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.

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.

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.

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.

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.

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.

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.

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.

Ruthenium derivatives attenuate LPS-induced inflammatory responses and liver injury via suppressing NF-κB signaling and free radical production.

Ruthenium metal complex has been shown to exert several chemical and biological activities. A series of three novel ruthenium derivatives (TQ 1, 2 and 4) were synthesized to evaluate the anti-inflammatory and hepatoprotective activities in lipopolysaccharide (LPS)-stimulated macrophages and mice liver injury. The hydroxyl radical (OH°) scavenging activity of these derivatives has also been evaluated. The results revealed that among the tested compounds, TQ-4 effectively attenuated LPS-induced abnormal alteration in liver histoarchistructure via reducing alanine transaminase (ALT) and aspartate transaminase (AST). This compound exhibited significant inhibition of inflammatory cytokines (TNF-α and IL-1ß), inflammatory enzyme (iNOS), the component of NF-κB signaling pathway (p65) and JNK phosphorylation in LPS-induced mice liver tissues. In vitro results showed that TQ-4 had the best inhibition of NO production and iNOS expression in LPS-induced RAW 264.7 cells. Mechanistic approach indicated that TQ-4 inhibited the LPS-induced JNK phosphorylation, IκBα degradation, NF-κB p65 phosphorylation and its nuclear translocation, and hydroxyl radical (OH°) productions in RAW 264.7 cells. However, the compounds TQ-1 and 2 had no effects in this study. TQ-4 also inhibited LPS-induced OH° production. This study reveals the protective effect of TQ-4 against LPS-induced acute liver injury, inflammation, and oxidative reaction by destructing JNK/NF-κB signaling pathways. The result of this study may infer that TQ-4 might be a promising ruthenium metal derivative and/or therapeutic agent for treating liver injury.

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.

HDAC6 dysfunction contributes to impaired maturation of adult neurogenesis in vivo: vital role on functional recovery after ischemic stroke.

BACKGROUND: Promoting post-stroke neurogenesis has long been proposed to be a therapeutic strategy for the enhancement of functional recovery after cerebral ischemic stroke. Despite numerous approaches have been widely reported the proliferation or differentiation of the neurogenic population therapeutic strategies by targeting adult neurogenesis not yet to be successfully clarified in clinical settings. Here, we hypothesized that alterations in microenvironment of the ischemic brain might impede the functional maturation of adult newly generated neurons that limits functional recovery after stroke. METHODS: The in vivo retroviral based labeling model was applied to directly birth-date and trace the maturation process of adult newly generating neurons after hypoxic challenge. A rehabilitation therapy procedure was adopted through the combination of task-specific motor rehabilitating training with environmental enrichment to promote functional recovery after stroke. In addition, a pharmacological or genetic suppression of HDAC6 was performed to evaluate the functional significance of HDAC6 in the pathology of ischemic stroke induced deficits. RESULTS: Serial morphological analyses at multiple stages along the maturation process showed significant retardation of the dendritic maturation on the newly generated neurons after stroke. Subsequent biochemical analyses revealed an aberrant nuclear translocation of HDAC6 that leads to the hyper-acetylation of α-tubulin (an indication of over-stabilized microtubules) after hypoxic challenge was observed at different time points after stroke. Furthermore, the mimicry experiments with either pharmacological or genetic suppression of HDAC6, phenocopied the stroke induced retardation in dendritic maturation of newly generating neurons in vivo. More importantly, we provide direct evidence showing the proper function of HDAC6 is required for rehabilitation therapy induced therapeutic benefits after stroke. CONCLUSION: Together, our current study unravels that dysfunction of HDAC6 contributes to stroke induced deficits in neurogenesis and provides an innovative therapeutic strategy that targets HDAC6 for promoting functional recovery toward the patients with stroke in clinic.

Suppression of Human Platelet Activation via Integrin αIIbß3 Outside-In Independent Signal and Reduction of the Mortality in Pulmonary Thrombosis by Auraptene.

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.

Esculetin, a Coumarin Derivative, Prevents Thrombosis: Inhibitory Signaling on PLCγ2-PKC-AKT Activation in Human Platelets.

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.

Possible Molecular Targets of Novel Ruthenium Complexes in Antiplatelet Therapy.

In oncotherapy, ruthenium (Ru) complexes are reflected as potential alternatives for platinum compounds and have been proved as encouraging anticancer drugs with high efficacy and low side effects. Cardiovascular diseases (CVDs) are mutually considered as the number one killer globally, and thrombosis is liable for the majority of CVD-related deaths. Platelets, an anuclear and small circulating blood cell, play key roles in hemostasis by inhibiting unnecessary blood loss of vascular damage by making blood clot. Platelet activation also plays a role in cancer metastasis and progression. Nevertheless, abnormal activation of platelets results in thrombosis under pathological settings such as the rupture of atherosclerotic plaques. Thrombosis diminishes the blood supply to the heart and brain resulting in heart attacks and strokes, respectively. While currently used anti-platelet drugs such as aspirin and clopidogrel demonstrate efficacy in many patients, they exert undesirable side effects. Therefore, the development of effective therapeutic strategies for the prevention and treatment of thrombotic diseases is a demanding priority. Recently, precious metal drugs have conquered the subject of metal-based drugs, and several investigators have motivated their attention on the synthesis of various ruthenium (Ru) complexes due to their prospective therapeutic values. Similarly, our recent studies established that novel ruthenium-based compounds suppressed platelet aggregation via inhibiting several signaling cascades. Our study also described the structure antiplatelet-activity relationship (SAR) of three newly synthesized ruthenium-based compounds. This review summarizes the antiplatelet activity of newly synthesized ruthenium-based compounds with their potential molecular mechanisms.

Structure⁻Activity Relationship Study of Newly Synthesized Iridium-III Complexes as Potential Series for Treating Thrombotic Diseases.

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 structure⁻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.

Hinokitiol Inhibits Migration of A549 Lung Cancer Cells via Suppression of MMPs and Induction of Antioxidant Enzymes and Apoptosis.

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.

Novel Therapeutic Agent against Platelet Activation In Vitro and Arterial Thrombosis In Vivo by Morin Hydrate.

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 PLCγ2⁻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.

Structure-Antiplatelet Activity Relationships of Novel Ruthenium (II) Complexes: Investigation of Its Molecular Targets.

The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven to be a positive approach to the prevention of thrombosis. Ruthenium complexes are fascinating for the development of new drugs, as they possess numerous chemical and biological properties. The present study aims to evaluate the structure-activity relationship (SAR) of newly synthesized ruthenium (II) complexes, TQ-1, TQ-2 and TQ-3 in agonists-induced washed human platelets. Silica gel column chromatography, aggregometry, immunoblotting, NMR, and X-ray analyses were performed in this study. Of the three tested compounds, TQ-3 showed a concentration (1-5 µM) dependent inhibitory effect on platelet aggregation induced by collagen (1 µg/mL) and thrombin (0.01 U/mL) in washed human platelets; however, TQ-1 and TQ-2 had no response even at 250 µM of collagen and thrombin-induced aggregation. TQ-3 was effective with inhibiting collagen-induced ATP release, calcium mobilization ([Ca2+]i) and P-selectin expression without cytotoxicity. Moreover, TQ-3 significantly abolished collagen-induced Lyn-Fyn-Syk, Akt-JNK and p38 mitogen-activated protein kinases (p38 MAPKs) phosphorylation. The compound TQ-3 containing an electron donating amino group with two phenyl groups of the quinoline core could be accounted for by its hydrophobicity and this nature might be the reason for the noted antiplatelet effects of TQ-3. The present results provide a molecular basis for the inhibition by TQ-3 in collagen-induced platelet aggregation, through the suppression of multiple machineries of the signaling pathway. These results may suggest that TQ-3 can be considered a potential agent for the treatment of vascular diseases.

Multi-Targeting Andrographolide, a Novel NF-κB Inhibitor, as a Potential Therapeutic Agent for Stroke.

A key focus in the field of drug discovery has been motivated by the neuroprotection of natural compounds. Cerebral ischemia is a multifaceted pathological process with a series of mechanisms, and a perspective for the development of neuroprotectants from traditional herbal medicine or natural products is a promising treatment for this disease. Natural compounds with the effects of anti-oxidation, anti-inflammation, anti-apoptosis, and neurofunctional regulation exhibit therapeutic effects on experimental ischemic brain injury. Conferring to the pharmacological mechanisms underlying neuroprotection, a study found that androgapholide, a diterpene lactone compound, exhibits varying degrees of neuroprotective activities in both in vitro and in vivo experimental models of stroke. The neuroprotective mechanisms of andrographolide are suggested as: (I) increasing nuclear factor E2-related factor 2-heme oxygenase (Nrf2-HO-1) expression through p38-mitogen activated protein kinase (MAPK) regulation, (II) inducing cerebral endothelial cells (CEC) apoptosis and caspase-3 activation, (III) down regulating Bax, inducible nitric oxide synthase (iNOS), and (IV) inhibiting hydroxyl radical (OH-) formation, and activating transcription factor NF-κB signaling pathways. Recently, several researchers have also been trying to unveil the principal mechanisms involved in the neuroprotective effects of andrographolide. Therefore, this review aims to summarize an overview on the neuroprotective effects of andrographolide and exemplifies the essential mechanisms involved. This paper can provide information that andrographolide drug discovery may be a promising strategy for the development of a novel class of neuroprotective drug.