| Atherosclerosis (AS) is a chronic progressive inflammatory disease. Thepathogeny and the mechanism are still unknown. The pathology changes of ASare the arterial intima incrassation, and the smooth muscle cell hyperplasia, andthe lipid deposition and the information of the foam cells after the macrophagesphagcytize lipids. In studies of AS, we find whether the animal experiments or theclinic researches indicate that vascular cell adhesion molecule-1 (VCAM-1),intercellular adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitortype-1 (PAI-1) may involve in the process of AS and statin drugs not only reducecholesterole, but also have the effects of anti-atherosclerosis. In order toinvestigate the expression of VCAM-1, ICAM-1 and PAI-1 in AS and anti-atherosclerosis mechanism of atorvastatin, we have studied as follow: 1. Building AS models and examining the expression of VCAM-1,ICAM-1 and PAI-1 in plasma and arterial walls in the normal diet and thecholesterol diet groups by means of ELISA, immunohistochemistry analysis andRT-PCR 1.1 Building AS models 1.1.1 Animals and groups Twenty four male rabbits were randomly divided into the normal diet, andthe cholesterol diet and the atorvastatin groups, and was fed for 16 weeks. 1.1.2 Doing the pathology specimens After 16 weeks, the rabbits were put to death and the aortas were harvestedfor the pathologic morphology observations.1.1.3 Establishing hypercholesterolemiaThere were no obvious differences between TC and LDL in the normal dietand the cholesterol diet groups in plasma in 0 week (P>0.01) and there were noobvious differences between TC and LDL in the normal diet group in 8 and 16weeks compared with 0 week (P>0.01). There were obvious increase of TC andLDL in the cholestrol diet group after 8 and 16 weeks and there were obviousdifferences compared with 0 week (P<0.01).1.1.4 Naked view of ASNaked observations were found that the structure of the arterial walls wasnormal and smooth, there was no formation of the atherosclerotic plaques in thenormal diet group. In the cholestrol diet group, generous flaw-whitestearo-matters projected over lumen and the arterial walls diffused intumescence.All those connected with each other and formed atherosclerotic plaques .The ratioof the plaque area to the intima was 28.12±3.77%.1.1.5 Microscope view of ASIn the normal diet group, endothelial cells were integrated and lined up inorder, and intra-elasticity tunics were distinct and integrated, and the volume ofthe media smooth muscle cells and the arrangement were normal, and no foamcells and lipidoses were found in the intimas and the mediums, also the intimalthickness was 4.45±0.58μm and the ratio of the intima to the medium was 0.0537±0.007. In the cholesterol diet group, the endotheliocytes in the aortic intimasbecame swelling and degenerated, and the intimas were obviously thick with thesmooth muscle cell proliferation and the rearrangement, and lipidoses of ecto-celland the macrophages can be find in the intimas and the mediums, and the intimalthickness was 67.47±7.13μm which obviously became thick compared with thenormal diet group and the ratio of the intima to the medium increased comparedwith the normal diet group and it was 0.878±0.370 (P<0.01).1.2 Measuring the expression of VCAM-1, ICAM-1 and PAI-1 in thenormal diet and the cholesterol diet groups in plasma by mean of ELISA1.2.1 Expression of VCAM-1There were no significant differences in VCAM-1 level in the normal dietgroup in 0, 8 and 16 weeks (P>0.01). VCAM-1 level increased in the cholesteroldiet group in 8 weeks compared with 0 week (P<0.01) and obviously increased in16 weeks compared with 8 weeks (P<0.01).1.2.2 Expression of ICAM-1There were no significant differences in ICAM-1 levels in the normal dietgroup in 0, 8 and 16 weeks (P>0.01). ICAM-1 level in the cholesterol diet groupincreased in 8 weeks compared with 0 week, but there were no significantdifferences (P>0.05). ICAM-1 level in the cholesterol diet group obviouslyincreased in 16 weeks compared with 8 weeks (P<0.01).1.2.3 Expression of PAI-1There were no significant differences in PAI-1 level in the normal diet groupin 0, 8 and 16 weeks (P>0.01). PAI-1 level increased in the cholesterol diet groupin 8 weeks compared with 0 week (P<0.01) and obviously increased in 16 weekscompared with 8 weeks (P<0.01).1.3 Measuring the aorta expression of VCAM-1, ICAM-1 and PAI-1 in thenormal diet and the cholesterol diet groups by mean of immunohistochemistryanalysis1.3.1 Expression of VCAM-1A little brown positive stained materials were found in the intimas and themediums in the normal diet group and the positive percentage was 2.92±0.31%.Lots of brown positive stained materials were found in the intimas and themediums in the cholesterol diet group and the positive percentage was 18.38±2.55%. The positive percentage of the materials in the cholesterol diet groupobviously increased compared with the normal diet group (P<0.01).1.3.2 Expression of ICAM-1A little brown positive stained materials were found in the intimas and themediums in the normal diet group and the positive percentage was 1.67±0.23%.Lots of brown positive stained materials were found in the intimas and themediums in the cholesterol diet group and the positive percentage was 12.23±0.56%. The positive percentage in the cholesterol diet group obviously increasedcompared with the normal diet group (P<0.01).1.3.3 Expression of PAI-1A little brown positive stained materials were found in the intimas and themediums in the normal diet group and the positive percentage was 3.24±0.21%.Lots of brown positive stained materials were found in the intimas and themediums in the cholesterol diet group and the positive percentage was 15.46±2.23%.The positive percentage in the cholesterol diet group obviously increasedcompared with the normal diet group (P<0.01).1.4 Measuring expression of VCAM-1 and ICAM-1 in the normal diet andthe cholesterol diet groups by mean of RT-PCR1.4.1 Expression of VCAM-1mRNACorrespondence OD value of VCAM-1mRNA in the normal diet and thecholesterol diet groups were 0.684±0.006 and 1.116±0.017 (P<0.01), indicatingVCAM-1 excess expression in AS .1.4.2 Expression of ICAM-1 mRNACorrespondence OD value of ICAM-1mRNA in the normal diet and thecholesterol diet groups was 0.214±0.003 and 0.572±0.004 (P<0.01), indicatingICAM-1 excess expression in AS.2. Decreasing lipid effect of atorvastatin and correlating VCAM-1,ICAM-1 and PAI-1 expressions with TC and LDL2.1 Decreasing lipid effectThere were no obvious differences of TC and LDL in three groups in plasmain 0 week (P>0.05), and there were no obvious differences of TC and LDL in thenormal diet group in 8 and 16 weeks compared with 0 week (P>0.05). TC andLDL of the cholesterol diet and the atorvastatin groups in 8 and 16 weeks wereobviously higher than normal diet group (P<0.01). TC and LDL of the atorvastatingroup were lower than the cholesterol diet group in 8 weeks (P>0.05) and weregreatly lower than the cholesterol diet group in 16 weeks (P<0.01).2.2 Correlation of VCAM-1, ICAM-1 and PAI-1 expression with TC andLDL in plasma2.2.1 Correlation of VCAM-1 expression with TC and LDLThe dependent variable was TC and LDL, and the independent variable wasVCAM-1 and linear regression analysis was used to distinguish the correlations ofTC and LDL with VCAM-1. It was found that TC and LDL were positivelycorrelated with VCAM-1 and the coeffocient of product-moment correlation r was0.712 and 0.792 (P<0.001).2.2.2 Correlation of ICAM-1 expression with TC and LDLThe dependent variable was TC and LDL, and the independent variable wasICAM-1 and linear regression analysis was used to distinguish the correlations ofTC and LDL with ICAM-1. It was found that TC and LDL were positivelycorrelated with ICAM-1 and the coeffocient of product-moment correlation r was0.5823 and 0.6547 (P<0.005).2.2.3 Correlation of PAI-1 expression with TC and LDLThe dependent variable was TC and LDL, and the independent variable wasPAI-1 and linear regression analysis was used to distinguish the correlations of TCand LDL with PAI-1. It was found that TC and LDL were positively correlatedwith PAI-1 and the coeffocient of product-moment correlation r was 0.602 and0.613 (P<0.001).3. Effects of atorvastatin in VCAM-1, ICAM-1and PAI-13.1 Effect in plasma3.1.1 Effect in VCAM-1There were no obvious differences in the three groups in 0 week (P>0.01).VCAM-1 in the cholesterol diet and the atorvastatin groups in 8 and 16 weeks washigher than in the normal diet group in 0 week (P<0.01). VCAM-1of theatorvastatin group was lower than the cholesterol diet group in 16 weeks(P<0.01).3.1.2 Effect in ICAM-1There were no obvious differences in the three groups in 0 week (P>0.01).ICAM-1 in the cholesterol diet and the atorvastatin groups in 16 weeks was higherthan normal diet group in 0 week (P<0.01). ICAM-1of the atorvastatin group waslower than the cholesterol diet group in 16 weeks (P<0.01).3.1.3 Effect in PAI-1There were no obvious differences in the three groups in 0 week (P>0.01);PAI-1 in the cholesterol diet and the atorvastatin group in 8 and 16 weeks washigher than the normal diet group in 0 week (P<0.01). PAI-1 of the atorvastatingroup was lower than the cholesterol diet group in 16 weeks (P<0.01).3.2 Effect of atorvastatin in the positive percentage of VCAM-1,ICAM-1and PAI-1 in the aorta walls3.2.1 Effect of VCAM-1Lots of brown positive stained materials were found in the intimas and themediums in the cholesterol diet and the atorvastatin groups and they were 18.38±2.55% and 11.18±1.95%. The quantities of the materials in the atorvastatin groupwere lower than the cholesterol diet group (P<0.01).3.2.2 Effect of ICAM-1Lots of brown positive stained materials were found in the intimas and themediums in the cholesterol diet and the atorvastatin groups and they were 12.23±0.56% and 8.26±0.34%. The quantities of the materials in atorvastatin groupwere lower than the cholesterol diet group (P<0.01).3.2.3 Effect of PAI-1Lots of brown positive stained materials were found in the intimas and themediums in the cholesterol diet and the atorvastatin groups and they were 15.46±2.23% and 9.75±1.49%. The quantities of the materials in the atorvastatin groupwere lower than the cholesterol diet group (P<0.01).3.3 Effect of atorvastatin in VCAM-1 mRNA and ICAM-1mRNA in aortas3.3.1 Effect in VCAM-1mRNACorrespondence OD value in the normal diet, the cholesterol diet and theatorvastatin groups were 0.684±0.006, 1.116±0.017 and 0.856±0.011 (P<0.01),indicating that VCAM-1 express excesively in AS and atorvastatin can inhibit theexpression of VCAM-1.3.3.2 Effect in ICAM-1mRNACorrespondence OD value in the normal diet, the cholesterol diet and theatorvastatin groups were 0.214±0.003,0.572±0.004,0.406±0.011 (P<0.01) ,indicating that ICAM-1 express excesively in AS and atorvastatin can inhibit theexpression of ICAM-1.4. Effect of atorvastatin in inhibiting AS4.1 Naked viewWe can see from the naked observations that the structure of the arterial wallswere normal and smooth and there was no formation of atherosclerotic plaques inthe normal diet group. In the cholestrol diet group, generous flaw-whitestearo-matters projected over lumen and the arterial walls diffused intumescence.All those connected with each other and formed atherosclerotic plaques. The ratioof plaque area to intima was 28.12±3.77%. The plaques greatly reduced in theatorvastatin group, and ratio of plaque area to intima was 16.09%±2.77% and waslower than the cholestrol diet group (P<0.01).4.2 Microscope viewIn the normal diet group, endothelial cells were integrated and lined up inorder, and intra-elasticity tunic was distinct and integrated, and media smoothmuscle cell volume and arrangement were normal, and no foam cells and lipidoseswere found in the intimas and the mediums, and the intimal thickness was4.45±0.58μm and the ratio of intima to media was 0.053±0.007μm. In thecholesterol diet group, the aortic intima endotheliocytes became swelling anddegenerated with the smooth muscle cell proliferation and the rearrangement, andthe mediums became thicker, and lipidoses of ecto-cells and the macrophages canbe find in the intimas and the mediums, and the intimal thickness was67.47±7.13μm and the ratio of the intima to media was 0.878±0.370. In theatorvastatin group, the turgidity and the degeneration of endotheliocytes wererelieved, and the smooth muscle cell proliferation and rearrangement wereimproved, and the lipidoses and the foam cells were lower, and the intimalthickness was 38.11±6.02μm and ratio of the intima to medium was 0.391±0.213and it was higher than the control group (P<0.01) and lower than the cholesterolgroup (P<0.01).The dates were presented as mean ± SE, and statistical comparsions betweengroups were followed by the Student t test. The corresponding control value withP<0.05 considered statistically significant.Our studies have shown:1. The expression of VCAM-1, ICAM-1 and PAI-1 in serum increase in therabbits of AS;the percentage of the positive stained of VCAM-1, ICAM-1 andPAI-1 is much higher in the rabbits of AS;the expression of VCAM-1 mRNA andICAM-1mRNA increase in the aortic walls.2. TC and LDL not only can lead to VCAM-1, ICAM-1 and PAI-1escessive expression, but also have the positive correlation with the expression ofVCAM-1, ICAM-1 and PAI-1.3. Atorvastatin reduces the expression of VCAM-1, ICAM-1 and PAI-1 inserum in the rabbits of AS, and inhibits the percentage of positive stained ofVCAM-1, ICAM-1 and PAI-1 and inhibits the expression of VCAM-1 mRNA andICAM-1mRNA in the aortic walls.4. Atorvastatin has greatly effect of decreasing TC and LDL, and thedecreasing tendency is at 8 weeks and the significant low is at 16 weeks.5. Atorvastatin reduces plaques, intima thickness and foam cells andinhibits and relieves AS.The significance of studies:1. It has explored the relationship of VCAM-1, ICAM-1 and PAI-1 withAS, and it is important to reveal the mechanism of AS.2. It has offered markers in laboratory in order to diagnose and detect AS.3. It has infirmed that atorvastatin not only has greatly effect of decreasingTC and LDL, but also inhibits AS.4. It has indicated that inhibiting VCAM-1, ICAM-1 and PAI-1 expressionsmay be one of anti-atherosclerosis mechanism of atorvastatin. |
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