Effect Of Dehydroepiandrosterone Retarding Atherosclerosis Formation By Inhibiting The Expression Of Vascular Cell Adhesion Molecule-1

A number of investigations have demonstrated that atherosclerosis (AS) is a chronic inflammatory disease. The recruitment of blood monocytes into arterial subendothelial space is one of the important early events in the initiation and development of atherosclerosis. And there are many chemokines, adhesion molecules and growth factors playing important role in this process. Vascular cell adhesion molecule-1(VCAM-1)is one kind of important adhesion molecules, which mediates strong adhesion of monocytes to endothelial and attracted monocytes to migrate into subendothelial space.Dehydroepiandrosterone (DHEA) is one kind of weak androgens secreted by adrenal gland. Its concentration reaches peak in adolescent age and then decrease quickly following with ageing. It is reported that the low concentration of DHEA in blood is related with happening of the cardiac and cerebral vascular accident. So people began to investigate the effect of DHEA retarding AS, but its specific mechanism has not been demonstrated distinctly. The purpose of this paper is to observe the effect of DHEA in the expression of VCAM-1 mRNA and protein both in AS model animals and THP-1 monocytes induced by Ox-LDL. Meanwhile it is also an objection to observe whether the retarding effect can be enhanced through supplement of alltransretinoic acid (the excitomotor of retinoic acid receptor). And by means of this, its possible mechanism of retarding AS can be interpreted.Materials and methods1. The construction of animal model and grouping 40 male New Zealand White rabbits were divided into 5 groups. And the rabbits were fed with the following diets for 10 weeks: Group①: a regular rabbit diet plus 1% cholesterol +3% lard. Group②: 0.25% DHEA was incorporated into the diet of group 1. Group③: the diet of group 1 plus 0.25% DHEA and all-trans retinoic acid(0.6mg·kg-1·d-1). Group④0.25% DHEA was incorporated into the regular diet. Group⑤: a regular diet.2. The detection of biochemical indicator in every group After 10 weeks, every group rabbits were forbidden to feed for 12 hours. Then draw about 5ml blood of every rabbit from the heart in drugged state. The plasma lipoprotein levels were measured,including:cholesterol total(TC),triglyceride(TG),high density lipoprotein cholesterol (HDL-C)and low density lipoprotein cholesterol(LDL-C).3. The histopathologic change of aortaThe abdominal aorta of every group was abruptioed, fixed and stained by Sudan IV. The pathological changes were observed in gross specimens. And then the specimens were embedded, cut and HE stained. The aortic fatty streak areas, the thickness of intimae and the thickness of tunica media were morphologically observed and measured.4. Cell culture and groupingTHP-1 monocytes were suspension cultured with PRMI1640 medium containing 10% fetal bovine serum. And the cells were separated into seven groups following the experimental plan:①control group;②ox-LDL stimulated group;③ox-LDL+ low concentration DHEA group;④ox-LDL+ middle concentration DHEA group;⑤ox-LDL+ high concentration DHEA group;⑥ox-LDL+ DHEA + all-trans retinoic acid group;⑦DHEA group. Every group were collected and preserved following different experimental methods after induced by drug for 24 hours.5. Immunohistochemistry and immunocytochemistry After imbedded in paraffin and cut, the specimens of every group was stained according to the description of SP kit. Then the slides were collected pictures and analyzed by HMIAS-2000 high definition Image Analytic System.After made to be uniform film preparation, Cultured THP-1 monocytes on glass slides were fixed in fixative (ethanol: acetone=1:1)for 20 minutes at room temperature. VCAM-1 protein was detected using an immunocytochemical kit according to the convention SP method.6. RT-PCRTotal RNA was extracted from the freezing aorta and the cultured THP-1 monocytes using Trizol reagent, one microgram of which was reverse transcribed into cDNA and then entered into the polymerase chain reaction (PCR). Ten microliter of PCR products were carried out 1.5% agarose gel electrophoresis. The results were photographed under ultraviolet, scanned by SQ9636 scanner and analyzed by Image Analytic system. Values were expressed as the relative absorbance normalized to GAPDH levels.7.Statistical analysisData were analyzed by ANOVA and t-test of the SPSS12.0 software.Results1. The detection of biochemical indicator in every group The TC and LDL-C levels of rabbits in AS model group were higher than that of control group obviously. After intervention by DHEA there was little decrease in the LDL-C level(p<0.05). And after intervention by DHEA and ATRA there was further decrease in the LDL-C and TC level(p<0.05).2. The gross observation of aortaThe wall of aorta was thin and the intima was smooth in control group, and on which there were no fatty streak and plaque. But brunneus fatty plaque could be seen obviously and the intima was thickening in the wall of aorta of rabbits whose diet plus 1% cholesterol +3% lard. The fatty plaque was scatter and areas of it were decreased evidently in the rabbits whose diet incorporated with DHEA. And the walls of aorta were thinner compared with the rabbits whose diet plus 1% cholesterol and 3% lard.3. The histological observation and measurement of aortaCompared with the control group, Aortic tunica intima was thickening and subendothelial space was widening obviously in the rabbits whose diet plus 1% cholesterol +3% lard. And there were multiplicity accumulative foam cells under the endothelium. After intervention by DHEA, the thickness of aortic tunica intima and the area of fatty plaque were decreased 59% and 48% respectively Compared with the AS model rabbits. And the accumulative foam cells and smooth muscle cells were somewhat lightened.4. Observation of VCAM-1 protein expressionVCAM-1 protein showed by immunohistochemistry staining was brown substance,which located in aortic tunica intima of every group. Compared with the control group and the DHEA group, Aortic tunica intima was thickening obviously in the rabbits whose diet plus 1% cholesterol and 3% lard. And there were multiplicity granular brown substances in it. In the rabbits whose diet incorporated with DHEA, the thickness of aortic tunica intima and granular brown substance were all decreased obviously.VCAM-1 protein of THP-1 monocyts showed by immunocytochemistry staining was granular brown substance,which located mainly in cell membrane. Compared with the control group, the granular brown substances were increased obviously in OX-LDL induced THP-1 monocyts. After supplied DHEA in different concentration, the expressions of VCAM-1 protein were weakened evidently.5. Observation of VCAM-1 mRNA expression The expression of VCAM-1 mRNA was more in aortic tunica intima of AS model rabbits(0.9651±0.0992)compared with that in control group(0.2178±0.0495), and which was lowered in the rabbits whose diet incorporated with DHEA(0.6499±0.1434). But there were no significant deviation in the expression of VCAM-1 mRNA between the rabbits whose diet incorporated with ATRA and the rabbits who have no ATRA supplied in diet(P>0.05).The expression of VCAM-1 mRNA was more in OX-LDL induced THP-1 monocyts(0.6236±0.0237)compared with that in control group(0.1693±0.0068), which was lowered After supplied DHEA in different concentration. And The expression of VCAM-1 mRNA was the lowest when the concentration of DHEA was 5μmol·L-1 (0.2988±0.0312) .Conclusions1. DHEA can retard the formation of atherosclerotic plaque in high cholesterol-fed rabbits.2. DHEA has the effect of retarding atherosclerosis in high cholesterol-fed rabbits, which was probably involved with the inhibition of VCAM-1 mRNA and protein expression.3. DHEA also showed inhibiting effects on the VCAM-1 mRNA and protein expression in Ox-LDL-induced THP-1 monocytes.4. All-trans retinoic acid has no obviously fortified effect on the VCAM-1 expression in this action of DHEA.