Involvement of p38 MAPK phosphorylation and nitrate formation in aristolochic acid-mediated antiplatelet activity.

Aristolochic acid (AsA) is produced from Aristolochia fangchi, and has been used as a Chinese herbal medicine. AsA possesses various biological activities including antiplatelet, antifungal, and anti-inflammatory properties. The aim of this study was to examine the mechanisms of AsA in inhibiting platelet aggregation. AsA (75 - 150 microM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen (1 microg/mL) than other agonists. AsA (115 and 150 microM) inhibited collagen-induced platelet activation accompanied by [Ca+2)]i mobilization, thromboxane A2 (TxA2) formation and phosphoinositide breakdown. On the other hand, AsA also markedly increased levels of NO/cyclic GMP, and cyclic GMP-induced vasodilator-stimulated phosphoprotein phosphorylation. AsA inhibited p38 MAPK but not ERK1/2 phosphorylation in washed platelets. In conclusion, the most important findings of this study suggest that the inhibitory effects of AsA possibly involve the (1) inhibition of the p38 MAPK-cytosolic phospholipase A2-arachidonic acid-TxA2-[Ca+2)]i cascade, and (2) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C. These results imply that Aristolochia fangchi treatment alone or in combination with other antiplatelet drugs, may result in alteration of hemostasis in vivo.

Antiplatelet activity of caffeic acid phenethyl ester is mediated through a cyclic GMP-dependent pathway in human platelets.

The aim of this study was to examine the inhibitory mechanisms of caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee, in platelet activation. In this study, CAPE (15 and 25 microM) markedly inhibited platelet aggregation stimulated by collagen (2 microg/ml). CAPE (15 and 25 microM) increased cyclic GMP level, and cyclic GMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser157 phosphorylation, but did not increase cyclic AMP in washed human platelets. Rapid phosphorylation of a platelet protein of Mw. 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (150 nM). This phosphorylation was markedly inhibited by CAPE (15 and 25 microM). The present study reports a novel and potent antiplatelet agent, CAPE, which involved in the following inhibitory pathways: CAPE increases cyclic GMP/VASP Ser157 phosphorylation, and subsequently inhibits protein kinase C activity, resulting in inhibition of P47 phosphorylation, which ultimately inhibits platelet aggregation. These results strongly indicate that CAPE appears to represent a novel and potent antiplatelet agent for treatment of arterial thromboembolism.

Tetramethylpyrazine suppresses HIF-1alpha, TNF-alpha, and activated caspase-3 expression in middle cerebral artery occlusion-induced brain ischemia in rats.

AIM: To examine the detailed mechanisms underlying the inhibitory effect of tetramethylpyrazine (TMPZ) in inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats. METHODS: MCAO-induced focal cerebral ischemia in rats was used in this study. The hypoxia-inducible factor-1alpha(HIF-1alpha), activation of caspase-3, and TNF-alpha mRNA transcription in ischemic regions were detected by immunoblotting and RT-PCR, respectively. Anti-oxidative activity was investigated using a thiobarbituric acid-reactive substance (TBARS) test in rat brain homogenate preparations. RESULTS: We showed the statistical results of the infarct areas of solvent- and TMPZ (20 mg/kg)-treated groups at various distances from the frontal pole in MCAO-induced focal cerebral ischemia in rats. Treatment with TMPZ (20 mg/kg) markedly reduced the infarct area in all regions, especially in the third to fifth sections. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1alpha and the activation of caspase-3, as well as TNF-alpha transcription in ischemic regions. These expressions were markedly inhibited by treatment with TMPZ (20 mg/kg). However, TMPZ (0.5-5 mmol/L) did not significantly inhibit TBARS reaction in rat brain homogenates. CONCLUSION: The neuroprotective effect of TMPZ may be mediated at least by a portion of the inhibition of HIF-1alpha and TNF-alpha activations, followed by the inhibition of apoptosis formation (active caspase-3), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, TMPZ treatment may represent an ideal approach to lowering the risk of or improving function in ischemia- reperfusion brain injury-related disorders.

Neuroprotective effects of PMC, a potent alpha-tocopherol derivative, in brain ischemia-reperfusion: reduced neutrophil activation and anti-oxidant actions.

2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC) is the most potent analogue of alpha-tocopherol for anti-oxidation. It is more hydrophilic than other alpha-tocopherol derivatives and has potent free radical-scavenging activity. In the present study, PMC significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of PMC at 20mg/kg, showed marked reductions in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1alpha, active caspase-3, iNOS, and nitrotyrosine expressions in ischemic regions. These expressions were markedly inhibited by treatment with PMC (20mg/kg). In addition, PMC (4-12 microM) inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). Furthermore, PMC (6, 12, and 60 microM) also significantly inhibited neutrophil migration stimulated by leukotriene B(4) (160 nM). An electron spin resonance (ESR) method was conducted on the scavenging activity of PMC on the free radicals formed. PMC (12 microM) greatly reduced the ESR signal intensities of superoxide anion, hydroxyl radical, and methyl radical formation. In conclusion, we demonstrate a potent neuroprotective effect of PMC on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by inhibition of free radical formation, followed by inhibition of HIF-1alpha activation, apoptosis formation (active caspase-3), neutrophil activation, and inflammatory responses (i.e., iNOS and nitrotyrosine expressions), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, PMC treatment may represent a novel approach to lowering the risk or improving function in ischemia-reperfusion brain injury-related disorders.

Inhibitory mechanisms of tetramethylpyrazine in middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats.

Tetramethylpyrazine (TMPZ) is an active ingredient isolated from a commonly used Chinese herb, Ligusticum wallichii Franchat, which has long been used in China for the treatment of vascular diseases. In the present study, TMPZ significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of TMPZ at 10 and 20 mg/kg produced concentration-dependent reductions in infarct size compared to that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in both nitrotyrosine and inducible nitric oxide synthase (iNOS) expression in ischemic regions. The expressions of nitrotyrosine and iNOS were markedly inhibited by TMPZ (20 mg/kg) treatment. Furthermore, TMPZ (100-250 microM) concentration-dependently inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). TMPZ (100-250 microM) also significantly inhibited neutrophil migration stimulated by fMLP (800 nM) and LTB4 (160 nM). An electron spin resonance (ESR) method was used to further study the scavenging activity of TMPZ on free radicals formed in human neutrophils. TMPZ (100 and 200 microM) greatly reduced the ESR signal intensity of hydroxyl radical formation. In conclusion, we demonstrate a neuroprotective effect of TMPZ in MCAO-induced focal cerebral ischemia in vivo. TMPZ mediates at least part of the free radical-scavenging activity and inhibits neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, TMPZ treatment may represent an ideal approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.

Inhibitory effects of lycopene on in vitro platelet activation and in vivo prevention of thrombus formation.

Lycopene is a natural carotenoid antioxidant that is present in tomatoes and tomato products. The pharmacologic function of lycopene in platelets is not yet understood. Therefore, in this study we sought to systematically examine the effects of lycopene in the prevention of platelet aggregation and thrombus formation. We found that lycopene concentration-dependently (2-12 micromol/L) inhibited platelet aggregation in human platelets stimulated by agonists. Lycopene (6 and 12 micromol/L) inhibited phosphoinositide breakdown in platelets labeled with tritiated inositol, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane B2 formation stimulated by collagen. In addition, lycopene (6 and 12 micromol/L) significantly increased the formations of cyclic GMP and nitrate but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of 47,000 Da (P47), a marker of protein kinase C activation, was triggered by PDBu (60 nmol/L). This phosphorylation was markedly inhibited by lycopene (12 micromol/L) in phosphorus-32-labeled platelets. In an in vivo study, thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Lycopene (5, 10, and 20 mg/kg) significantly prolonged the latency period for the induction of platelet-plug formation in mesenteric venules. These results indicate that the antiplatelet activity of lycopene may involve the following pathways: (1) Lycopene may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane B2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization. (2) Lycopene also activated the formations of cyclic GMP/nitrate in human platelets, resulting in the inhibition of platelet aggregation. The results may imply that tomato-based foods are especially beneficial in the prevention of platelet aggregation and thrombosis.

Gene stacking in Phalaenopsis orchid enhances dual tolerance to pathogen attack.

Cymbidium Mosaic Virus (CymMV) and Erwinia carotovora have been reported to cause severe damage to orchid plants. To enhance the resistance of orchids to both viral and bacterial phytopathogens, gene stacking was applied on Phalaenopsis orchid by double transformation. PLBs originally transformed with CymMV coat protein cDNA (CP) were then re-transformed with sweet pepper ferredoxin-like protein cDNA (Pflp) by Agrobacterium tumefaciens, to enable expression of dual (viral and bacterial) disease resistant traits. A non-antibiotic selection procedure in the second transformation minimized the potential rate of 'stacking' antibiotic genes in the orchid gene pool. Transgene integration in transgenic Phalaenopsis lines was confirmed by Southern blot analysis for both CP and pflp genes. Expression of transgenes was detected by northern blot analysis, and disease resistant assays revealed that transgenic lines exhibited enhanced resistance to CymMV and E. carotovora. This is the first report describing a transgenic Phalaenopsis orchid with dual resistance to phytopathogens.