Wan-Jung Lu

Neuroprotective effects of xanthohumol, a prenylated flavonoid from hops (humulus lupulus), in ischemic stroke of rats

Xanthohumol is the principal prenylated flavonoid in hops (Humulus lupulus L.), an ingredient of beer. Xanthohumol was found to be a potent chemopreventive agent; however, no data are available concerning its neuroprotective effects. In the present study, the neuroprotective activity and mechanisms of xanthohumol in rats with middle cerebral artery occlusion (MCAO)-induced cerebral ischemia were examined. Treatment with xanthohumol (0.2 and 0.4 mg/kg; intraperitoneally) 10 min before MCAO dose-dependently attenuated focal cerebral ischemia and improved neurobehavioral deficits in cerebral ischemic rats. Xanthohumol treatment produced a marked reduction in infarct size compared to that in control rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor (HIF)-1α, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), and active caspase-3 protein expressions in ischemic regions. These expressions were obviously inhibited by treatment with xanthohumol. In addition, xanthohumol (3-70 μM) concentration-dependently inhibited platelet aggregation stimulated by collagen (1 μg/mL) in human platelet-rich plasma. An electron spin resonance (ESR) method was used to examine the scavenging activity of xanthohumol on free radicals which had formed. Xanthohumol (1.5 and 3 μM) markedly reduced the ESR signal intensity of hydroxyl radical (OH•) formation in the H₂O₂/NaOH/DMSO system. In conclusion, this study demonstrates for the first time that in addition to its originally being considered an agent preventing tumor growth, xanthohumol possesses potent neuroprotective activity. This activity is mediated, at least in part, by inhibition of inflammatory responses (i.e., HIF-1α, iNOS expression, and free radical formation), apoptosis (i.e., TNF-α, active caspase-3), and platelet activation, resulting in a reduction of infarct volume and improvement in neurobehavior in rats with cerebral ischemia. Therefore, this novel role of xanthohumol may represent high therapeutic potential for treatment or prevention of ischemia-reperfusion injury-related disorders.

Suppression of NF-κB signaling by andrographolide with a novel mechanism in human platelets: regulatory roles of the p38 MAPK-hydroxyl radical-ERK2 cascade.

Andrographolide, a novel nuclear factor-κB (NF-κB) inhibitor, is isolated from leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by activating the endothelial nitric oxide synthase (eNOS)-NO-cyclic GMP pathway. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of andrographolide in NF-κB-mediated events in platelets. In this study, NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were time-dependently activated by collagen in human platelets, and these signaling events were attenuated by andrographolide (35 and 75 μM). ODQ and KT5823, respective inhibitors of guanylate cyclase and cyclic GMP-dependent kinase (PKG), strongly reversed andrographolide-mediated inhibition of platelet aggregation, relative [Ca(2+)]i mobilization, and IKKβ, and p65 phosphorylation. In addition, SB203580 (an inhibitor of p38 MAPK), but not PD98059 (an inhibitor of ERKs), markedly abolished IKKβ and p65 phosphorylation. SB203580, NAC (a free-radical scavenger), and BAY11-7082 (an inhibitor of NF-κB) all diminished ERK2 phosphorylation, whereas PD98059, BAY11-7082, and NAC had no effects on p38 MAPK phosphorylation. Furthermore, SB203580, but not BAY11-7082 or PD98059, reduced collagen-induced hydroxyl radical ((·)HO) formation. KT5823 also markedly reversed andrographolide-mediated inhibition of p38 MAPK and ERK2 phosphorylation, and hydroxyl radical formation in platelets. In conclusion, this study demonstrated that andrographolide may involve an increase in cyclic GMP/PKG, followed by inhibition of the p38 MAPK/(·)HO-NF-κB-ERK2 cascade in activated platelets. Therefore, andrographolide may have a high therapeutic potential to treat thromboembolic disorders and may also be considered for treating various inflammatory diseases.

A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

BackgroundPlatelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function.MethodsPlatelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study.ResultsNF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM)-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization.ConclusionsSesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when these types of drugs are considered for the treatment of cancer and various inflammatory diseases.

Hinokitiol inhibits platelet activation ex vivo and thrombus formation in vivo

Hinokitiol is a tropolone-related bioactive compound that has been used in hair tonics, cosmetics, and food as an antimicrobial agent. Recently, hinokitiol has attracted considerable interest because of its anticancer activities. Platelet activation plays a crucial role in atherothrombotic processes. We examined the effects of hinokitiol treatment on platelet activation using human platelets. In the present study, hinokitiol (1 and 2 μM) inhibited the collagen-induced aggregation of human platelets, but did not inhibit the activation of platelets by other agonists, including thrombin, arachidonic acid, and ADP. Hinokitiol inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Akt in collagen-activated human platelets, and significantly reduced intracellular calcium mobilization and hydroxyl radical (OH·) formation. Hinokitiol also reduced the PKC activation and platelet aggregation stimulated by PDBu. In addition, hinokitiol significantly prolonged thrombogenesis in mice. Hinokitiol did not influence the binding of a fluorescent triflavin probe to the αIIbβ3 integrin on platelet membrane, and neither ODQ nor SQ22536 significantly reversed the hinokitiol-mediated inhibition of platelet aggregation. In conclusion, hinokitiol may inhibit platelet activation by inhibiting the PLCγ2-PKC cascade and hydroxyl radical formation, followed by suppressing the activation of MAPKs and Akt. Our study suggests that hinokitiol may represent a potential therapeutic agent for the prevention or treatment of thromboembolic disorders.

Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase

Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5-5 μM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca(2+)](i) mobilization, thromboxane A(2) (TxA(2)) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH(●)) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA(2) cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.

Comparison of the relative activities of inducing platelet apoptosis stimulated by various platelet-activating agents

Apoptosis-like events are known to occur in anuclear platelets. Although the mechanisms responsible for these events are still not completely understood, studies suggested that some platelet agonists can activate platelet apoptosis. However, the relative activities of various platelet agonists in inducing apoptosis have not yet been investigated. In the present study we explored this issue, and attempted to identify the correlation between platelet activation and apoptosis. In a platelet aggregation study, there were no significant differences respectively stimulated by arachidonic acid (AA; 100 µM), ADP (20 µM), collagen (10 µg/mL), thrombin (0.1 U/mL), U46619 (10 µM), and A23187 (5 µM). In a subsequent study, we fixed these concentrations of agonists to further compare their relative activities in inducing platelet apoptosis. Our results found that thrombin, U46619, and A23187 possess stronger activities than the other agonists in inducing platelet apoptosis (i.e., phosphatidylserine exposure, mitochondrial membrane potential depolarization, eukaryotic initiation factor (eIF)2α, and caspase activation). On the other hand, AA induced no apoptotic events in platelets. Based on this approach, we demonstrated for the first time that thrombin, U46619, and A23187, but not AA, possess stronger activity in inducing platelet apoptosis. In addition, we also found that platelet activation might not necessarily be associated with the occurrence of platelet apoptosis. The in vivo physiological function of the apoptotic machinery in platelets is not yet clearly understood, and needs to be further investigated in the future.

Role of a Janus kinase 2-dependent signaling pathway in platelet activation

INTRODUCTION Janus kinases (JAKs) are intracellular non-receptor tyrosine kinases that transduce cytokine-mediated signals through a pathway mediated by JAK and the signal transducer and activator of transcription (STAT) proteins. The JAK-STAT pathway is involved in immune response, inflammation, and tumorigenesis. Platelets are anuclear blood cells that play a central role in hemostasis. METHODS The aggregometry, immunoblotting, and platelet functional analysis used in this study. RESULTS We found that the JAK2 inhibitor AG490 (25 and 50μM) attenuated collagen-induced platelet aggregation and calcium mobilization in a concentration-dependent manner. In the presence of AG490, the phosphorylation of PLCγ2, protein kinase C (PKC), Akt or JNK in collagen-activated aggregation of human platelets was also inhibited. In addition, we found that various inhibitors, such as the PLCγ2 inhibitor U73122, the PKC inhibitor Ro318220, the phospoinositide 3-kinase inhibitor LY294002, the p38 mitogen-activated protein kinase inhibitor SB203580, the ERK inhibitor PD98059, and the JNK inhibitor SP600125, had no effects on collagen-induced JAK2 activity. However, U73122, Ro318220 and SP600125 significantly diminished collagen-induced STAT3 phosphorylation. These findings suggest that PLCγ2-PKC and JNK are involved in JAK2-STAT3 signaling in collagen-activated platelets. CONCLUSION Our results demonstrate that the JAK2-STAT3 pathway is involved in collagen-induced platelet activation through the activation of JAK2-JNK/PKC-STAT3 signaling. The inhibition of JAK2 may represent a potential therapeutic strategy for the preventing or treating thromboembolic disorders.

Mechanisms of Andrographolide-Induced Platelet Apoptosis in Human Platelets: Regulatory Roles of the Extrinsic Apoptotic Pathway

Andrographolide, a novel nuclear factor‐κB (NF‐κB) inhibitor, is isolated from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by inhibition of the p38 MAPK/●HO‐NF‐κB‐ERK2 cascade. Although platelets are anucleated cells, apoptotic machinery apparatus recently has been found to regulate platelet activation and limit platelet lifespan. Therefore, we further investigated the regulatory effects of andrographolide on platelet apoptotic events. In this study, apoptotic signaling events for caspase‐3, ‐8, and Bid were time (10–60 min)‐ and dose (25–100 μΜ)‐dependently activated by andrographolide in human platelets. Andrographolide could also disrupt mitrochondrial membrane potential. In addition, caspase‐8 inhibitor (z‐IETD‐fmk, 50 μΜ) was found to reverse andrographolide‐induced caspase‐8 activation, whereas the antagonistic anti‐Fas receptor (ZB4, 500 ng/mL) and anti‐tumor necrosis factor‐R1 (H398, 10 µg/mL) monoclonal antibodies did not. In conclusion, this study for the first time demonstrated that andrographolide might limit platelet lifespan by initiating the caspase‐8‐dependent extrinsic apoptotic pathway, in spite of no direct evidence that death receptors are involved in this process proved. Overall, the various medicinal properties of andrographolide suggest its potential value in treating patients with thromboembolic disorders. Copyright

Prevention of arterial thrombosis by nobiletin: in vitro and in vivo studies.

Nobiletin, a bioactive polymethoxylated flavone isolated from citrus fruits, has been proven to prevent cancer and inflammation. Dietary flavonoids have been shown to reduce the risk of cardiovascular diseases (CVDs), and platelet activation plays a crucial role in CVDs. This study investigated the effect of nobiletin on platelet activation in vitro and in vivo. Nobiletin (10-30μM) inhibited collagen- and arachidonic acid-induced platelet aggregation in washed human platelets, but it did not inhibit platelet aggregation induced by other agonists such as thrombin and 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin. Nobiletin inhibited the phosphorylation of phospholipase PLCγ2, protein kinase PKC, Akt and mitogen-activated protein kinase MAPKs in collagen-activated human platelets and markedly reduced intracellular calcium mobilization and hydroxyl radical (OH(·)) formation. Nobiletin did not affect either phorbol-12,13-dibutyrate-stimulated PKC activation or platelet aggregation. In addition, neither SQ22536, an adenylate cyclase inhibitor nor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, significantly reversed the nobiletin-mediated inhibition of platelet aggregation. Moreover, nobiletin substantially prolonged the closure time in whole blood according to platelet function analysis and increased the occlusion time of thrombotic platelet plug formation in mice. In conclusion, this study demonstrates for the first time that, in addition to being a potential agent for preventing tumor growth and inflammation, nobiletin exhibits potent antiplatelet activity, which initially inhibits the PLCγ2/PKC cascade and hydroxyl radical formation, subsequently suppresses the activation of Akt and MAPKs and ultimately inhibits platelet activation. Our study suggests that nobiletin represents a potential therapeutic agent for preventing or treating thromboembolic disorders.

Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus), Prevents Platelet Activation in Human Platelets

Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH●) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.