| BACKGROUNDDendranthema morifolium (Ramat.) Tzvel. cv. Hangju (DMH), belonging to the large chrysanthemum family, is widely used in China, especially in the south, either as a fragrant floral tea or as a cooling herb in traditional Chinese medicine. Components of DMH that have been identified include volatile oils, glycosides, adenine, amino acids, vitamins A and B, flavonoids and polyphenols. We have shown that DMH increases contractility and coronary flow in perfused rat heart, and therefore protects against the reduction in contractility and coronary flow induced by ischemia/reperfusion. However, the possible pharmacological effects of DMH on blood vessels have not been elucidated. We therefore hypothesized that total flavones purified from the aqueous extract of DMH (FDM) might exert a direct vasorelaxant effect, contributing to the increase in coronary flow seen in the isolated perfusedheart. After confirming the hypothesis, we investigated the mechanisms underlying the flavone-induced vasorelaxation.METHODS1. Total flavones extracted from dendranthema morifolium (Ramat.) Tzvel. cv. HangjuDried flowers of chrysanthemum (Dendranthema morifolium (Ramat.) Tzvel. cv. Hangju) were obtained from Tongxiang City, Zhejiang Province. Total flavones (FDM) were extracted by Prof H.D. Jiang of the College of Pharmaceutical Sciences, Zhejiang University.2. Preparation of rat aortic ringsMale Sprague-Dawley rats (240-300g) were killed by stunning followed by cervical dislocation. The thoracic aorta was rapidly removed, cleaned of fat and connective tissue, and cut into 3 mm rings. The rings were mounted in organ chambers containing 10 ml Kreb's solution at 37 , bubbled with 95% O2 and 5% CO2. The rings were equilibrated for 1 h and the Kreb's solution was changed every 15 min. Changes in isometric tension were recorded by force transducers connected to a data acquisition system (PowerLab, AD Instruments). Before each experiment, rings were stimulated with 60 mmol/L KC1 at least 3 times until a reproducible contractile response was obtained. The presence of functional endothelium was verified by the ability of acetylcholine (10-5 mol/L) to induce more than 70% relaxation in rings precontracted with phenylephrine (PE, 10-6 mol/L).3. Vascular smooth muscle cell cultureBriefly, after the rats weighting 120-150g were killed, the aorta was dissected and the adventitia and the endothelium of the aorta were removed. Then the aorta was cut into pieces about Ixlmm. The pieces of the tissue were cultured in the DMEM containing 20% fetal calf serum.After the vascular smooth muscle cells were confluent, 0.25% trypsin with 0.02% EDTA was used to isolate cells from the culture dish. The mixed suspension was centrifuged and the cells in culture bottle were cultured again.4. The colorimetry of MTTThe vascular smooth muscle cells in fifth and sixth generations were transferred to the 96-well plate. After 20 hours of culture, 15 l 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) (5 mg/ml) was added into every well. Four hours later, the culture medium was replaced with DMSO. The absorbency of every well at wave length of 570nm was observed.RESULTS1. Vasorelaxant effect of FDMAfter 10-6mol/L PE induced a steady contraction, FDM was added cumulatively and induced a concentration-dependent relaxation. FDM caused complete relaxation ofendothelium-intact and -denuded aortic rings. Functional removal of the endothelium did not affect the FDM-induced relaxation. Similarly, FDM produced a concentration-dependent reduction of high K+ (60 mmol/L) induced contraction both in endothelium-intact and in endothelium-denuded arteries.2. Effect of FDM on Ca2+-induced contractionCa2+ induced a concentration-dependent contraction of rat aortic rings without endothelium depolarized by 60 mmol/L KC1. Incubation of the aortic rings with 5x10" g/L FDM significantly inhibited the Ca2+-induced contraction.3. Effect of FDM on PE-induced contraction in Ca2+-free solutionIn endothelium-de...
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