ABSTRACT
This study was designed to investigate inhibitory effects and possible mechanisms of snake venom tripeptide (pENW) on platelet adhesion in order to promote the development of a novel anti-platelet therapy. To study the inhibitory effects of pENW on platelet adhesion, washed platelets pre-incubated with pENW (116.5-466.2 μmol x L(-1)) were used to test the ability of platelet adhesion to fibrinogen. Effect of pENW on fibrin clot retraction was also tested. Effect of pENW on platelets viability was tested by MTT assay. Effect of pENW on reactive-oxygen species (ROS) levels of platelet was studied by flow cytometry assay. Calcium mobilization in Fura-2/AM-loaded platelets was monitored with a spectrofluorimeter. Cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), thromboxane A2 (determined as its metabolite thromboxane B2) were measured using enzyme immunoassay kits. Akt, ERK and p38 phosphorylation were tested by Western blot. The results showed that pENW inhibited platelet adhesion and fibrin clot retraction in a concentration-dependent manner without cytotoxicity. Intracellular cGMP and cAMP in both resting and thrombin-activated platelets were increased by pENW. In addition, pENW attenuated intracellular Ca2+ mobilization and TXA2 production in platelets stimulated by thrombin. As shown by Western blot assay, Akt, ERK and p38 phosphorylation in thrombin-induced platelet were attenuated by pENW. However, inhibitory effects of pENW had nothing to do with ROS. Thus, pENW exhibited a significant inhibition on platelet adhesion to fibrinogen, which means pENW could block the first step of thrombosis as while as retard the more stable clot formation. The mechanisms of pENW on inhibition platelet adhesion might be related to instant regulations, such as protein kinases.
Subject(s)
Blood Platelets , Blotting, Western , Calcium , Metabolism , Cyclic AMP , Metabolism , Cyclic GMP , Metabolism , Flow Cytometry , Phosphorylation , Platelet Aggregation , Reactive Oxygen Species , Metabolism , Snake Venoms , Chemistry , Thromboxane A2 , Metabolism , Thromboxane B2 , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the effects of Naomaitai capsule on the learning and memory abilities and the content of acetylcholine(Ach) in the brain tissue of the model rat.</p><p><b>METHOD</b>One vascular dementia(VD) model was produced by occlusion of bilateral common carotid arteries in rats with following steps: ischemia 20 minutes-reperfusion 10 minutes-ischemia 20 minutes. The learning and memory abilities were tested by water maze. The other VD model was formed by 4-vessel occlusion-reperfusion injury in rats. The time of right reflect recovery and the content of Ach in the brain tissue were determined.</p><p><b>RESULT</b>Naomaitai capsule can significantly improve the learning and memory abilities of the dementia rats( P < 0.05, P < 0.01), increase the content of Ach (P < 0.05, P < 0.01) and reduce the time of right reflect recovery(P < 0.01).</p><p><b>CONCLUSION</b>Naomaitai capsule can improve the learning and memory abilities by increasing the content of Ach of the VD rats.</p>