicos/farmacologia , Fosfolipase C gama/metabolismo , Ácido Araquidônico/farmacologia , Ácido Araquidônico/metabolismo , Fosfolipases/metabolismo , Fosfolipases/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Ativação Plaquetária , Plaquetas/metabolismo , Agregação Plaquetária , Proteína Quinase C/metabolismo , Fosforilação , Colágeno/metabolismoRESUMO
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.
Assuntos
Glycyrrhiza , Selectina-P , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/metabolismo , Colágeno/metabolismo , Flavonoides/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Isoflavonas , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Selectina-P/metabolismo , Fenóis , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Ativação Plaquetária , Agregação Plaquetária , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trombina/metabolismo , Tromboxanos/metabolismoRESUMO
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.
Assuntos
Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B , Animais , Ciclo-Oxigenase 2/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Transdução de SinaisRESUMO
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.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , NF-kappa B , Animais , Anti-Inflamatórios/farmacologia , Ciclo-Oxigenase 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Alcaloides Indólicos/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Quinazolinas , Células RAW 264.7 , Ácidos Teicoicos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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.
Assuntos
Fibrinolíticos/farmacologia , Alcaloides Indólicos/farmacologia , NF-kappa B/metabolismo , Nitrilas/farmacologia , Quinazolinas/farmacologia , Sulfonas/farmacologia , Trombose/tratamento farmacológico , Trombose/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Modelos Animais de Doenças , Fibrinolíticos/uso terapêutico , Sequestradores de Radicais Livres/farmacologia , Sequestradores de Radicais Livres/uso terapêutico , Radicais Livres/antagonistas & inibidores , Humanos , Quinase I-kappa B/antagonistas & inibidores , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Alcaloides Indólicos/uso terapêutico , Masculino , Camundongos Endogâmicos ICR , NF-kappa B/antagonistas & inibidores , Nitrilas/uso terapêutico , Selectina-P/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Quinazolinas/uso terapêutico , Sulfonas/uso terapêutico , Fator de Transcrição RelA/metabolismoRESUMO
OBJECTIVE: Oxidative stress-mediated inflammatory events involve in the progress of several diseases such as asthma, cancers, and multiple sclerosis. Auraptene (AU), a natural prenyloxycoumarin, possesses numerous pharmacological activities. Here, the anti-inflammatory effects of AU were investigated in lipoteichoic acid- (LTA-) induced macrophage cells (RAW 264.7). METHODS: The expression of cyclooxygenase (COX-2), tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase (iNOS) and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, c-Jun N-terminal kinase (JNK), heme oxygenase (HO-1), p65, and IκBα were all identified by western blotting assay. The level of nitric oxide (NO) was measured by spectrometer analysis. The nuclear translocation of p65 nuclear factor kappa B (NF-κB) was assessed by the confocal microscopic staining method. Native polyacrylamide gel electrophoresis was performed to perceive the activity of antioxidant enzyme catalase (CAT). RESULTS: AU expressively reduced NO production and COX-2, TNF-α, IL-1 ß, and iNOS expression in LTA-stimulated cells. AU at higher concentration (10 µM) inhibited ERK and JNK, but not p38 phosphorylation induced by LTA. Moreover, AU blocked IκB and p65 phosphorylation, and p65 nuclear translocation. However, AU pretreatment was not effective on antioxidant HO-1 expression, CAT activity, and reduced glutathione (GSH, a nonenzymatic antioxidant), in LTA-induced RAW 264.7 cells. CONCLUSION: The findings of this study advocate that AU shows anti-inflammatory effects via reducing NF-κB/MAPKs signaling pathways.
RESUMO
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.
Assuntos
Alcaloides Indólicos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Quinazolinas/farmacologia , Trombose/prevenção & controle , Alcaloides/química , Alcaloides/farmacologia , Animais , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Evodia/química , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/metabolismo , Nucleotídeos Cíclicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/isolamento & purificação , Quinazolinas/uso terapêutico , Quinolinas/química , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trombose/metabolismo , Trombose/patologiaRESUMO
Platelets play a crucial role in cardiovascular disorders (CVDs); thus, development of a therapeutic target that prevents platelet activation reduces CVDs. Pterostilbene (PTE) has several remarkable pharmacological activities, including anticancer and neuroprotection. Herein, we examined the inhibitory mechanisms of PTE in human platelets and its role in the prevention of vascular thrombosis in mice. At very low concentrations (1-5 µmol/L), PTE strongly inhibited collagen-induced platelet aggregation, but it did not have significant effects against thrombin and 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin (U46619). PTE markedly reduced P-selectin expression on isolated α-granules by a novel microchip. Moreover, PTE inhibited adenosine triphosphate (ATP) release, intracellular ([Ca2+]i) mobilization (resting, 216.6 ± 14.0 nmol/L; collagen-activated platelets, 396.5 ± 25.7 nmol/L; 2.5 µmol/L PTE, 259.4 ± 8.8 nmol/L; 5 µmol/L PTE, 231.8 ± 9.7 nmol/L), phospholipase C (PLC)γ2/protein kinase C (PKC), Akt, and mitogen-activated protein kinase (MAPK) phosphorylation. Neither 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) nor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed platelet aggregation inhibited by PTE. PTE did not affect vasodilator-stimulated phosphoprotein phosphorylation. In mice, PTE obviously reduced the mortality (from 100 to 37.5%) associated with acute pulmonary thromboembolism without increasing the bleeding time. Thus, PTE could be used to prevent CVDs.
Assuntos
Ativação Plaquetária , Trombose , Animais , Plaquetas , Humanos , Camundongos , Fosforilação , Agregação Plaquetária , Resveratrol , Estilbenos , Trombose/prevenção & controleRESUMO
Columbianadin (CBN), a natural coumarin isolated from Angelica decursiva, is reported to have numerous biological activities, including anticancer and platelet aggregation inhibiting properties. Here, we investigated CBN's anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell activation and deciphered the signaling process, which could be targeted by CBN as part of the mechanisms. Using a mouse model of LPS-induced acute liver inflammation, the CBN effects were examined by distinct histologic methods using trichrome, reticulin, and Weigert's resorcin fuchsin staining. The result showed that CBN decreased LPS-induced expressions of TNF-α, IL-1ß, and iNOS and NO production in RAW 264.7 cells and mouse liver. CBN inhibited LPS-induced ERK and JNK phosphorylation, increased IκBα levels, and inhibited NF-κB p65 phosphorylation and its nuclear translocation. Application of inhibitors for ERK (PD98059) and JNK (SP600125) abolished the LPS-induced effect on NF-κB p65 phosphorylation, which indicated that ERK and JNK signaling pathways were involved in CBN-mediated inhibition of NF-κB activation. Treatment with CBN decreased hydroxyl radical (â¢OH) generation and increased HO-1 expression in RAW 264.7 cells. Furthermore, LPS-induced liver injury, as indicated by elevated serum levels of liver marker enzymes (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) and histopathological alterations, were reversed by CBN. This work demonstrates the utility of CBN against LPS-induced inflammation, liver injury, and oxidative stress by targeting JNK/ERK and NF-κB signaling pathways.
RESUMO
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.
Assuntos
Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/efeitos dos fármacos , Estilbenos/metabolismo , Animais , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Retração do Coágulo/efeitos dos fármacos , Colágeno , Fibrinogênio/metabolismo , Hemostasia/efeitos dos fármacos , Humanos , Integrina alfa2/efeitos dos fármacos , Integrina alfa2/metabolismo , Integrina beta3/efeitos dos fármacos , Integrina beta3/metabolismo , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Camundongos , Selectina-P/metabolismo , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Trombose/metabolismoRESUMO
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.
Assuntos
Anti-Inflamatórios/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Alcaloides Indólicos/farmacologia , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Quinazolinas/farmacologia , Animais , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Inibidor de NF-kappaB alfa/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Cumarínicos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Substâncias Protetoras/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trombose/tratamento farmacológico , Animais , Humanos , CamundongosRESUMO
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.
Assuntos
Cumarínicos/uso terapêutico , Ativação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/mortalidade , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Cumarínicos/química , Cumarínicos/farmacologia , Humanos , Camundongos , Nucleotídeos Cíclicos/metabolismo , Selectina-P/metabolismo , Fosforilação/efeitos dos fármacos , Embolia Pulmonar/sangue , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Plaquetas/metabolismo , Trombose/etiologia , Trombose/prevenção & controle , Umbeliferonas/uso terapêutico , Biomarcadores , Plaquetas/efeitos dos fármacos , Humanos , Fosfolipase C gama/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Umbeliferonas/química , Umbeliferonas/farmacologiaRESUMO
After the publication of the above paper, the authors noted that an incomplete version of the address was presented for the second author affiliation; essentially, 'School of Medicine' had been omitted from the address. Therefore, the author and affiliation details for this paper should have been presented as follows: KouGi Shyu1,2, Marappan Velusamy3, ChihWei Hsia2, ChihHao Yang2, ChihHsuan Hsia2, DuenSuey Chou2, Thanasekaran Jayakumar2, JoenRong Sheu2 And JiunYi Li2,4. 1Division of Cardiology, Shin Kong Wu HoSu Memorial Hospital, Taipei 111; 2Graduate Institute of Medical Sciences and Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan, R.O.C.; 3Department of Chemistry, North Eastern Hill University, Shillong, Meghalaya 793022, India; 4Department of Cardiovascular Surgery, Mackay Memorial Hospital, and Mackay Medical College, Taipei 104, Taiwan, R.O.C. The authors regret that the error with the second author affiliation was not noticed prior to the publication of their paper, and apologize for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 41: 25892600, 2018; DOI: 10.3892/ijmm.2018.3472].
RESUMO
A newly synthesized ruthenium metal complex, TQ5, exhibited antithrombotic and antiplatelet effects in our previous study. In the present study, the antiinï¬ammatory/hepatoprotective effects and mechanisms of action of TQ5 were investigated in lipopolysaccharide (LPS)induced RAW 264.7 macrophages in vitro and in acute liver injury in mice in vivo. The results demonstrated that TQ5 suppressed the LPSinduced production of nitric oxide, tumor necrosis factorα (TNFα), interleukin1ß (IL1ß) and inducible nitric oxide synthase (iNOS), without inducing cytotoxicity or damaging the morphology of the RAW 264.7 macrophages. In addition, the role of TQ5 in mediating mitogenactivated protein kinases and nuclear factor κB (NFκB) pathways involved in the inï¬ammation process of LPSstimulated RAW264.7 cells was investigated. Although TQ5 did not alter the phosphorylation of extracellular signalrelated kinase, p38 or cJun Nterminal kinase in LPStreated cells, it suppressed the phosphorylation of Akt in a concentrationdependent manner. TQ5 significantly reversed the LPSinduced degradation of inhibitor of NFκBα and phosphorylation of p65. The mRNA expression levels of iNOS, TNFα and IL1ß were also suppressed by TQ5. TQ5 improved LPSinduced liver injury in mice by inhibiting the expression of TNFα, IL1ß and iNOS and phosphorylation of NFκBp65. These findings suggest that Akt/NFκB signaling may be a promising target for TQ5 to combat LPSinduced inflammation. Therefore, TQ5 may act as a potential agent for the development of antiinï¬ammatory drugs to treat acute liver failure.
Assuntos
Complexos de Coordenação , Regulação para Baixo/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda , Macrófagos/metabolismo , NF-kappa B/metabolismo , Rutênio , Transdução de Sinais/efeitos dos fármacos , Animais , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/tratamento farmacológico , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/patologia , Macrófagos/patologia , Masculino , Camundongos , Células RAW 264.7 , Rutênio/química , Rutênio/farmacologiaRESUMO
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.
Assuntos
Complexos de Coordenação/química , Complexos de Coordenação/uso terapêutico , Irídio/uso terapêutico , Trombose/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Adulto , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Colágeno/farmacologia , Complexos de Coordenação/farmacologia , Feminino , Hemorragia/patologia , Humanos , Proteínas Imobilizadas/farmacologia , Irídio/química , Irídio/farmacologia , Ligantes , Masculino , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/patologia , Relação Estrutura-Atividade , Trombose/patologia , Fatores de Tempo , Adulto JovemRESUMO
Several studies have reported that metal complexes exhibit anti-inflammatory activities; however, the molecular mechanism is not well understood. In this study, we used a potent ruthenium (II)-derived compound, [Ru(η6-cymene)2-(1H-benzoimidazol-2-yl)-quinoline Cl]BF4 (TQ-6), to investigate the molecular mechanisms underlying the anti-inflammatory effects against lipopolysaccharide (LPS)-induced macrophage activation and liver injury in mice. Treating LPS-stimulated RAW 264.7 cells with TQ-6 suppressed nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in a concentration-dependent manner. The LPS-induced expression of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) were reduced in TQ-6-treated cells. TQ-6 suppressed, LPS-stimulated p38 MAPK phosphorylation, IκBα degradation, and p65 nuclear translocation in cells. Consistent with the in vitro studies, TQ-6 also suppressed the expression of iNOS, TNF-α, and p65 in the mouse model with acute liver injury induced by LPS. The present study showed that TQ-6 could protect against LPS-induced in vitro inflammation in macrophage and in vivo liver injury in mice, and suggested that NF-κB could be a promising target for protecting against LPS-induced inflammation and liver injury by TQ-6. Therefore, TQ-6 can be a potential therapeutic agent for treating inflammatory diseases.
RESUMO
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.