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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.

Tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) are elevated in pleural fluids of tuberculous pleuritis (TBP) where pleural mesothelial cells (PMCs) conduct the first-line defense against Mycobacterium tuberculosis (MTB). However, the clinical implication of TNF-α and MMPs in TBP and the response of PMCs to MTB infection remain unclear. We measured pleural fluid levels of TNF-α and MMPs in patients with TBP (n = 18) or heart failure (n = 18) as controls. Radiological scores for initial effusion amount and residual pleural fibrosis at 6-month follow-up were assessed. In vitro human PMC experiments were performed to assess the effect of heat-killed M. tuberculosis H37Ra (MTBRa) on the expression of TNF-α and MMPs. As compared with controls, the effusion levels of TNF-α, MMP-1 and MMP-9 were significantly higher and correlated positively with initial effusion amount in patients with TBP, while TNF-α and MMP-1, but not MMP-9, were positively associated with residual pleural fibrosis of TBP. Moreover, effusion levels of TNF-α had positive correlation with those of MMP-1 and MMP-9 in TBP. In cultured PMCs, MTBRa enhanced TLR2 and TLR4 expression, activated ERK signaling, and upregulated TNF-α mRNA and protein expression. Furthermore, knockdown of TLR2, but not TLR4, significantly inhibited ERK phosphorylation and TNF-α expression. Additionally, both MTBRa and TNF-α markedly induced MMP-1 and MMP-9 synthesis in human PMCs, and TNF-α neutralization substantially reduced the production of MMP-1, but not MMP-9, in response to MTBRa stimulation. MTBRa activates TLR2/ERK signalings to induce TNF-α and elicit MMP-1 and MMP-9 in human PMCs, which are associated with effusion volume and pleural fibrosis and may contribute to pathogenesis of TBP. Further investigation of manipulation of TNF-α and MMP expression in pleural mesothelium may provide new insights into the mechanisms and rational treatment strategies for TBP.

Hinokitiol, a natural monoterpenoid from the heartwood of Calocedrus formosana, has been reported to have anticancer effects against various cancer cell lines. However, the detailed molecular mechanisms and the inhibiting roles of hinokitiol on adenocarcinoma A549 cells remain to be fully elucidated. Thus, the current study was designed to evaluate the effect of hinokitiol on the migration of human lung adenocarcinoma A549 cells in vitro. The data demonstrates that hinokitiol does not effectively inhibit the viability of A549 cells at up to a 10 µM concentration. When treated with non-toxic doses (1–5 µM) of hinokitiol, the cell migration is markedly suppressed at 5 µM. Hinokitiol significantly reduced p53 expression, followed by attenuation of Bax in A549 cells. A dose-dependent inhibition of activated caspase-9 and -3 was observed in the presence of hinokitiol. An observed increase in protein expression of matrix metalloproteinases (MMPs) -2/-9 in A549 cells was significantly inhibited by hinokitiol. Remarkably, when A549 cells were subjected to hinokitiol (1–5 µM), there was an increase in the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in cells. In addition, the incubation of A549 cells with hinokitiol significantly activated the cytochrome c expression, which may be triggered by activation of caspase-9 followed by caspase-3. These observations indicate that hinokitiol inhibited the migration of lung cancer A549 cells through several mechanisms, including the activation of caspases-9 and -3, induction of p53/Bax and antioxidant CAT and SOD, and reduction of MMP-2 and -9 activities. It also induces cytochrome c expression. These findings demonstrate a new therapeutic potential for hinokitiol in lung cancer chemoprevention.

Current αIIbβ3 antagonists are potent antithrombotic agents, their clinical use is limited by the risk of life-threatening bleeding. Emerging evidence has highlighted key mechanistic differences between thrombosis and hemostasis, opening avenues for safer antithrombotic strategies. Targeting integrin αIIbβ3 outside-in signaling has been proposed to mitigate bleeding risk; however, the short half-life of peptide-based therapeutics remains a major challenge. In this study, we developed an optimized αIIbβ3 antagonist, KGDRR—a recombinant mutant protein derived from snake venom disintegrin, incorporating an Arg55 residue within the KGD loop—through systematic structure–activity relationship (SAR) analysis. Molecular docking revealed a critical cation–π interaction between Arg55 of KGDRR and Tyr122 of the β3 subunit, stabilizing integrin αIIbβ3 in an unliganded-closed conformation. Functionally, KGDRR selectively inhibited thrombus propagation by blocking ligand binding and downstream Gα13-mediated outside-in signaling while preserving initial thrombus core formation, which is a limitation of current αIIbβ3 inhibitors. Unlike conventional antagonists, KGDRR maintained αIIbβ3 in an unliganded-closed conformation without inducing the integrin activation and conformational change that lead to immune-mediated platelet clearance and thrombocytopenia. In animal models, KGDRR effectively suppressed thrombus growth without causing thrombocytopenia or prolonging bleeding time. Furthermore, intramuscular administration of KGDRR achieved a functional half-life 3.5 times longer than that of the clinically used antithrombotic eptifibatide at equivalent antithrombotic efficacy. In conclusion, KGDRR exhibits potent antithrombotic activity with a favorable safety profile and enhanced pharmacokinetic stability. These findings position KGDRR as a promising next generation αIIbβ3 antagonist with the potential to improve clinical outcomes in antithrombotic therapy.

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.

Platelets are anucleate cells whose dysregulation contributes to thrombocytopenia during sepsis. Thrombocytopenia is an early complication of Gram-negative infection, in which lipopolysaccharide (LPS) serves as a principal mediator; however, its precise contribution remains unclear. In this study, LPS, at concentration 10 µg/mL, did not induce human platelet aggregation but significantly potentiated low-dose collagen (0.5 μg/mL)-induced aggregation, ATP release, intracellular calcium levels ([Ca2+]i) and P-selectin expression. Scanning electron microscopy revealed that either collagen or LPS activated filopodia elongation in human platelets, whereas LPS combined with collagen further activated the phenotype of platelet activation (lamellipodia formation). Beyond these activation responses, LPS also increased TLR4 expression and triggered hallmark apoptotic events, including mitochondrial depolarization, Bax expression, caspase-8 and caspase-3 activation, and phosphatidylserine exposure, concomitant with downregulation of Bcl-2. Moreover, LPS-induced apoptotic platelets displayed ultrastructural changes, characterized by membrane blebbing and filopodia loss. Thus, these findings present the first evidence that LPS enhances platelet aggregation in association with apoptosis through the TLR4–Bax/Bcl-2–mitochondrial dysfunction–caspase-8/3 activation signaling pathway, providing mechanistic insight into sepsis-associated thrombocytopenia.

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‐3β 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.

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 ([Ca 2+ ]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 α IIb β 3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin α IIb β 3 -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 β 3 , 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.

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.

Honokiol, derived from Magnolia officinalis , has various pharmacological properties. Platelet activation plays a critical role in cardiovascular diseases. Honokiol has been reported to inhibit collagen-stimulated rabbit platelet aggregation. However, detailed further studies on the characteristics and functional activity of honokiol in platelet activation are relatively lacking. In the present study, honokiol specifically inhibited platelet aggregation and Ca +2 ion mobilization stimulated with collagen or convulxin, an agonist of glycoprotein (GP) VI, but not with aggretin, an agonist of integrin α 2 β 1 . Honokiol also attenuated the phosphorylation of Lyn, PLCγ2, PKC, MAPKs, and Akt after convulxin stimulation. Honokiol have no cytotoxicity in zebrafish embryos. Honokiol diminished the binding of anti-GP VI (FITC-JAQ1) mAb to human platelets, and it also reduced the coimmunoprecipitation of GP VI-bound Lyn after convulxin stimulation. The surface plasmon resonance results revealed that honokiol binds directly to GP VI, with a K D of 289 μM. Platelet function analysis revealed that honokiol substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation in mice. In conclusion, honokiol acts as a potent antagonist of collagen GP VI in human platelets, and it has therapeutic potential in the prevention of the pathological thrombosis.