Development Of A Model Of Atherosclerosis In Rats And Experimental Study Of Nitric Oxide In Atherosclerosis

Objective: In study of atherosclerosis, plaque, especial composition of plaque is more and more emphasized. Composition of plaque correlates intensely with development of atherosclerosis. There are many questions to be solved, a appropriate animal model is important for study of atherosclerosis. Nitric oxide (NO) plays an important role in development of atherosclerosis. In this study, a model of atherosclerosis was developed by a high-cholesterol diet, vitamin DS and endothelium denudate. With the new model, characteristics of atherosclerosis and hyperlipidemia were compared through NO and nitric oxide synthase (NOS). In order to evaluate the role of NO and NOS in platelets in development of atherosclerosis, NO and NOS in platelets as well as that in plaques were both studied. Three interference factors L-arginine (influencing on NO and NOS), chitosan (influencing on serum lipids) and simvastatin (influencing on both serum lipids and NO, NOS) were employed to study the roles in atherosclerosis.Methods: 1. Rats in the study were divided into six groups: normal control group, hyperlipidemia group (fed with high-cholesterol diet), atherosclerosis group (vitamin DS intramuscular injection, endothelium denudate and fed with high-cholesterol diet with vitamin DS), L-arginine group (besides treatment as atherosclerosis group, 2.25% L-arginine was added in the drinking water), chitosan group (besides treatment as atherosclerosis group, 5% chitosan was added in the diet), simvastatin group (besides treatment as atherosclerosis group, 0.03% simvastatin was added in the diet). The study lasted 90 days. 2. Blood was drawn from abdominal aortas to separate serum for quantitation of serum lipids. 3. With nitric acid reducdase assay for quantitation of NO2-/NO3-. 4. High performance liquid chromatographic (HPLC) assay for quantitation of L-arginine in serum. 5. Thoracic aorta hematoxylin-eosin (HE) staining to observe the structure. 6. Thoracic aorta immunohistochemistry assay to identify macrophages in plaques in the first three groups. 7. Thoracic aortaimmunohistochemistry assay to identify inducible nitric oxide synthase (iNOS) in plaques. 8. Western Blot to analyze endothelial nitric oxide synthase (eNOS) and iNOS in thoracic aortas. 9. Assay of activities of eNOS and iNOS in thoracic aortas. 10. Western Blot to analyze eNOS and iNOS in platelets. 11. Assay of activities of eNOS and iNOS in platelets. 12. To evaluate the influence of L-arginine, chitosan and simvastatin on above markers.Results: 1. Thoracic aorta HE staining: distinct plaques with abundant lipids, inflammatory cells aggregation and calcification formed in thoracic aortas of atheroslerosis group; structure of aortas in hyperlipidemia group was normal; there were small plaques and calcification in L-arginine group; besides small regions or spots of calcification, structure of aortas in chitosan and simvastatin groups was almost normal. 2. Thoracic aorta immunohistochemistry assay to identify macrophages in plaques in atherosclerosis group, results was positive. 3. Thoracic aorta immunohistochemistry assay to identify iNOS in plaques: both normal control group and hyperlipidemia group were negative; atherosclerosis group was intensively positive; L-arginine group was positive; chitosan group was weakly positive; simvastatin group was negative. 4. Serum TC, TG and LDL-C: hyperlipidemia group, atherosclerosis group and L-arginine group all increased (P<0.01) ; chitosan group decreased markedly (PO.01 or PO.05 VS atherosclerosis group); simvastatin group decreased markedly too (PO.01 VS atherosclerosis group). HDL-C: hyperlipidemia group, atherosclerosis group and L-arginine group all decreased (P<0.01) . In the four markers, there were no differences between hyperlipidemia and atherosclerosis group. 5. Serum L-arginine: All hyperlipidemia group, atherosclerosis group, chitosan group and simvastatin group were same; whereas supplying L-arginine could increase serum L-arginine (PO.01 VS the other five groups). 6. Serum NO2"/NO3": VS normal control group...