Effects Of Doxazosin And Its Enantiomers On Serum Lipid Levels And Histopathological Changes In Rabbits Fed An Atherogenic Diet And In Aged Mice

Lipid disorder is a major risk factor of cardiovascular disease. With improvement in living standards, large amounts of fatty acids are taken in, which promotes the increases in total serum cholesterol (TC) and low density lipoprotein cholesterol (LDL-C). Clinical evidences show that the elevated TC and LDL-C levels are closely related to the cardiovascular diseases, and the elevated TC level is one of the most important risk factors for atherosclerosis (AS) and coronary arterial diseases. Decreases in the levels of TC, TG and LDL-C and an inecrease in HDL-C level could be helpful in protecting the formation and development of AS.Doxazosin (DOX), a long lasting selective antagonist ofα1- adrenoceptors, has been widely used in treatment of hypertension and benign prostatic hyperplasia (BPH), and its racemic form of (±)DOX has been used in clinical practice at present. Basic and clinical experiments have proved that (±)DOX is able to antagonize the contractile responses to noradrenaline in vascular smooth muscle. In the treatment of BPH, adverse effects induced by (±)DOX were reported such as dizziness, postural hypotension and syncope. It was reported that (?)doxazosin [(?)DOX] and (+)doxazosin [(+)DOX] were prepared using chiral mobile phase HPLC and high performance capillary electrophoresis. The blocking effect of (?)DOX onα1-adrenoceptor was significantly weaker than that of (±)DOX and (+)DOX in the isolated rabbit thoracic aorta, carotid artery and mesenteric artery. The effect of (?)DOX on arterial blood pressure in the anesthetized rats was also significantly weaker than that of (±)DOX and (+)DOX administered intravenously or intraduo- denally. However, the effect of decreasing urinary bladder pressure by (?)DOX in the anesthetized rats and guinea-pig was the same as that of (±)DOX. These findings suggested that the pharmacological activity of (?)DOX had chiral selective effect between the cardiovascular system and lower urinary tract system, and (?)DOX might become a potential agent for the treatment of BPH/LUTS.It was reported that (±)DOX decreased serum TC, TG and LDL-C levels, and inhibited the aortic fatty streak formation in the hypercholesterolemic hamster model. In the rabbit fed an atherogenic diet, (±)DOX administered by oral gavage daily at 5mg for 9 wk did not affect the serum lipid levels significantly, but it was able to protect the arteries from atherosclerosis.González-Juanatey and colleagues reported that (±)DOX induced cell apoptosis in the primary rat cardiomyocytes, primary neonatal rat atrial myocytes and HL-1 mouse cardiomyocytes, and the apoptosis induction by (±)DOX was not dependent on its effect ofα1-adrenoceptor blockade.So far it is not clarified whether (-)DOX and (+)DOX affect the serum lipid levels and histopathological changes in arteries, heart and kidney in the rabbits fed an atherogenic diet and serum lipid levels in the aged mice. In the present study, the hyperlipidemic rabbits and aged mice were used to observe the effects of (-)DOX, (+)DOX and (±)DOX on serum lipid levels and animal mortality. Histopathological changes in arteries, heart and kidney were observed in the rabbit fed an atherogenic diet, and cell proliferation and apoptosis of the heart ventricle of the hyperlipidemic rabbits were detected by flow cytometry (FCM).PartⅠEffect of doxazosin and its enantiomers on serum lipid levels and animal mortality in rabbits fed an atherogenic dietExcept for the rabbits in normal diet group, all the other animals were fed an atherogenic diet. Serum lipids were determined in the rabbits fed a normal or atherogenic diet for 4 wk. Then, the rabbits fed an atherogenic diet were randomly divided into 4 groups (10 rabbits per group) according to their serum TC level, except for the animals whose serum TC level less than 10 mmol?L-1. Four groups included ather- ogenic diet group, atherogenic diet with (-)DOX group, atherogenic diet with (+)DOX group and atherogenic diet with (±)DOX group. Ten rabbits fed a normal diet were taken as the normal diet group in the study at the same time. Effects of (-)DOX, (+)DOX and (±)DOX on serum lipid levels and animal mortality were observed.1 Effects of doxazosin and its enantiomers on rabbit body weightBody weights of the rabbits before atherogenic diet feeding and before administration of (-)DOX, (+)DOX and (±)DOX in the 5 experimental groups were not significantly different from each other (P>0.05). Body weights of the rabbits after 3wk, 6wk and 9wk of daily administration of the three agents in the 5 experimental groups were not significantly different from each other as well (P>0.05).Body weights of the rabbits after 4~13wk of normal diet, 7~13wk of atherogenic diet, 4~13wk of atherogenic diet with (+)DOX, and 10~13wk of atherogenic diet with (±)DOX were increased significantly (P<0.05 and P<0.01) in comparison with the body weights before atherogenic diet feeding. However, rabbit body weight in the group of atherogenic diet with (-)DOX was not significantly increased during 13wk of atherogenic diet feeding (P>0.05).2 Effects of doxazosin and its enantiomers on rabbit mortalityDuring the experimental period, one rabbit died in normal diet group (mortality rate, 10%), 4 rabbits died in atherogenic diet group (mortality rate, 40%), and the mortality rates were 10% to 30% in the groups treated with (-)DOX, (+)DOX and (±)DOX. Although mortality rates in the drug-treated groups did not change significantly in comparison with atherogenic diet group (P>0.05), the mortality rates in the drug-treated groups were lower than the mortality rate in atherogenic diet group. The mortality rate in the group of atherogenic diet with (±)DOX was the same as that in normal diet group.3 Effects of doxazosin and its enantiomers on serum lipid levels of rabbits fed an atherogenic diet3.1 Effects of doxazosin and its enantiomers on serum TC levels of rabbits fed an atherogenic dietSerum TC levels of the rabbits before atherogenic diet feeding in the 5 experimental groups were not significantly different from each other (P>0.05). Serum TC levels of the rabbits before administration of (-)DOX, (+)DOX and (±)DOX and after 3wk, 6wk and 9wk of daily administration of the three agents (including atherogenic diet group) were significantly elevated in comparison with serum TC level of the rabbits fed a normal diet (P<0.01). However, serum TG levels of the rabbits in atherogenic diet with (-)DOX group, atherogenic diet with (+)DOX group and atherogenic diet with (±)DOX group were not significantly different from the serum TG level of the rabbits in atherogenic diet group (P>0.05).Serum TC levels of the rabbits after 10~13wk of atherogenic diet and 7~13wk of atherogenic diet with (-)DOX were further increased significantly (P<0.01) in comparison with those of the rabbits before administration of solvent and (-)DOX, respectively. On the other hand, serum TC levels of the rabbits after 7~13wk of atherogenic diet with (+)DOX and 7~13wk of atherogenic diet with (±)DOX did not change significantly (P>0.05) in comparison with those of the rabbits before administration of (+)DOX and (±)DOX, respectively.3.2 Effects of doxazosin and its enantiomers on serum TG levels of rabbits fed an atherogenic dietSerum TG levels of the rabbits before atherogenic diet feeding in the 5 experimental groups were not significantly different from each other (P>0.05). Serum TG levels of the rabbits before administration of (-)DOX, (+)DOX and (±)DOX and after 3wk, 6wk and 9wk of daily administration of the three agents (including atherogenic diet group) were significantly elevated in comparison with serum TG level of the rabbits fed an normal diet (P<0.01). However, serum TG levels of the rabbits in atherogenic diet with (-)DOX group, atherogenic diet with (+)DOX group and atherogenic diet with (±)DOX group were not significantly different from the serum TG level of the rabbits in atherogenic diet group (P>0.05).Serum TG levels of the rabbits after 7~13wk of normal diet were decreased significantly (P<0.05 and P<0.01) in comparison with serum TG level of the rabbits before administration of solvent. On the other hand, serum TC levels of the rabbits after 7~13wk of atherogenic diet, 7~13wk of atherogenic diet with (-)DOX, 7~13wk of atherogenic diet with (+)DOX and 7~13wk of atherogenic diet with (±)DOX did not change significantly (P>0.05) in comparison with those of the rabbits before administration of solvent, (-)DOX, (+)DOX and (±)DOX, respectively.3.3 Effects of doxazosin and its enantiomers on serum HDL-C levels of rabbits fed an atherogenic dietResults from statistical comparisons among the 5 groups of the data before atherogenic diet feeding and of the data after administration of the three agents indicate that changes in serum HDL-C levels were similar to those in serum TG levels mentioned above.Results from the statistical comparison between the data obtained before and after administration of (-)DOX, (+)DOX, (±)DOX and solvent indicate that changes in serum HDL-C levels were similar to those in serum TG levels mentioned above except for a significant decrease in serum HDL-C level after 6wk of daily administration of (±)DOX (P<0.05).3.4 Effects of doxazosin and its enantiomers on serum LDL-C levels of rabbits fed an atherogenic dietResults from statistical comparisons among the 5 groups of the data before atherogenic diet feeding and of the data after administration of the three agents indicate that changes in serum LDL-C levels were similar to those in serum TG levels mentioned above.Serum LDL-C levels of the rabbits after 7~13wk of normal diet were decreased significantly (P<0.01) in comparison with serum LDL-C level of the rabbits before administration of solvent. On the other hand, serum LDL-C levels of the rabbits after 7~13wk of atherogenic diet and 10wk of atherogenic diet with (±)DOX were increased significantly (P<0.05 and P<0.01) in comparison with those of the rabbits before administration of solvent and (±)DOX, respectively. However, serum LDL-C levels of the rabbits after 7~13wk of atherogenic diet with (-)DOX and 7~13wk of atherogenic diet with (+)DOX did not change significantly (P>0.05) in comparison with those of the rabbits before administration of (-)DOX and (+)DOX, respectively.PartⅡEffects of doxazosin and its enantiomers on histopathological changes in aorta and kidney in rabbits fed an atherogenic dietThe effects of (±)DOX and its enantiomers on histopathological changes in thoracic and abdominal aorta and in kidney of the rabbits fed an atherogenic diet were observed, and a possible mechanism underlying the decreased mortality in hyperlipemic rabbits was investigated.1 Effects of doxazosin and its enantiomers on morphological changes in the thoracic aorta of rabbits fed an atherogenic dietMorphological abnormalities had not been seen in the thoracic aorta of rabbits fed a normal diet. In the rabbits fed an atherogenic diet for 13wk, the luminal surface of intima of thoracic arota became rough, and AS plague could be seen clearly in different sizes. The ratio of aortic plaque area to total intima area was examined, and the percentage of total plaque area was 75.4±17.21% in the rabbits fed an atherogenic diet for 13wk. Percentages of total plaque area in the rabbits treated with (-)DOX, (+)DOX and (±)DOX were significantly decreased in comparison with the value in the rabbits fed an atherogenic diet (P<0.05 and P<0.01). There was no significant difference in percentages of total plaque area among the three groups treated with (-)DOX, (+)DOX and (±)DOX (P>0.05).2 Effects of doxazosin and its enantiomers on histomorphological changes in the thoracic aorta of rabbits fed an atherogenic dietHistomorphological abnormalities had not been seen in the thoracic aorta of rabbits fed a normal diet under an ordinary optical microscope. In 6 rabbits fed an atherogenic diet for 13wk, typical atherosclerotic lesions in the thoracic aorta were found in 3 rabbits, such as arterial wall thickness, intima thickness, roughness of the luminal surface, endothelial cell damage, foam cells accumulated in the subintimal space, decrease of acidophilic cytoplasm SMCs in the tunica media and disorganized smooth muscle orientation. Fibrous plague was found in 2 rabbits, and fatty streak was found in one rabbit. Results from the statistical analysis (Ridit test) indicate that (-)DOX and (+)DOX significantly reduced the formation of fibrous plaque and atherosclerotic plaque.3 Effects of doxazosin and its enantiomers on histomorphological changes in the abdominal aorta of rabbits fed an atherogenic dietHistomorphological abnormalities had not been seen in the abdominal aorta of rabbits fed a normal diet under an ordinary optical microscope. In 6 rabbits fed an atherogenic diet for 13wk, atherosclerotic lesions in the abdominal aorta were found in one rabbits, such as intima thickness, roughness of the luminal surface, foam cells accumulated in the subintimal space, decrease of acidophilic cytoplasm SMCs in the tunica media and disorganized smooth muscle orientation. A few of fibrous plaques were found in 5 rabbits. Results from the statistical analysis (Ridit test) indicate that (-)DOX significantly reduced the formation of heavy atherosclerotic plaque.4 Effects of doxazosin and its enantiomers on histomorphological changes in the kidney of rabbits fed an atherogenic dietHistomorphological abnormalities had not been seen in the kidney of rabbits fed a normal diet under an ordinary optical microscope. In 6 rabbits fed an atherogenic diet for 13wk, lipids were accumulated in the renal tubular epithelial cells in all animals, and ranular degeneration and vacuolar degeneration of the tubular epithelial cells were observed. A large number of foam cells were found in renal interstitium accompanying with necrotic fibrous tissue. Other pathological changes included the glomeralar enlargement, foam cell formation in glomerular mesangial cells, and thickening or stiffening of capillary wall. Results from the statistical analysis (Ridit test) indicate that (-)DOX and (±)DOX significantly inhibited the foam cell formation in renal interstitium, and (±)DOX had a protective effect on renal tubular epithelial cells in the rabbits fed an atherogenic diet.PartⅢEffects of doxazosin and its enantiomers on cell cycle and apoptosis of the ventricular heart cells and histopathological changes of heart ventricle in rabbits fed an atherogenic dietCell cycle and apoptosis in the ventricular heart cells detected by flow cytometry and histopathological changes in heart ventricle were investigated in the rabbits fed an atherogenic diet for 13wk. Then, the effects of (±)DOX and its enantiomers on cell cycle and apoptosis in the ventricular heart cells and on histopathological changes of heart ventricle were investigated in the rabbits fed an atherogenic diet for 13wk.1 Effects of doxazosin and its enantiomers on cell cycle and apoptosis of the ventricular heart cells in rabbits fed an atherogenic dietThe proportion of cells in G0/G1 phase was increased significantly (P<0.05), and that in S phase and PI value were decreased significantly (P<0.05) in the rabbit fed an atherogenic diet for 13wk in comparison with those in the rabbits fed an normal diet. Although the proportion of cells in G2/M phase was decreased, there was no significant difference between the two groups (P>0.05).The proportions of cells in G0/G1 phase and S phase and PI values of the ventricular heart cells in the rabbits after 7~13wk of atherogenic diet with (-)DOX or (±)DOX were significantly different from that in the rabbits fed an atherogenic diet (P<0.05 and P<0.01), and reached the levels in the rabbits fed a normal diet. The proportions of cells in G2/M phase of the ventricular heart cells in the rabbits after 7~13wk of atherogenic diet with (-)DOX or (±)DOX were significantly increased in comparison with that in the rabbits fed an atherogenic diet (P<0.05). There were no significant changes in cell cycle and PI value of the ventricular heart cells in the rabbits fed an atherogenic diet with (+)DOX in comparison with that in the rabbits fed an atherogenic diet (P>0.05). Cell apoptosis and apoptotic rate of the ventricular heart cells in the rabbits treated with the three agents and in the rabbits fed an atherogenic diet did not change significantly in comparison with that in the rabbits fed a normal diet (P>0.05).2 Effects of doxazosin and its enantiomers on histomorphological changes in the heart ventricle of rabbits fed an atherogenic dietHistomorphological abnormalities had not been seen in the heart ventricle of the rabbits fed a normal diet under an ordinary optical microscope. In 6 rabbits fed an atherogenic diet for 13wk, pathological changes, such as vacuolar degeneration, fatty degeneration, focal lymphocyte infiltration, focal myocardial necrosis, focal myocardial fibrosis and disorganized smooth muscle orientation, were observed. Results from the statistical analysis (Ridit test) indicate that (-)DOX and (±)DOX significantly reduced the pathological changes in heart ventricle of the rabbits fed an atherogenic diet, but (+)DOX worsened the pathological changes.PartⅣEffects of doxazosin and its enantiomers on the serum lipid levels after long-term administration in aged miceIn order to establish a new animal model for further investigation of lipid regulation action by (±)DOX, we observed the changes in serum lipid levels in the male mouse at 13 mouths of age and the effects of (±)DOX and its enantiomers on serum lipid levels in aged mice.1 Effects of doxazosin and its enantiomers on the body weight of aged miceBefore drug administration, there was no significant difference in the body weight among all groups (P>0.05). After 12wk administration of solvent, the body weight of aged mice was decreased slightly without statistical significance (P>0.05). In comparison with the aged mice without drug-treatment, the body weight of aged mice treated by (+)DOX at 0.6 mg?kg-1 for 12wk was significantly increased (P<0.05), and significant changes in the body weight were not observed in the aged mice of other groups (P>0.05). In comparison with the body weight of aged mice before drug-treatment, the body weight of aged mice treated by (±)DOX at 0.6 mg?kg-1 for 12wk was significantly decreased only (P<0.05).2 Effects of doxazosin and its enantiomers on the survival rate of aged miceIn the group of aged mice withou drug-treatment, 5 mice died during 12wk experiments, and the mortality rate was 45.4%. The mortality rate in each of drug-treated groups was similar to that in the group of aged mice, and the mortality rates were 36.4%~54.6%. The mortality rate of aged mice treated with (-)DOX (+)DOX or (±)DOX was not significantly different from that of aged mice without drug-treatment (P>0.05).3 Effects of doxazosin and its enantiomers on the serum lipid levels of aged miceThe levels of serum TC, TG, LDL-C and VLDL-C in aged mice without drug-treatment were higher than those in young mice, and there were significant differences in VLDL-C and HDL-C levels between the two groups (P <0.01).In comparison with the level of serum TC of aged mice without drug-treatment, serum TC levels of aged mice in all drug-treated groups were decreased, and there were significant differences in the groups treated with (-)DOX 1.8 mg?kg-1, 0.6 mg?kg-1, (+) DOX 6.0 mg?kg-1 and 0.6 mg?kg-1 (P<0.05 and P<0.01). In comparison with the levels of serum TG and LDL-C of aged mice without drug-treatment, serum TG and LDL-C levels of aged mice in all drug-treated groups were decreased slightly without statistical significance (P>0.05). The serum HDL-C levels were increased in all drug-treated groups, but a statistical significance was only existed at (-)DOX 6.0 mg?kg-1 group (P<0.05). The serum VLDL-C levels of aged mice were significant decreased by treatments with (-)DOX (+)DOX and (±)DOX (P<0.05 and P<0.01) except for (±)DOX 0.6 mg?kg-1 group. Conclusion1 (-)DOX and (±)DOX increase the survival rate of rabbits fed an atherogenic diet and improve LDL-C disorder mildly. Rabbit fed an atherogenic diet is not a suitable animal model for investigating the lipid-regulating action byα1-adrenoceptor antagonists.2 (-)DOX and (±)DOX inhibit the formation and development of athero- sclerosis in thoracic and abdominal aorta, and foam cell formation in glomerular mesangial cells obviously. Moreover, (±)DOX offers a significant protective effect on the renal tubular epithelial cells. Those effects might contribute to their action of survival rate increase of the rabbits fed an atherogenic diet.3 Atherogenic diet is able to reduce cell proliferation of the rabbit ventricular heart cells and induce a histopathological damage in the heart ventricle. (-)DOX improves histopathological damage in the heart ventricle induced by atherogenic diet. (-)DOX and (+)DOX have chiral selective effects on cell proliferation and histopathological damage in the rabbits with hyperlipidemia.4 The VLDL-C levels are significantly increased, and HDL-C levels are significantly decreased in aged male Kunming mice. Long-term administrations of (-)DOX, (+)DOX and (±)DOX significantly decrease serum VLDL-C and TC levels, and there is no significant difference in the effects of lipid-regulation between the two enantiomers. Therefore, the aged male Kunming mice might be a suitable animal model for investigating the lipid-regulating action byα1-adrenoceptor antagonists.