Effect Of Doxazosin And Its Enantiomers On The Proliferation Cycle And Apoptosis In The Rat Aortic Smooth Muscle Cells

Atherosclerosis (AS) is harmful to human health. In recent years, the incidence of this disease is obvious upward in our country. And restenosis (RS) is a thorny matter after the surgery of percutaneous transluminal coronary angioplasty (PTCA). It followed to proliferation of vascular smooth muscle cells(VSMCs). Cell cycle is the last passageway of cell proliferation. Many researches have indicated that apoptosis of VSMCs played more and more important role in AS and RS. Over- proliferation and apoptosis on VSMCs are not only the major basis of pathology that AS forms but also the important causes that RS forms. Therefore, adjusting apoptosis of VSMCs may become a way that it is able to prevent AS and RS. Doxazosin (racemic-doxazosin, (±)Doxazosin) is a highly selectiveα1-adrenoceptor antagonist, Some tests have showed that (±)doxazosin prevented possibly the formation of RS. But it also produces several side effects in cardiovascular system. It was reported that (-)doxazosin and (+)doxazosin were prepared using chiral mobile phase HPLC. We have suggested in our preceding experiment that (±)doxazosin and its enantiomers could inhibit proliferation in the rat aortic VSMCs, but also had chiral selective effects, that had nothing to do with theα1-adrenoceptor. In the experiments, We explore the mechanism of (±)doxazosin and its enantiomers on the proliferation inhibition in the rat aortic VSMCs using Giemsa and flow cytometry methods.Objective: To culture the rat aortic VSMCs in vitro, observe the effect of (-)doxazosin, (+) doxazosin and (±) doxazosin on the cell cycle and apoptosis in the rat aortic VSMCs, analyse the proliferation inhibition mechanism of (±) doxazosin and its enantiomers in VSMCs.Methods: Sprague-Dawley (SD) rat aortic VSMCs was cultured in vitro, by the passage and synchronization, Then, We gave respectively (-)doxazosin, (+)doxazosin and (±)doxazosin (30μmol.L-1 and 25μmol.L-1), and cultivated them in the sterile bottle for 96 h and 72 h. We observe morphological changes of apoptosis using Giemsa, and we determined the apoptosis rate and values of Bcl-2 and Bax protein of the rat aortic VSMCs treated by (-)doxazosin, (+)doxazosin and (±) doxazosin using flow cytometry(FCM).Results:1 Effects of (-)doxazosin, (+)doxazosin and (±)doxazosin on VSMCs proliferation cycleAfter VSMCs was treated by (-)doxazosin, (+)doxazosin and (±)doxazosin(25μmol.L-1) for 72 h, cell cycle distribution and proliferation index occurred some changes: compaired to the solvent group, G0/G1 phase cells in (±)doxazosin and (-)doxazosin group significantly increased (P<0.01), and S phase cells, G2/M phase cells and proliferation index were significantly lower (P<0.01). The treatment group of (+)doxazosin only S phase cells decreased significantly (P<0.01). Compairing with (-)doxazosin treatment group, G0/G1 phase cells of (+) doxazosin group decreased significantly (P<0.01), S phase cells, G2/M phase cells and proliferation index significantly increased (P<0.05), and (±)doxazosin treatment group were not significantly different.2 Effects of apoptosis induced by (±)doxazosin and its enantiom- ers on the rat aortic VSMCsThe VSMCs that were treated by(-)doxazosin, (+)doxazo -sinand (±)doxazosin for 96 h displayed morphological changes of apoptosis, the role of (-)doxazosin was stronger than of (+)doxazosin. The apoptosis rates of VSMCs treated by (-)doxazosin, (+)doxazosin and (±)doxazosin for 72 h were 6.06±0.31%, 1.36±0.30% and 3.63±0.99% measured by FCM machine. (-)Doxazosin and (±)doxazosin could induce apoptosis of VSMCs. The apoptosis rates by (-)doxazosin was significantly higher than that by (+)doxazosin and (±)doxazosin (P<0.01).3 Effects of (±)doxazosin and its enantiomers on expressin of Bcl-2 and Bax protein in the rat aorta VSMCsThe rat aorta VSMCs were treated by (-)doxazosin, (+) doxazosin and (±)doxazosin for 72 h, FCM machine measured that the fluorescence index(FI) of Bcl-2 protein were 10.83±4.30, 9.50±1.22 and 8.53±2.20, that of Bax protein were 15.77±6.00, 10.33±1.12 and 10.30±1.83. With the solvent control group compared to the experimental group, there were not significant difference on expressin of Bcl-2 and Bax protein (P>0.05).4 Effects of (±)doxazosin and its enantiomers on Bcl-2/Bax value in the rat aorta VSMCsBcl-2/Bax protein ratio of VSMCs that were treated by (-) doxazosin, (+)doxazosin and (±)doxazosin for 72 h were 0.68±0.01, 0.92±0.03 and 0.82±0.10. With the solvent control group (0.99±0.06) compared to the experimental group, (-)doxazosin and (±)doxazosin reduced significantly the ratio of Bcl-2/Bax protein (P<0.01 and P<0.05), and there was also significant difference compared to (+)doxazosin (P<0.05).Conclusions: (±)doxazosin and its enantiomers can inhibit the transformation from G0 to S on the proliferation cycle in VSMCs, and also have chiral selective effect. The inhibition by (-)doxazosin is stronger than that by (+)doxazosin, but the role is the same to the (±)doxazosin. VSMCs is treated by (-)doxazosin, (+)doxazosin and (±)doxazosin, displaying morphological changes of apoptosis, (±)doxazosin and its enantiomers are able to induce the apoptosis of the rat aorta VSMCs, and also have chiral selective effect. apoptosis by (-)doxazosin is stronger than that by (+)doxazosin, compared to (±)doxazosin was no significant difference. The mechanism that apoptosis induced by (-)doxazosin, (+)doxazosin and (±)doxazosin in the rat aorta VSMCs is probably related to decreasing the ratio of Bcl-2/Bax protein.