Improvement and Application of Acute Blood Stasis Rat Model Aligned with the 3Rs (Reduction, Refinement and Replacement) of Humane Animal Experimentation.

OBJECTIVE: To establish a novel cardiocentesis method for withdrawing venous blood from the right atrium, and to improve an acute blood stasis rat model using an ice bath and epinephrine hydrochloride (Epi) while considering the 3Rs (reduction, refinement, and replacement) of humane animal experimentation. METHODS: An acute blood stasis model was established in male Sprague-Dawley rats by subcutaneous injection (s.c.) Epi (1.2 mg/kg) administration at 0 h, followed by a 5-min exposure to an ice-bath at 2 h and s.c. Epi administration at 4 h. Control rats received physiological saline. Rats were fasted overnight and treated with Angelicae Sinensis Lateralis Radix (ASLR) and Pheretima the following day. Venous blood was collected using our novel cardiocentesis method and used to test whole blood viscosity (WBV), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) content. RESULTS: The rats survived the novel cardiocentesis technique; WBV value returned to normal while hematological parameters such as hemoglobin level and red blood cell count were restored to >94% of the corresponding values in normal rats following a 14-day recovery. Epi (1.2 mg/kg, s.c.) combined with a 5-min exposure to the ice bath replicated the acute blood stasis rat model and was associated with the highest WBV value. In rats showing acute blood stasis, ASLR treatment [4 g/(kg·d) for 8 days] decreased WBV by 9.98%, 11.09%, 9.34%, 9.00%, 7.66%, and 7.03% (P<0.05), while Pheretima treatment [2.6 g/(kg·d), for 8 days] decreased WBV by 25.49%, 25.94%, 16.28%, 17.76%, 11.07%, and 7.89% (P<0.01) at shear rates of 1, 3, 10, 30, 100, and 180 s-1, respectively. Furthermore, Pheretima treatment increased APTT significantly (P<0.01). CONCLUSIONS: We presented a stable, reproducible, and improved acute blood stasis rat model, which could be applied to screen drugs for promoting blood circulation and eliminating blood stasis.

Calcium channel blocking activity of calycosin, a major active component of Astragali Radix, on rat aorta.

AIM: To investigate the vasoactivity of calycosin, a major active component of Astragali Radix. METHODS: Experiments were performed on isolated rat thoracic aortic rings pre-contracted with phenylephrine (PHE) or KCl. RESULTS: Calycosin produced a concentration-dependent relaxation on the tissue pre-contracted using PHE with 4.46+/-0.13 of pD(2) and 95.85%+/-2.67% of E(max); or using KCl with 4.27+/-0.05 of pD2 and 99.06%+/-2.15% of Emax, and displaced downwards the concentration-response curves of aortic rings to PHE or KCl. The relaxant effect of calycosin on denuded endothelium aortic rings was the same as on intact endothelium aortic rings, and its vasorelaxant effect was not influenced by L-NAME or indomethacin. In Ca(2+)-free solution, calycosin (30 micromol/L) did not have an effect on PHE (1 micromol/L)-induced aortic ring contraction. The effects of calycosin and nifedipine where somewhat different; calycosin decreased aortic ring contractions induced by the two agonists, but nifedipine displayed a more potent inhibitory effect on KCl-induced contractions than on PHE-induced contractions, and the vascular relaxing effects of calycosin and nifidipine were additive on PHE-induced contraction but not KCl-induced. CONCLUSION: Calycosin is a vasorelaxant. Its action is endothelium-independent and is unrelated to intracellular Ca(2+) release. It is a noncompetitive Ca(2+) channel blocker. The effect of calycosin on Ca(2+) channel blockade may be different from that of dihydropyridines. This study demonstrated a novel pharmacological activity of calycosin, and supplied a theoretic foundation for Astragali Radix application.

Honokiol inhibits arterial thrombosis through endothelial cell protection and stimulation of prostacyclin.

AIM: To study the effect of honokiol on arterial thrombosis and endothelial cells. METHODS: Rabbit platelet aggregation was performed with Borns turbid method. Thrombosis was produced by the endothelial injury stimulated with electric current. Rat aortic endothelial cells (RAEC) were cultured and cell viability was assessed using the MTT assay. Nitric oxide (NO) concentrations in serum-free media of RAEC were determined using the kinetic cadmium-reduction method. The stable metabolite prostacyclin was measured in serum-free media of RAEC by radioimmunoassay. RESULTS: Honokiol (37.6-376 micromol/L) decreased rabbit platelet aggregation in vitro in a concentration-dependent manner, while intravenously injection of honokiol (0.12-12 microg/kg) significantly inhibited rabbit platelet aggregation induced by collagen ex vivo. In the electrical current-stimulated carotid thrombosis model in rats, honokiol (5-50 microg/kg, iv) prolonged the thrombus occlusion time in a does-dependent manner. In vitro honokiol (0.376-37.6 micromol/L) effectively protected cultured RAEC against oxidized low density lipoprotein (ox-LDL) injury, and significantly increased 6-keto-PGF1alpha (the stable metabolite of prostacyclin) in serum-free media of RAEC. Honokiol also increased NO level in RAEC serum-free medium at a lower concentration range (0.0376-0.376 micromol/L), but honokiol 3.76 micromol/L decreased NO level. CONCLUSION: Honokiol is a potent arterial thrombosis inhibitor. Endothelial cell protection and the stimulation of prostacyclin release may be its main anti-thrombosis mechanism. Stimulation of NO release in endothelial cells may play a role, but it is not a key factor.

Inhibition of platelet aggregation by polyaspartoyl L-arginine and its mechanism.

AIM: To observe the oral anti-platelet efficacy and the potential action mechanism of polyaspartoyl L-arginine (PDR), a new L-arginine rich compound. METHODS: Platelet aggregation was conducted by Born's method; bleeding time was determined using tail's bleeding time in mice; platelet adhesion was carried out with glass bottle method; nitric oxide (NO) was tested with Griess's method; and cAMP, thromboxane B(2) (TXB(2)) and 6-keto-PGF(1 alpha ) were assessed with commercial kits. RESULTS: The inhibition by PDR (15-60 mg/kg i.g. or 10 mg/kg i.v.) of platelet aggregation induced by adenosine diphosphate (ADP), collagen or thrombin at 1 h after oral administration or at 20 min after i.v. injection for rats (P<0.01), and its (15 mg/kg, i.g.) inhibition of ADP-induced platelet aggregation for rabbits during 6 h after administration were observed. PDR (15-60 mg/kg) prolonged the bleeding time of mice (P<0.05) and (30 mg/kg) increased NO concentration in plasma. On the other hand PDR did not change the contents of cAMP in platelet and TXB2 or 6-keto-PGF(1 alpha) in plasma. CONCLUSION: PDR is a novel, oral effective platelet aggregation inhibitor and its action mechanism possibly related to increasing NO generation.

[Studies on anti-inflammatory and immunologic enhancement of kangning granules].

OBJECTIVE: To investigate the effect of Kangning granules on inflammatory and immunology. METHOD: Pedal swelling induced by 1% agar in mice, dermal capillary hyper-permeability induced by histamine in rats were used to evaluate the anti-inflammatory of Kangning granules, and the clearance rate of intravenous charcoal particles was used to study its effect on non-specific immunologic functions. Serum haemolysin level and the number of antibody secretory cells in spleen were determined to assess its effects of specific immunologic functions. MTT colored method was used to determine the effect of Kangning granules on T- or B-Lymphocytes proliferation. RESULT: 5, 10, 20 g x kg(-1) of Kangning Granules significantly inhibited pedal swelling induced by agar in mice (P < 0.05, P < 0.01). 2.4, 4.8, 9.6 g x kg(-1) of Kangning Granules evidently decreased dermal capillary hyper-permeability induced by histamine in rats (P < 0.05, P < 0.01). 10, 20 g x kg(-1) of Kangning Granules enhanced the phagocytic function and phagocytosing velocity in mice (P < 0.05). 5, 10, 20 g x kg(-1) of Kangning Granules obviously increased serum haemolysin level and the number of antibody secretory cells in spleen (P < 0.05, P < 0.01). 20 g x kg(-1) of Kangning Granules promoted B-Lymphocytes proliferation (P < 0.05). CONCLUSION: Kangning granules significantly inhibited inflammatory reaction and increase immune functions in animals.

[Variation of endogeneous ET, CGRP and NO in myocardial ischemia in rats and the regulatory effect of xinshuping].

OBJECTIVE: To observe the variation of ET and CGRP contents in ischemic heart, and NO level in serum of myocardial damaged rats, and their regulation when with the protection of Xinshuping, a traditional Chinese medicine compound. METHOD: The models of myocardial ischemia were prepared by subcutaneous injection of isoproterenol or by ligation of coronary artery. RESULT: ET content in myocardium was significantly increased (P < 0.01), and CGRP content as well as NO level in serum was not changed obviously in the model induced by isoproterenal. However, NO level in serum of rats treated with Xinshuping (ig bid x 2.5 d) was markedly raised (P < 0.01), neither ET not CGRP contents were affected by it. LDH and CK levels in serum of rats were evidently lowered by Xinshuping treatment. S-T segment's elevation of ECG was significantly inhibited and myocardial infarction size was reduced markedly by Xinshuping treatment in rats subjected to coronary artery ligature. CONCLUSION: ET, CGRP or NO is involved in myocardial infarction caused by isoproterenol. The ischemic damage or dysfunction in different models is obviously protected by Xinshuping. The promotion of NO release from vascular endothelium is probably related with this protective effect.