Cyclovirobuxine D （CVB-D） is a compound extracted from Chinese traditional plant Buxus microphylla, which has been used for treating arrhythmia and myocardial ischemia in China. In this study, we investigated its effect on blood coagulation and thrombotic formation in mouse and rat models. The doses of CVB-D used in this study （5-20 mg/kg） prolonged clotting time （CT） in a dose-dependent manner （P〈0.01）. It also significantly prolonged thrombin time （TT）, prothrombin time （PT） and activated partial thromboplast time （aPTT） （P〈0.05 or P〈0.01） at the doses of 10-20 mg/kg. CVB-D did not affect the bleeding time （BT） compared with the control group, while warfarin significantly prolonged the bleeding time. CVB-D at the doses of 5-20 mg/kg reduced wet weight of thrombosis （P〈0.01）. This study demonstrated the anti-coagulation effect and anti-thrombosis effect of orally administered CVB-D without substantially increasing bleeding. These findings suggest that CVB-D probably can be used as an oral anti-coagulant in addition to its current applications.
Methoxyl methyl ether isoamylene quercetin （MIAQ） is one of the newly synthesized quercetin derivatives. The present study investigated the effect of MIAQ on rat aorta endothelial cells （RAECs） injured by hydrogen peroxide （H2O2）, as well as the potential mechanisms. We observed that MIAQ at 2.5-10μmol/L significantly enhanced the viability of injured RAECs, and the effect was more potent than quercetin and ct-tocopherol. However, M1AQ at the same concentration failed to show anti-oxidant activity in a cell-free system. In H2O2-injured endothelial cells treated with MIAQ （5-10μmol/L）, the level of nitric oxide （NO） and malondialdehyde was decreased, and the activities of superoxide dismutase and glutathione peroxidase was enhanced. In addition, RAECs treated with MIAQ （2.5-10 μmol/L） exhibited significant inhibiting apoptosis. In conclusion, MIAQ had protective effect on RAECs, possibly through increasing NO production and antioxidases activities, as well as inhibiting apoptosis. These findings suggest that MIAQ is possibly beneficial in the prevention of atherosclerosis and other diseases related to endothelial injury.
Calf spleen extract(CSE) has been clinically used as an adjuvant agent in malignant tumor therapy.It can improve the physical status of patients.However,its mechanism of action remains relatively unclear.In this study,we investigated the effect of CSE on leucopenia in mice and on promyelocytic leukemia cells(HL60).CSE in 10 mL/kg(2.5 mg bioactive polypeptides and 190μg ribose/mL) significantly increased leukocyte numbers in leucopenia mice.The effect on neutrophil numbers,among all leukocytes,was the most evident.CSE stimulated the proliferation of bone marrow cells in vitro and decreased the GM-CSF level in serum.In addition,CSE significantly reduced the viability of HL60 cells,decreased the production of lactate and adenosine triphosphate(ATP) of these cells.CSE induced the apoptosis and S-phase arrest in HL60 cells.In conclusion,CSE can enhance leukocyte numbers,which may be attributed to its direct stimulatory effect on bone marrow cells.CSE is an inhibitor of promyelocytic leukemia cell viability,which may be attributed to the induction of the apoptosis,the arrest of cell cycle and the inhibition of the glycolysis of cells.
Aim L-Arginine· L-aspartate, a double salt, has been recently reported toinhibit platelet aggregation and thrombosis, but its action mechanism is not clear yet. This studywas conducted to investigate its effect on FITC-PAC-1, an anti-glycoprotein IIb/IIIa monoclonalantibody binding to activated platelets, and on correlative autacoid levels in plasma or inplatelets in order to explore its potential pathway of inhibiting platelet aggregation andthrombosis. Methods Monoclonal antibody binding to activated platelets was assayed by flowcytometry; NO was assessed by colorimetric method. cAMP, TXB_2 or 6-keto-PGF_(1α) levels wereassessed by radioimmunoassay. Results Gavaged 30 mg·kg^(-1) of L-arginine·L-aspartate increasedboth concentration of NO in plasma and 6-keto-PGF_(1) in incubated supernatant of aortic segment ofrats ex vivo (P < 0.05), but it did not influence cAMP content in platelets and the level of TXB_2or 6-keto-PGF_(1) in plasma of rats, whereas ASA significantly lowered TXB_2 or 6-keto-PGF_(1α) inplasma. Both 100 μmol-L^(-1) of L-arginine ·L-aspartate and ASA inhibited FITC-PAC-1 binding toactivated platelets in vitro. Conclusion The increase in NO and PGI_2 release from endo-thelialcells and consequent inhibition of platelet activation may contribute to the inhibition of plateletaggregation and thrombosis by L-arginine· L-aspartate; whereas arachidonic acid or cAMP metabolicpathway is not closely correlative with the studied effect.
Recombinant ancylostoma caninum anticoagulant peptide-5 （rAcAP5） has been reported to inhibit thrombin-activatable fibrinolysis inhibitor （TAFIa） activity and have thrombolytic effect. The present study was to screen a strain expressing high-yield of rAcAP5 and to assess its thrombolytic effect on embolic middle cerebral artery occlusion （MCAO） model in rats. Codons encoding for AcAP5 were optimized. Six expression plasmids and eleven E. coli strains with different characteristics were used, a total of 66 recombinant expression strains were generated and the one with the highest yield was selected to express rAcAP5, which was purified through anion- and cation-exchange chromatography. The purity of rAcAP5 and its molecular weight were determined by HPLC and mass spectrometry, respectively. The thrombolytic effect of rAcAP5 was evaluated on embolic MCAO model in rats; regional cerebral blood flow （rCBF） was monitored with a Laser-Doppler flowmetry to test the occlusion and recanalization of MCA. The highest yield recombinant strain was C2566H/pTYB 1-rAcAP5. AcAP5 （28 mg） with 90% of purity was obtained from 1 L of cell culture. In rat embolic MCAO model, vehicle （normal saline） treatment did not change the rCBF, while treatment with rAcAP5 （50-200 μg/kg, i.v.） increased the rCBF in a dose-dependent manner. In conclusion, we prepared and characterized the rAcAP5 peptide and revealed its thrombolytic effect in embolic MCAO model and our results suggested that this peptide had the potential to be used as a thrombolytic agent.
