Eugenol: A Potential Modulator of Human Platelet Activation and Mouse Mesenteric Vascular Thrombosis via an Innovative cPLA2-NF-κB Signaling Axis.

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.

Ginkgetin effectively mitigates collagen and AA-induced platelet activation via PLCγ2 but not cyclic nucleotide-dependent pathway in human.

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.

Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

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.

Myricetin as a promising inhibitor of platelet fibrinogen receptor in humans.

Platelets play a vital role in the formation of dangerous arterial thrombosis. Platelets are activated by adhesive proteins or soluble agonists through their specific receptors. The receptor-mediated signaling pathways lead to common signaling events, which result in shape changes and inside-out signaling, leading fibrinogen binding to glycoprotein IIb/IIIa complex (integrin αIIbß3). This interaction initiates integrin αIIbß3-mediated outside-in signaling, subsequently culminating in granule secretion and aggregation. Myricetin is a flavonoid that occurs in a variety of plants. Although myricetin has been demonstrated to have several bioactive properties, its role in platelet activation has not been extensively studied. The present study demonstrated the ability of myricetin to inhibit platelet aggregation stimulated by collagen, thrombin, and U46619. Myricetin reduced the ATP-release, cytosolic Ca2+ mobilization, and P-selectin expression and the activation of PLCγ2/PKC, PI3K/Akt/GSK3ß, and MAPK. Myricetin exerted a direct inhibitory effect on the activation of integrin αIIbß3 by disrupting the binding between FITC-PAC-1 and the integrin. Moreover, myricetin suppressed integrin αIIbß3-mediated outside-in signaling, such as integrin ß3, Src, and Syk phosphorylation on immobilized fibrinogen. In animal studies, myricetin significantly prolonged the occlusion time of thrombotic platelet plug formation in mesenteric microvessels without extending bleeding time. This study concludes that myricetin is a natural integrin αIIbß3 inhibitor and a novel antithrombotic agent.

Mechanisms of glabridin inhibition of integrin αIIbß3 inside-out signals and NF-κB activation in human platelets.

BACKGROUND: Platelets play a crucial role in cardiovascular diseases (CVDs) and are activated by endogenous agonists like collagen. These agonists initiate signal transduction through specific platelet receptors, resulting in platelet aggregation. Glabridin, a prenylated isoflavonoid found in licorice root, is known for its significance in metabolic abnormalities. Glabridin has been observed to inhibit collagen-induced platelet aggregation, but the precise mechanisms, specifically concerning NF-κB activation and integrin αIIbß3 signaling, are not yet fully understood. METHODS: In this study, platelet suspensions were prepared from healthy human blood donors, and the aggregation ability was observed using a lumi-aggregometer. The inhibitory mechanisms of glabridin in human platelets were evaluated through immunoblotting and confocal microscopy. The anti-thrombotic effects of glabridin were assessed by histological analysis of lung sections in acute pulmonary thromboembolism and by examining fluorescein-induced platelet plug formation in mesenteric microvessels in mice. RESULTS: Glabridin inhibited integrin αIIbß3 inside-out signals such as Lyn, Fyn, Syk, and integrin ß3 activation and NF-κB-mediated signal events, with similar potency to classical inhibitors BAY11-7082 and Ro106-9920. Glabridin and BAY11-7082 inhibited IKK, IκBα, and p65 phosphorylation and reversed IκBα degradation, while Ro106-9920 only reduced p65 phosphorylation and reversed IκBα degradation. BAY11-7082 reduced Lyn, Fyn, Syk, integrin ß3, phospholipase Cγ2 and protein kinase C activation. Glabridin reduced platelet plug formation in mesenteric microvessels and occluded vessels in thromboembolic lungs of mice. CONCLUSION: Our study revealed a new pathway for activating integrin αIIbß3 inside-out signals and NF-κB, which contributes to the antiplatelet aggregation effect of glabridin. Glabridin could be a valuable prophylactic or clinical treatment option for CVDs.

Garcinol acts as a novel integrin αIIbß3 inhibitor in human platelets.

AIMS: Platelet activation plays a central role in arterial thrombosis. Platelets are activated by adhesive proteins (i.e., collagen) or soluble agonists (i.e., thrombin), the respective receptor-specific signaling cause inside-out signaling, leading to the binding of fibrinogen to integrin αIIbß3. This binding triggers outside-in signaling, resulting in platelet aggregation. Garcinol, a polyisoprenylated benzophenone, is extracted from the fruit rind of Garcinia indica. Although garcinol exhibits considerable bioactivities, few studies have investigated the effect of garcinol on platelet activation. MAIN METHODS: Aggregometry, immunoblotting, flow cytometer, confocal microscopic analysis, fibrin clot retraction, animal studies such as fluorescein-induced platelet plug formation in mesenteric microvessels, acute pulmonary thromboembolism, and tail bleeding time were performed in this study. KEY FINDINGS: This study indicates that garcinol inhibited platelet aggregation stimulated by collagen, thrombin, arachidonic acid, and U46619. Garcinol reduced integrin αIIbß3 inside-out signaling, including ATP release; cytosolic Ca2+ mobilization; P-selectin expression; and Syk, PLCγ2/PKC, PI3K/Akt/GSK3ß, MAPKs, and NF-κB activation stimulated by collagen. Garcinol directly inhibited integrin αIIbß3 activation by interfering with FITC-PAC-1 and FITC-triflavin by collagen. Additionally, garcinol affected integrin αIIbß3-mediated outside-in signaling, such as decreasing platelet adhesion and the single-platelet spreading area; suppressing integrin ß3, Src, FAK, and Syk phosphorylation on immobilized fibrinogen; and inhibiting thrombin-stimulated fibrin clot retraction. Garcinol substantially reduced mortality caused by pulmonary thromboembolism and prolonged the occlusion time of thrombotic platelet plug formation without extending bleeding time in mice. SIGNIFICANCE: This study identified that garcinol, a novel antithrombotic agent, acts as a naturally occurring integrin αIIbß3 inhibitor.

Influence of Garlic (Allium sativum) Clove-Based Selenium Nanoparticles on Status of Nutritional, Biochemical, Enzymological, and Gene Expressions in the Freshwater Prawn Macrobrachium rosenbergii (De Man, 1879).

Selenium (Se) is one of the essential micronutrients for performing vital body functions. This study aims at examining the influence of dietary supplementation of garlic clove-based green-synthesized selenium nanoparticles (GBGS-SeNPs, 48-87 nm) on carcass minerals and trace elements, and growth, biochemical, enzymological, and gene expression analyses in the freshwater prawn, Macrobrachium rosenbergii post larvae (PL). The 96 h LC50 of this GBGS-SeNPs to M. rosenbergii PL was 52.23 mg L-1. Five different artificial diets without supplementation of GBGS-SeNPs (control, 0.0 mg kg-1) and with supplementations of GBGS-SeNPs starting from 100 times lower than the LC50 value (0.5, 1.0, 1.5, and 2.0 mg kg-1) were prepared and fed to M. rosenbergii PL for 90 days. A dose-dependent accumulation of Se was observed in the carcass of experimental prawns. GBGS-SeNPs, up to 1.5 mg kg-1 significantly influenced the absorption of other trace elements (Ca, Cu, and Fe) and mineral salts (K, Mg, Na, and Zn). GBGS-SeNPs-supplemented diets showed efficient food conversion ratio (FCR) of 1.32 g against 2.71 g, and therefore enhanced the survival rate (85.6% against 78.8% in control) and weight gain (WG) of 1.41 g against 0.46 g of control prawn. GBGS-SeNPs significantly elevated the activities of protease, amylase, and lipase, and the contents of total protein, essential amino acids (EAA), total carbohydrate, total lipid, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), and ash. These indicate the growth promoting potential of GBGS-SeNPs in prawn. The insignificantly altered activities of glutamic oxaloacetate transaminase (GOT), glutamic pyruvate transaminase (GPT), superoxide dismutase (SOD), and catalase, and the content of malondialdehyde (MDA) up to 1.5 mg kg-1 suggest its acceptability in prawn. Moreover, a respective down- and upregulated myostatin (MSTN) and crustacean hyperglycemic hormone (CHH) genes confirmed the influence of GBGS-SeNPs on the growth of prawn. In contrast, 2.0 mg kg-1 GBGS-SeNPs supplementation starts to produce negative effects on prawn (FCR, 1.76 g; survival rate, 82.2%; WG, 0.84 g against respective values of 1.32 g, 85.6%; and 1.41 g observed in 1.5 mg kg-1 of GBGS-SeNPs-supplemented diet fed prawn). This study recommends a maximum of 1.5 mg kg-1 GBGS-SeNPs as dietary supplement to attain sustainable growth of M. rosenbergii. This was confirmed through polynomial and linear regression analyses.

Glabridin, a Bioactive Flavonoid from Licorice, Effectively Inhibits Platelet Activation in Humans and Mice.

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.

Activation of Nrf2 by Esculetin Mitigates Inflammatory Responses through Suppression of NF-κB Signaling Cascade in RAW 264.7 Cells.

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.

Anti-Inflammatory Mechanism of An Alkaloid Rutaecarpine in LTA-Stimulated RAW 264.7 Cells: Pivotal Role on NF-κB and ERK/p38 Signaling Molecules.

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.

Corrigendum to "Auraptene, a Monoterpene Coumarin, Inhibits LTA-Induced Inflammatory Mediators via Modulating NF-κB/MAPKs Signaling Pathways".

[This corrects the article DOI: 10.1155/2021/5319584.].

Decreased Human Platelet Activation and Mouse Pulmonary Thrombosis by Rutaecarpine and Comparison of the Relative Effectiveness with BAY11-7082: Crucial Signals of p38-NF-κB.

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.

Auraptene, a Monoterpene Coumarin, Inhibits LTA-Induced Inflammatory Mediators via Modulating NF-κB/MAPKs Signaling Pathways.

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.

Rutaecarpine, an Alkaloid from Evodia rutaecarpa, Can Prevent Platelet Activation in Humans and Reduce Microvascular Thrombosis in Mice: Crucial Role of the PI3K/Akt/GSK3ß Signal Axis through a Cyclic Nucleotides/VASP-Independent Mechanism.

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.

Columbianadin Dampens In Vitro Inflammatory Actions and Inhibits Liver Injury via Inhibition of NF-κB/MAPKs: Impacts on ∙OH Radicals and HO-1 Expression.

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 (•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.

Platelet autophagic machinery involved in thrombosis through a novel linkage of AMPK-MTOR to sphingolipid metabolism.

Basal macroautophagy/autophagy has recently been found in anucleate platelets. Platelet autophagy is involved in platelet activation and thrombus formation. However, the mechanism underlying autophagy in anucleate platelets require further clarification. Our data revealed that LC3-II formation and SQSTM1/p62 degradation were noted in H2O2-activated human platelets, which could be blocked by 3-methyladenine and bafilomycin A1, indicating that platelet activation may cause platelet autophagy. AMPK phosphorylation and MTOR dephosphorylation were also detected, and block of AMPK activity by the AMPK inhibitor dorsomorphin reversed SQSTM1 degradation and LC3-II formation. Moreover, autophagosome formation was observed through transmission electron microscopy and deconvolution microscopy. These findings suggest that platelet autophagy was induced partly through the AMPK-MTOR pathway. In addition, increased LC3-II expression occurred only in H2O2-treated Atg5f/f platelets, but not in H2O2-treated atg5-/- platelets, suggesting that platelet autophagy occurs during platelet activation. atg5-/- platelets also exhibited a lower aggregation in response to agonists, and platelet-specific atg5-/- mice exhibited delayed thrombus formation in mesenteric microvessles and decreased mortality rate due to pulmonary thrombosis. Notably, metabolic analysis revealed that sphingolipid metabolism is involved in platelet activation, as evidenced by observed several altered metabolites, which could be reversed by dorsomorphin. Therefore, platelet autophagy and platelet activation are positively correlated, partly through the interconnected network of sphingolipid metabolism. In conclusion, this study for the first time demonstrated that AMPK-MTOR signaling could regulate platelet autophagy. A novel linkage between AMPK-MTOR and sphingolipid metabolism in anucleate platelet autophagy was also identified: platelet autophagy and platelet activation are positively correlated.Abbreviations: 3-MA: 3-methyladenine; A.C.D.: citric acid/sod. citrate/glucose; ADP: adenosine diphosphate; AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy-related; B4GALT/LacCS: beta-1,4-galactosyltransferase; Baf-A1: bafilomycin A1; BECN1: beclin 1; BHT: butylate hydrooxytoluene; BSA: bovine serum albumin; DAG: diacylglycerol; ECL: enhanced chemiluminescence; EDTA: ethylenediamine tetraacetic acid; ELISA: enzyme-linked immunosorbent assay; GALC/GCDase: galactosylceramidase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/GluSDase: glucosylceramidase beta; GPI: glycosylphosphatidylinositol; H2O2: hydrogen peroxide; HMDB: human metabolome database; HRP: horseradish peroxidase; IF: immunofluorescence; IgG: immunoglobulin G; KEGG: Kyoto Encyclopedia of Genes and Genomes; LAMP1: lysosomal associated membrane protein 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MPV: mean platelet volume; MTOR: mechanistic target of rapamycin kinase; ox-LDL: oxidized low-density lipoprotein; pAb: polyclonal antibody; PC: phosphatidylcholine; PCR: polymerase chain reaction; PI3K: phosphoinositide 3-kinase; PLS-DA: partial least-squares discriminant analysis; PRP: platelet-rich plasma; Q-TOF: quadrupole-time of flight; RBC: red blood cell; ROS: reactive oxygen species; RPS6KB/p70S6K: ribosomal protein S6 kinase B; SDS: sodium dodecyl sulfate; S.E.M.: standard error of the mean; SEM: scanning electron microscopy; SGMS: sphingomyelin synthase; SM: sphingomyelin; SMPD/SMase: sphingomyelin phosphodiesterase; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; UGT8/CGT: UDP glycosyltransferase 8; UGCG/GCS: UDP-glucose ceramide glucosyltransferase; ULK1: unc-51 like autophagy activating kinase 1; UPLC: ultra-performance liquid chromatography; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; WBC: white blood cell; WT: wild type.

Targeting MAPK/NF-κB Pathways in Anti-Inflammatory Potential of Rutaecarpine: Impact on Src/FAK-Mediated Macrophage Migration.

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.

Molecular Targets of Natural Products for Chondroprotection in Destructive Joint Diseases.

Osteoarthritis (OA) is the most common type of arthritis that occurs in an aged population. It affects any joints in the body and degenerates the articular cartilage and the subchondral bone. Despite the pathophysiology of OA being different, cartilage resorption is still a symbol of osteoarthritis. Matrix metalloproteinases (MMPs) are important proteolytic enzymes that degrade extra-cellular matrix proteins (ECM) in the body. MMPs contribute to the turnover of cartilage and its break down; their levels have increased in the joint tissues of OA patients. Application of chondroprotective drugs neutralize the activities of MMPs. Natural products derived from herbs and plants developed as traditional medicine have been paid attention to, due to their potential biological effects. The therapeutic value of natural products in OA has increased in reputation due to their clinical impact and insignificant side effects. Several MMPs inhibitor have been used as therapeutic drugs, for a long time. Recently, different types of compounds were reviewed for their biological activities. In this review, we summarize numerous natural products for the development of MMPs inhibitors in arthritic diseases and describe the major signaling targets that were involved for the treatments of these destructive joint diseases.

Carbon Dot Nanoparticles Exert Inhibitory Effects on Human Platelets and Reduce Mortality in Mice with Acute Pulmonary Thromboembolism.

The inhibition of platelet activation is considered a potential therapeutic strategy for the treatment of arterial thrombotic diseases; therefore, maintaining platelets in their inactive state has garnered much attention. In recent years, nanoparticles have emerged as important players in modern medicine, but potential interactions between them and platelets remain to be extensively investigated. Herein, we synthesized a new type of carbon dot (CDOT) nanoparticle and investigated its potential as a new antiplatelet agent. This nanoparticle exerted a potent inhibitory effect in collagen-stimulated human platelet aggregation. Further, it did not induce cytotoxic effects, as evidenced in a lactate dehydrogenase assay, and inhibited collagen-activated protein kinase C (PKC) activation and Akt (protein kinase B), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. The bleeding time, a major side-effect of using antiplatelet agents, was unaffected in CDOT-treated mice. Moreover, our CDOT could reduce mortality in mice with ADP-induced acute pulmonary thromboembolism. Overall, CDOT is effective against platelet activation in vitro via reduction of the phospholipase C/PKC cascade, consequently suppressing the activation of MAPK. Accordingly, this study affords the validation that CDOT has the potential to serve as a therapeutic agent for the treatment of arterial thromboembolic disorders.

Modulation of human platelet activation and in vivo vascular thrombosis by columbianadin: regulation by integrin αIIbß3 inside-out but not outside-in signals.

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.