Chiral Selective Effects Of Doxazosin And Its Enantiomers On Cell Proliferation In Cultured Vascular Smooth Muscle Cells Of The Rat Aorta

Objective: vascular smooth muscle cells (VSMCs) proliferation is the common pathological basis of high blood pressure,atherosclerosis and percutaneous coronary angioplasty (PTCA)restenosis and other diseases .Probably inhibiting VSMC proliferation is one of the effective ways to prevent the formation of atherosclerotic plaque and restenosis after PTCA .Doxazosin is a new type of long-lasting and strong selectiveα1 adrenergic receptor antagonist ,having functions of controlling blood pressure, improving microcirculation and lipid metabolism, and has become the first-line drugs that treatment benign prostatic hyperplasia(BPH) complicated with lower urinary tract symptoms (LUTS).Their adverse reactions are less and safety. Doxazosin handle separation into L doxazosin [(-) doxazosin] and R doxazosin [(+) doxazosin].Our research results show that (-) doxazosin may be the drug, better than (+) doxazosin, (±)doxazosin on the treatment of BPH / LUTS.But the role of vascular smooth muscle cells is unclear. Therefore, to explore doxazosin and its enantiomers in rat vascular smooth muscle cells of the inhibitory effect of proliferation is necessary.Methods: Choosing 120 ~ 150g healthy SD rats, using tissue block affixed culture method to culture rat aortic vascular smooth muscle cells, trypsin digestion method subcultured, and applicating phase-contrast microscopy and Mouse monoclonal anti-smooth muscleα-actin antibody on cell morphology and immunohistochemical identification, according to cell growth rendering cell growth curve, and using MTT colorimetric assay to determine different concentrations of (-) doxazosin, (+) doxazosin and (±) doxazosin on vascular smooth muscle cell proliferation. Measured by flow cytometry (-) doxazosin, (+) doxazosin and (±) doxazosin on vascular smooth muscle cell proliferation cycle. Observed under an optical microscope the morphological changes of VSMCs by treatment of (-) doxazosin, (+) doxazosin and (±) doxazosin.1. VSMC morphology observation and immune cytochemical identification: under the inverted phase contrast microscope single smooth muscle cells are spindle or ribbon, having a number of cell processes, abundant cytoplasm, oval central nuclear,and there is one or more nucleoli. Dense cell growth expressed as a typical "Valley - peak" shape growth.α-actin that the chemical reaction of immune cells showed that, VSMC were stained brown-yellow spindle-type cells, and the cytoplasm of cells along the longitudinal axis arranged in dense bundles filaments were dyed brown.2. VSMC growth curve: VSMC growth curve is similar to "S" shape, one day after generation of cells has decreased, then enter the logarithmic phase, peaked in the fourth days, and then enter the plateau.3. (-) Doxazosin, (+) doxazosin and (±) doxazosin on VSMC proliferation: comparison with the solvent group, 10 ~ 30mmol·L-1 concentration of (-) Doxazosin and (±) doxazosin role in VSMCs 48h or 72h, significantly inhibited the proliferation of rat aortic VSMCs; and (+)doxazosin alone at 30 mmol·L-1 with inhibition. With the extension of the role of drugs on VSMCs to 96h, (-) doxazosin, (+) doxazosin and (±) doxazosin at 3~30 mmol·L-1 concentrations significantly inhibit the proliferation of aortic VSMCs; However, (-) doxazosin inhibition is significantly stronger than the same concentration (+) doxazosin (n = 6, P <0.05 or 0.01), with the same concentration of (±) doxazosin inhibition was no significant difference. Different concentrations of (-) doxazosin, (+) doxazosin and (±) doxazosin role on rat thoracic aorta VSMCs 48h, 72h, 96 h, and (-) doxazosin, (+) doxazosin and (±) doxazosin on VSMCs proliferation have a dose-dependent manner. The drugs of same concentrations treatment in rat thoracic aorta VSMCs 48h, 72h and 96h, then at 96h, inhibition rates of the three drugs on aortic VSMCs proliferation were significantly higher than 48h (n = 6, p <0.05 or 0.01) and 72h (n = 6, p <0.05 or 0.01) . The drugs of different concentrations treat with rat thoracic aorta VSMCs after 96 h, then the IC40 of(+) doxazosin was significantly greater than (±) doxazosin and (-) doxazosin (n = 3, P <0.01).4. Effects of (±)doxazosin and its enantiomers on cell proliferation in cultured VSMCs treated with phenoxy -enzamine:After VSMCs were treated by phenoxybenzamine, (-)doxazosin, (+)doxazosin and (±) doxazosin were also able to significantly inhibit the proliferation of the rat aortic VSMCs. Inhibition rates of (-)doxazosin, (+)doxazosin and (±)doxazosin on the rat aortic VSMCs were 33.24±7.79%, 24.12±10.14% and 30.60±5.11%. There was the trend of role that (-)doxazosin was stronger than (+)doxazosin, but there were not significant difference in the three drugs(P>0.05). The values of OD490nm were 0.24±0.03,0.27±0.04 and 0.25±0.02. After VSMCs were treated by SolventsⅠ( double distilled water), solventⅡ(alcohol) and phenoxybenzamine, OD490nm values of (-)doxazosin were 0.23±0.01,0.24±0.03 and 0.24±0.03, inhibition rates were 33.00±2.79%, 32.87±7.20% and 33.24±7.79%. There were not significant difference in them. Result of (+)doxazosin and (±)d- oxazosin were also usual.5. (-) Doxazosin, (+) doxazosin and (±) doxazosin on cell morphology of VSMC: After VSMCs have been treated by (-) Doxazosin, (+) doxazosin and (±) doxazosin, we can see that cell morphological changes, showing polygonal, with the seperation from the adjacent cells, and the cells have small granules within the emergence of cell wall thickening outline a clear, partly spherical cell shrinkage.Conclusion:It is the first study of doxazosin[(±) doxazosin]and its enantiomer to rat aortic VSMCs proliferation, the following conclusions: (±)Doxazosin,(+)doxazosin和 (-)doxazosin can inhibite cell proliferation in cultured VSMCs of the rat aorta, and have chiral selective effects . The inhibition of cell proliferation by (-)doxazosin is significantly stronger than that by (+)doxazosin,and the Inhibition effect and mechanism of doxazosin have nothing to do with theα1-adrenoceptor.