| Objective The differentially expressed genes are screened from the AS rats and AS rats with fluvastatin intervention through gene chip technology, by establishing a SD rat model of atherosclerosis. Thus the anti-atherosclserotic mechanism of fluvastatin may be explored and a novel theoretical evidences and strategy for prevention and treatment of AS may be provided.Materials and Methods (1) Forty-five male SD rats were randomly divided into 3 groups (15 rats in every group): group A (normal control), group B (atherosclerosis) and group C (atherosclerosis rats treated with fluvastatin). Group A were fed with common feedstuff. Group B were fed with high lipid feedstuff (including 3% cholesterol, 0.5% cholic acid natrium, 0.2% propylthiouracil, 5% refined sugar, 10% lard oil) plus vitamin D3 to model AS. And group C were fed as group B, followed with fluvastatin (20 mg/kg/d, intragastric administration).The study lasts for 12 weeks. (2) During the experiment, the levels of serum TC, TG and calcium were examined. (3) After being fed for 12 weeks,the rats were executed to take specimens from aorta for staining. The rest were placed in liquid nitrogen for preservation. (4) Specimens from aorta in all three groups were appllied to gene chip to screen the differentially expressed genes. (5) Fron the results of gene expression profile, the upregulated genes, such as HSP60 and ADMR, are selected. (6)RT-PCR and Western blot were performed to investigate the expression of HSP60 and ADMR in all three groups.Results (1) By the end of experiment one died in group C,one failed to model atherosclerosis and one died in group B. (2) The values of body weight, blood lipids and blood calcium show no significant difference statistically in the three groups at the beginning. It was found that the weights of group A and C keep increasing with time and the weights of group A increase significantly. The weights of Group B increases faster in the first eight weeks mainly and slower in the following 4 weeks. No statistically significant differences are found in the three groups. The triglyceride and cholesterol levels in group C were reduced significantly compared with group B (p < 0.01) and those in group B were increased significantly compared with group A (p < 0.05). The blood serum calcium levels in group B and group C were increased significantly compared with group A (p < 0.05). (3) Thoracic aorta HE staining: The three-layered structure of the vessel wall of the group A was distinct; the aortic tunica intima was smooth and thin, growth of plaque was not seen; arrangemen of the smooth muscle cell was regular. The three-layers structure of the vessel wall of the group B was not distinct; the aortic tunica intima was rough, damaged and incomplete; the muscular tunic was thick, arrangemen of the smooth muscle cell was irregular, calcification could be seen diffusively. The group C lied between the group A and the group B: three-layered structure of the vessel wall were distinct, intima integrated; the aortic tunica intima was rough; a few calcification could be seen, the smooth muscle cell lined up in order. (4) The results of gene expression profile: compared with group A, 610 genes were over-expressed and 1018 genes were under-expressed in group B. Among the over-expressed genes, there were molecules regulating inflammation genes, growth factors genes, proliferation genes, stress related molecules genes, and so on. Compared with group B, 2255 genes were over-expressed and 844 genes were under-expressed in group C. Inflammation genes, growth factors genes, proliferation genes and so on are included in the under-expressed genes; while ADMR,Pgm1 are included in the down-expressed ones. (5) The expression of HSP60 mRNA: compared with group A, the expression in group B significantly increased (P<0.05); and there was no significant change between group B and C. (6) The expressions of ADMR mRNA and protein: compared with group A, the expression in group B significantly increased; and compared with group B, the expression in group C significantly increased (both P<0.05).Conclusions(1) Atherosclerotic model is successfully duplicated by means of high-fat diet and vitamin D3 overload. (2) Severe disturbance of genes expression profile in atherosclerosis rat is a common phenomenon. The over-expressed and under-expressed genes extensively and intensively affect the inflammation, blood coagulation, oxidative stress, cell proliferation, and so on. It indicates that the pathogenesis of AS is very complex and many kinds of factors may involve. (3) Gene expression profile in group C shows that compared with group B, the expressions of many genes have been changed. The genes involved in inflammation, blood coagulation, cell proliferation etc are down-expressed. Thus, the multiple effects of statins are validated further. (4) The up-regulated HSP60 mRNA has been detected in the processing of atherosclerosis-forming. And the HSP60 expression changed unsignicantly with the intervance of fluvastatin. (5) HSP60 may be a predict factor of atherosclerosis, independent of the level of serum cholesterol, which may not participate into the molecular mechanisms of anti-atherosclerosis. (6) The up-regulated ADMR has been detected in the processing of atherosclerosis-forming and ADMR expression increased further with the intervence of fluvastatin. It suggests that ADMR might play an important role in the anti-atherosclerosis mechanisms of fluvastatin.In brief, by using gene chip technology we certificates that the pathogenesis of atherosclerosis is complex, caused by combination of multiple factors and a variety of genes. Rat can be used as an atherosclerotic model. Statins may involve the cholesterol-independent effects. These will provide novel strategy and theoretic evidence to prevention and treatment of atherosclerosis. |
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