| Atherosclerosis as well as cardiovascular and cerebrovascular diseases caused by it are the main risks for human health and important cause of human death. the study shows that the pathogenesis are: inflammatory response to injury theory, theory of fatty infiltration, platelet aggregation and thrombus formation theory as well as the theory of cloning smooth muscle cells. An idea was brought up that atherosclerosis is an inflammatory immune response, is the balance disorders of damage (pro-inflammatory, immune and oxidative damage) and restoration of protection (anti-inflammatory, immunomodulatory and anti- oxidation) mechanisms, including T, B lymphocytes, antibodies and other effector cells in atherosclerosis plays an important role in progress. It was also suggested that bone marrow or vascular smooth muscle progenitor cells of the outer membrane is also involved in atherosclerosis and development. in the mid and late period smooth muscle cells become proliferative and form muscle- derived foam cells, and smooth muscle cell inward membrane under the migra- tion and proliferate there. Recent studies have found that the Rho proteins of small molecular G proteins family and Rho/Rho kinase pathway plays an important role in the pathogenesis of cardiovascular disease. Rho/Rho kinase plays an important role in cell activities such as in regulating the cytoskeleton, maintaining shape, migrating, and contracting smooth muscle cells, also import- ant in a variety of biological activities such as vascular smooth muscle cell proliferation, cell adhesion, platelet aggregation, contact inhibition, growth and wither death. Research has shown that Rho/Rho kinase pathway involved in high blood pressure, variant angina, heart failure, myocardial remodeling after myocardial infarction and other pathological processes, our previous studies have proved that the Rho/Rho kinase pathway by promoting MCP-1 over- expression involved in atherosclerosis formation, but current study are less on their specific molecular mechanism. This experiment is mainly concerned with the research in this regard and to explore the atherosclerotic vascular smooth muscle cell proliferation at the possible mechanisms.This experiment based on the establishment of rat model of atherosclerosis, given fasudil hydrochloride (Rho kinase inhibitor) drug intervention, observed Rho kinase and p27 (KIP1) protein of aortic wall by immunohistochemistry, discuss the effect of p27 (KIP1) protein in rat atherosclerotic smooth muscle cell proliferation and fasudil hydrochloride intervention role.Empirical method:30 healthy male Wistar rat (weight 220-250g) were divided into 3 groups randomly: 1. normal control group (10), 2. angiosclerosis group (10) 3. Fasudil group (Rho kinase inhibitor; 10). The normal control group undertook fake sacculus proprius and then offered normal diet.Each rat of the other two groups given vitamin D3(3×105u/kg feed) intramuscular injection suffered artery drawing wound by sacculus proprius, with elementary feed incluing 2%choles- terol, 0.5%sodium cholate, 0.2%PTU, vitamin D3 (1.25×106u/kg feed) and 3% lard.Dose and pathway for administration: 5mg/kg for Fasudil, abdominal cavity injection 2 times every day. All the rats were weighed before the experiment and every 3 weeks in the experiment, until the ninth week they were detected lipid in their serum by drawing blood on empty stomach. Then they were killed and their artery tissue were fixed with 4% formaldehyde solution and carried out HE staining and immunohistochemical analysis. The process of sacculus-impairment was as follows: The rats were anesthetized with pentobarb- itol sodium 30mg/Kg by abdominal cavity. After conventional disinfection, the left skin of neck were cutdown above the breast bone for 1 cm, and disconnected the left carotid arteries, ligated the distal end of carotid artery, the proximal part blood stream was blocked by operation line. Nitroglycerin were dropped on the arteria carotis communis, cut the artery anterior wall, the balloon catheter with guide wire were inserted into the proximal part (the balloon inside diameter is 1.5mm, Long 20mm). The balloon was posted in the left carotid artery approximately 8cm, then the balloon was infunded 0.2ml Sodium Chloride. This procedure was repeated 3-4 times. Then the balloon was posted 8cm after draining.Draining and backswing were repeated 3 times. Then drawed the balloon, the carotid artery was ligated at proximal part. After sterilisating, subcutaneous tissue and skin were suturated. Notho-balloon-operation was cutting down the left skin of neck, and disconnected the left carotid arteries, ligated the proximal part of artery and distal end of carotid artery, shearing artery but did not insert balloon.Sample deal: The rats were anesthetized with pentobarbitol sodium 30mg/ Kg by abdominal cavity narcotization after 9 weeks, then were killed after hemospasia from crotch of arteria kidney. Got out all the artery after cutting the abdomen and breast, slice about 1cm below the aortic arch, then the arteries were fixed by 0.4% formaldehyde solution immediately 24 hours, conventional anhydration,clearing,soaking wax and embed for HE staining and immunohis- tochemistry detection. The expression of Rho kinase and p27 (KIP1) protein in the vascular tissues were detected by immunohistochemistry, and detected the effect to kinase and protein by fasudil group by semi-quantitative generation.Measurement data statistics used SPSS11.5 statistical software and was carried out by X2 test, the mean for every team were analysised by Student- Newman-Keuls, and demonstrated by x±s, P<0.05 was considered significant. Result:Lipid levels: compared with the normal group and the fasudil group, TC, TG, LDL-C of the atherosclerosis group significantly increased (P<0.01), and HDL-C decreased significantly (P<0.01), while blood lipids of the normal group and the fasudil group had no significant differences.HE pathological staining showed that in normal control group, the blood vessel wall was round with uniform thickness, internal and external elastic plate were clear and complete, endothelial cells of luminal side stained blue, neatly arranged, media smooth muscle cell was fusiform, no smooth muscle cell proliferation. In atherosclerosis group, however, vessel wall was severely damaged with typical plaque, intimal thickening and fibrosis, vascular intima was prominent to lumen, with visible lipid deposition and foam cells in plaque. A small number of inflammatory cells and proliferated smooth muscle cells could be seen in intima. With internal elastic plate broken, necrosis substances and calcium deposition could be seen in intima. Media smooth muscle cell was clearly proliferated with focal or flake calcification. In fasudil group, there were no significant proliferation in intima and media, and a small number of lipid deposition and foam cells and inflammatory cell infiltration could be seen in subendothelium.Semi-quantitative immunohistochemical analysis showed that Rho kinase expressed in the normal vessel wall, and increased significantly in atherosclerosis group compared with in other two groups (P<0.001), expression in fasudil group also increased compared with the normal expression (P<0.05). p27 (KIP1) protein expression in the normal vessel wall more, expression in atherosclerosis group decreased significantly compared with other two groups (P <0.001).Expression in fasudil group also decreased compared with that in the normal group (P <0.05).Conclusion:1,The expression of rho kinase obviously increased and the expression of p27 (KIP1) protein obviously reduced when Atherosclerosis happened in the vascular smooth muscle cells scleratheroma.2,That fasudil obviously inhibit proliferation of vascular smooth muscle cell,inhibit the up-regulation of rho kinase expresion and down-regulation of the p27(KIP1) protein.3,The mechanism of fasudil inhibit proliferation of vascular smooth muscle cell is related to the reduction of rho kinase expression and activity,increase p27 (KIP1) protein expression.4,The reduction of p27 (KIP1) protein is be likely to related to the activation of Rho kinase. |
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