Experimental Research Of Total Alkaloids Of Buxus Microphylla Leaves On Aorta Smooth Muscle Of Rats And Its Mechanisms

Objective:To investigate the effects of total alkaloids of buxus microphylla leaves (BML A) on isolated rats thoracic aorta rings. To explore the effective mechanisms in the level of cells.Methods:1. The isolated thoracic aortas being removed from Wistar rats,were cut into3mm rings and were mounted on the organ bath containing10ml Krebs-Henseleit solution at37℃,isometric tension of the rings were measured with BL-410biological function experimental system.The effects of BMLA at different concentration on the contraction of isolated thoracic aorta rings(with and without endothelium)in resting tension,or precontracted with KC1or PE were observed. The effects of BMLA on the dose-effect curves of PE, KC1and CaCl2were also recorded. The isolated thoracic aorta rings were preincubated by BMLA in Krebs-Henseleit solution without calcium,then were added with PE and CaCl2.2. Rats thoracic aortas were separated carefully and cut into fine tissue pieces.These pieces used for culture of vascular smooth muscle cells by method of adhesion to wall.Growing curve of culture cells was drew.Immunocytochemical stain with specific smooth muscle a-actin antibody was applied to identify the culture cells.3. To study the effects of BMLA on proliferation of vascular smooth muscle cells by MTT method.To observe the toxic effects of BMLA on vascular smooth muscle cells by LDH method.4. Using calcium sensitive fluorescence indicator Fura-2, the calcium concentration in isolated vascular smooth muscle cells was determined to investigate the effective mechanisms in the level of cells.Results:1. BMLA did not cause any relaxation or contraction responses in isolated thoracic aorta rings.But in aorta rings precontracted with PE or KC1, BMLA produced concentration-dependent relaxation in both endothelium-intact and denuded rings, and there was no difference in the inhibition between the endothelium-intact and denuded rings at the same concentration. BMLA induced greater relaxation than CVB-D, and there were no difference between acetic acid groups and controls. Exposure of isolated thoracic aorta rings to BMLA64、256μg/ml caused a significant reduction in the contractile response of PE、KCl and CaCl2,and shift their cumulative concentration-response curves rightward.2. BMLA inhibited not only the intracellular calcium release contraction induced by PE, but also the extracellular calcium influx contraction induced by PE. BMLA induced greater relaxation than the same concentration of CVB-D.3. The culture technique of tissue explants adherent method was used. After7to10days,vascular smooth muscle cells emigrated around the tissue explants and reached confluence after20days with a "hill-and-valley" pattern.There were lots of stained buffy bundles in cell cytoplasm after immunocytochemical stain, and almost all cells had those characters.4. Compared with control group, the low,middle and high dosage group significantly inhibited cells proliferation by MTT method.Using LDH method, compared with control group, the low dosage group significantly decreased LDH level which indicated it could protect cells, and there were no significant difference between the middle dosage group and control one. While the high dosage of BMLA had high LDH level which indicated that over high dosage of BMLA may damage cells.5. BMLA could significantly inhibit the extracellular calcium influx induced by PE and KCl under extracellular calcium concentrations1.5mmol/L, inhibition ratio was40.2%and49.9%.Under extracellular calcium concentrations Ommol/L, BMLA could significantly inhibit the intracellular calcium release induced by PE, inhibition ratio was72.4%,but had no significant difference on the intracellular calcium release induced by caffeine.Conclusion:1. BMLA produced significant concentration-dependent relaxation in aorta rings which were precontracted with PE or KCl.BMLA caused a significant reduction in the contractile response of PE、KCl and CaCl2,and shift their cumulative concentration-response curves rightward.2. BMLA can relax thoracic aorta vascular smooth muscle, and the mechanisms may be related to inhibiting [Ca2+]i of thoracic aorta vascular smooth muscle cells, which mainly occurs via the suppression of influx of extracellular calcium by voltage-dependent calcium channel and receptor-operated calcium channel and inhibition of IP3-sensitive intracellular stored calcium release.