Effects Of Dehydroepiandrosterone On Expression Of VCAM-1 In Rabbit Aorta And HUVEC Induced By High Level Lipid

Effects of Dehydroepiandrosterone on Expression of VCAM-1 in Rabbit Aorta and HUVEC Induced by High Level LipidAtherosclerosis is a chronic inflammatory disease, cytokines such as adhesion molecules and chemokines are involved in the pathologic process of atherosclerosis. Numerous risk factors including hyperlipidemia can induce arterial luminal endothelium dysfunction. This can increase the expression of cytokines such as vascular cell adhesion molecules-1(VCAM-1) on endothelium and VSMC. Thus promote the adherence of circulating monocytes to endothelium .Soon they also migrate into subendothelial space, then differentiate to macrophages there, subsequently convert to "foam cells", which is the primary character of early atherosclerosis. From above mentioned, we know that adhesion molecules have important actions in atherosclerosis. Accordingly, it is possible that blocking and slowing the formation of atherosclerosis by reducing the expression of correlative adhesion molecules.DHEA is the precurosor of testosterone; a few of them are transformed to estrogen. DHEA has relationship to several aged diseases. Recent researches indicate that DHEA has significant effect on atherosclerosis. Epidemiology researches indicate that low level DHEA in plasm was correlated closely with atherosclerosis and cardiac and encephalic disease which caused by atherosclerosis. DHEA is degenerated to estrogen which has the antiatherosclerotic effect by cytochrome P450 aromatase in vivo. Therefore, if we can increase the activity of cytochrome P450 aromatase, the antiatherosclerotic effect of DHEA could be enhanced in vessel. Some investigations indicate that cytochrome P450 aromatase has several promoters. The active promoter in endothelium and VSMC is promoterⅠ.6. The activated retinoic acid receptor could increase the expression of cytochrome P450 aromatase through promoterⅠ.6. Therfore, giving the activator of retinoic acid receptor --all-trans-retinoic acid (ATRA) can increase the expression of estrogen, which has the antiatherosclerotic effect .So ATRA may have the positive effect on the antiatherosclerotic effect of DHEA. This study carried on both in vitro and in vivo. Using ox-LDL as a stimulator in vitro and high level lipid as a stimulator to make the atherosclerotic animal model in vivo. Meanwhile, using DHEA,ATRA as interventors, examine the expression of VCAM-1.The aim is to open out the potential mechanism of antiatherosclerotic effect of DHEA.Part One Effects of Dehydroepiandrosterone on Expression of VCAM-1 in Rabbit Aorta Induced by High Level LipidObjectiveTo investigate the effects of dehydroepiandrosterone (DHEA) on expression of VCAM-1 in rabbit aorta induced by high level lipid, and the effects of all-trans-retinoic acid (ATRA) in this procedure.Methods1. Developed the AS and treatment animal modelSelected 40 male,2.0-2.5kg New Zealand big ear white rabbits. The rabbits were divided into 5 groups by random.1 control group: fed by normal feedstuff; 2 high level lipid group: fed by high level lipid feedstuff(added 1% cholesterol and 1% pig fat to normal feedstuff) ; 3 high level lipid+DHEA group: fed by the feedstuff what was added 0.125g/kg﹒d DHEA to the high level lipid feedstuff ; 4 high level lipid+DHEA+ATRA group: fed by the feedstuff what was added 0.125g/kg﹒d DHEA and 0.6mg/kg﹒d ATRA to the high level lipid feedstuff ;5 DHEA group: fed by the feedstuff what was added 0.125g/kg﹒d DHEA to the normal feedstuff. Rabbits of all groups were fed by corresponding feedstuff in 2 month, and then were killed. Collected the heart blood. Removed the main aorta from the heart to kidney aorta. Removed a piece of aorta from the aortic arch,reserved it in -80℃refrigerator. Then fixed the left main aorta in 4% PFA solution.2. Examined the level of blood lipidExamined the level of TC, TG, HDL-C, LDL-C in plasm by autobiochemistry analyser. 3. The ratio of plaque area and arterial area , the ratio of intima and tunica mediaEmbedded the fixed aorta in paraffin then sectioned and dyed. The ratio of plaque area and arterial area and the ratio of intima and tunica media were analyzed by HMIAS-2000 analysis system.4. immunohistochemistryEmbedded the fixed aorta in paraffin and sectioned. The aorta were probed with rab anti rab VCAM-1 antibody which was 1:400 diluted by PBS and the secondary antibody which was 1:1 diluted by PBS .VCAM-1 protein was detected by an immunohistochemical kit.VCAM-1 protein was quantified by the average absorbance values (A) in the cytoplasm and cell membrane.5. RT-PCRExtracted the total RNA from the aorta of all groupes. Reverse transcripted the RNA to cDNA. Cloned the cDNA by PCR. Separated all kinds of cDNA by electrophoresis. The absorbance values(A) of VCAM-1 and GAPDH were determined by HMIAS-2000 analysis system.VCAM-1 mRNA was quantified by AVCAM-1/AGAPDH.Results1. The level of blood lipidThe level of TC and LDL-C in high level lipid group was significantly increased as compared with control group(P<0.05). The level of TC and LDL-C in high level lipid+DHEA+ATRA group was decreased as compared with high level lipid group(P<0.05). The level of TC and LDL-C was similar in both DHEA group and control group(P>0.05).The level of TG and HDL-C :There was no remarkable difference between any two groups(P>0.05).2. The ratio of plaque area and arterial area, the ratio of intima and tunica mediaThe ratio of plaque area and arterial area and the ratio of intima and tunica mediain high level lipid group were significantly increased as compared with control group(P<0.05). Compared with high level lipid group the ratio of plaque area and arterial area and the ratio of intima and tunica media in high level lipid+DHEA group were obviously decreased(P<0.05). There were no remarkable difference in the ratio of plaque area and arterial area and the ratio of intima and tunica media between high level lipid+DHEA+ATRA group and high level lipid+DHEA group (P>0.05). Obvious difference in the ratio of plaque area and arterial area and the ratio of intima and tunica media between DHEA group and control group were not observed (P>0.05).3. immunohistochemistryThe positive signal is the brown granular substance located in the cytoplasm and cell membrane. The expression of VCAM-1 protein in high level lipid group was significantly increased as compared with control group(P<0.01).The brown granular substance in high level lipid group was more than control group. The colour of the brown granular substance in high level lipid group was heavier than control group. Compared with high level lipid group the expression of VCAM-1 protein in high level lipid+DHEA group was obviously decreased(P<0.01).The brown granular substance in high level lipid+DHEA group was less than high level lipid group. The colour of the brown granular substance in high level lipid+DHEA group was lighter than high level lipid group. There was no remarkable difference in the expression of VCAM-1 protein between high level lipid+DHEA+ATRA group and high level lipid+DHEA (P>0.05). Obvious difference in the expression of VCAM-1 protein between DHEA group and control group was not observed (P>0.05).4. RT-PCRThe expressions of VCAM-1 mRNA in main aorta in all groupes were observed. The expression of VCAM-1 mRNA in high level lipid group was significantly increased as compared with control group(P<0.05).Compared with high level lipid group the expression of VCAM-1 mRNA in high level lipid+DHEA group was obviously decreased(P<0.05).There was no remarkable difference in the expression of VCAM-1 mRNA between high level lipid+DHEA+ATRA group and high level lipid+DHEA group (P>0.05). Obvious difference in the expression of VCAM-1 mRNA between DHEA group and control group was not observed (P>0.05).ConclusionsDHEA showed inhibiting effects on high level lipid induced VCAM-1 expression in rabbit aorta. That may be one of the mechanisms of antiatherosclerotic effect of DHEA. But ATRA had no positive effect on DHEA function.Part Two Effects of Dehydroepiandrosterone on Expression of VCAM-1 in HUVEC Induced by ox-LDLObjectiveTo investigate the effects of dehydroepiandrosterone(DHEA) on expression of VCAM-1 in HUVEC induced by ox-LDL, and the effects of all-trans-retinoic acid (ATRA) in this procedure.Methods1. HUVEC culture:The human umbilical venous endothelial cells(HUVEC) were prepared from human umbilical core,provided by Tongji hospital. HUVEC were maintained in M199 medium with 15%FBS,0.03%Gln,50μg/ml ECGS, at 37℃.The cell were cultured to confluence and divided into 6 groups:1 control group; 2 ox-LDL group: added 25mg/L ox-LDL to the medium ; 3 ox-LDL+DHEA(low density) group: added 25mg/L ox-LDL and 100nmol/L DHEA to the medium ;4 ox-LDL+DHEA(high density) group: added 25mg/L ox-LDL and 1000nmol/L DHEA to the medium ; 5 ox-LDL+DHEA+ATRA group: added 25mg/L ox-LDL,1000nmol/L DHEA and 10-8mol/L ATRA to the medium ; 6 DHEA group: added 1000nmol/L DHEA to the medium .Cells of all groups were incubated with corresponding reagents in 24h.2. ELISAHUVEC of all groups were incubated with corresponding reagents in 24h.Then changed the medium which contained the reagents to normal medium. Collected the medium 12h later. The VCAM-1 protein in the medium were detected by enzyme-linked immunosorbent assay ( ELISA) according to the introduction of ELISA kit. The absorbance values were determined by ELIASA in 492nm.3. RT-PCRExtracted the total RNA from HUVEC of all groupes. Reverse transcripted the RNA to cDNA. Cloned the cDNA by PCR. Separated all kinds of cDNA by electrophoresis. The absorbance values(A) of VCAM-1 and GAPDH were determined by HMIAS-2000 analysis system.VCAM-1 mRNA was quantified by AVCAM-1/AGAPDH.Results1. ELISAThe expression of VCAM-1 protein in HUVEC in ox-LDL group was significantly increased 1.8 fold as compared with control group(P<0.01).the expression of VCAM-1 protein in ox-LDL+DHEA(low density) group and ox-LDL+DHEA(high density) group were obviously decreased in a dose-dependent manner as compared with ox-LDL group (P<0.05). They accounted for 73.5% and 60.4% respectively of that of the ox-LDL group . The expression of VCAM-1 protein was similar in both ox-LDL+DHEA+ATRA group and ox-LDL+DHEA(high density) group (P>0.05). The expression of VCAM-1 protein was similar in both DHEA group and control group (P>0.05).2. RT-PCRThe expressions of VCAM-1 mRNA in all groups HUVEC were observed. The expression of VCAM-1 mRNA in ox-LDL group was significantly increased as compared with control group(P<0.01).Compared with ox-LDL group the expression of VCAM-1 mRNA in ox-LDL+DHEA(low density) group and ox-LDL+DHEA(high density) group were obviously decreased in a dose-dependent manner (P<0.05). The expression of VCAM-1 mRNA was similar in both ox-LDL+DHEA+ATRA group and ox-LDL+DHEA(high density) group (P>0.05). The expression of VCAM-1 mRNA was similar in both DHEA group and control group(P>0.05). ConclusionsDHEA showed inhibiting effects on ox-LDL induced VCAM-1 expression in HUVEC. That may be one of the mechanisms of antiatherosclerotic effect of DHEA. But ATRA had no positive effect on DHEA function.