The Research Of Effect And Mechanism Of Rho/Rhokinase Pathway In The Formation Of Atherosclerosis

Recently, the morbidity and mortality of arteriosclerosis and cardia and cerebrovascular disorder is on the rise every year. People who develop the disease is become younger and younger. What is more, the disorder has become the main reason that lead to mutilation and death and the most common disease,they have severity influenced people'healthy. Many factors contributes to the emergence and development of artherosclerosis. the pathogenesy consist of inflammation damage response theory, adipose infiltration theory, platelet aggregation theory, thrombogenesis theory and smooth muscle cell clone theory. Recently, in some experts interview, chronic inflammation plays an important role in the formation of artherosclerosis plaque. In the earlier period of angiosclerosis, the disfunction of blood vessel endothelium provoke the formation of xanthoma cell that originate from macrophage and the conglutination and concentration of blood disk; In the middle and advanced stage, smooth muscle cell begin to hyperplasy, and migrate to generate foam cells that come from muscle. Smooth muscle cell migrate to the underside of inner membrane and hyperplasy here.Recently, investigation discover that Rho proteinum that derive from small G proteinum family and Rho/Rho kinase gateway play an important role in the nosogenesis of cardiovascular disease,. Such as, they participate the patho-procedure of hypertension, Prinzmetal angina, cardia failure, and cardiac muscle reconstitution after myocardial infarction. Few research has been done to study whether Rho/Rho kinase gateway plays an role in the nosogenesi of atherosclerosis. Rho/Rho kinase wasfound playing an important role in cystoskeleton adjustment, shape maintenance, immigration, smooth muscle cell shrinkage. Recently, experts find that Rho/Rho kinase also perform an critical function on various corpuscular action, such as vascular smooth muscle cell multiplication, cell adhesion, blood disc conglomeration, contact inhibition, growth and apoptosis. Fasudil is specific inhibitor of Rho kinase. The clinical pharmacologic action is to restrain phosphorylation of myosin light chain and lead to shrinkage of smooth muscle cell, so that blood vessel can dilate. This experiment approaches that Rho/Rho kinase take part in the course of atherosclerotic formation and it has an favourable effect on resisting atherosclerotic formation. We proved the theory by constructing model of atherosclerotic rat and administrating the model Fasudil to interfere.The experiment take atherosclerotic rat as model and they interfere by Valsartan (angiotensinⅡreceptor receptor) and Fasudil (Rho kinase inhibitor). Detect the expression of AT1R and MCP-1 by immunohistochemistry and demonstrate the involvement of Rho/Rhokinase pathway in the development of atherosclerotic. It proposed an original pathway of atherosclerotic formation and offer a new treatment prescription for cardiovascular disease.Empirical method: 40 health male Wistar rat (weight 220-250g) were divided into 4 groups randomly: 1. normal control group (10), 2. angiosclerosis group (10) 3. Fasudil group(Rho kinase inhibitor;10), 4. Valsartan group (10).The normal control group undertook fake sacculus proprius damage and then offered normal diet.Each rat of the other three groups gives vitamin D3(3×105u/kg feed) intramuscularinjection first.Then suffered artery drawing wound by sacculus proprius and were administrated with elementary feeder incluing 2%cholesterol, 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; 30mg/kg for Valsartan, lavage 1 timeevery day.All the rats were weighed before the experiment and in every 3 weeks in the experiment, until the ninth week they were detected serum lipid level by drawing blood on empty stomach. Then they were killed and their artery tissue were fixed with 4% ormaldehyde solution and carried out HE staining and immunohistochemical analysis. The process of sacculus-impairment was as follows: The rats were anesthetized with pentobarbitol sodium 30mg/Kg by abdominal cavity. After conventional disinfection, the left skin of neck were cutdown above the breast bone for 1 cm, and disconnect the left carotid arteries, ligate 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 open 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 disconnect the left carotid arteries, ligate the proximal partof artery and distal end of carotid artery, shearing artery and 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, conventional anhydration,clearing,soaking wax and embed for HE staining and immunohistochemistry detection. The expression of AT1R and MCP-1 in the vascular tissues were detected by immunohistochemistry. And compare fasudil group with Valsartan group of the influence on the production of AT1R and MCP-1 by semiquantitative method. 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:The level of blood-fat: compare with the normal control group ,TC,TG and LDL-C of the others three groups were singnificantly higher (P<0.01), HDL-C were singnificantly lower (P<0.01).There is no singnificantly difference in three groups beside the normal control group.HE staining: The arteriosclerosis group has the conspicuous plapue forming, the endomembrane was thickening with eccentricity, the plapue were filled with foam cells, small amounts inflammatory cells and the hyperplatic smooth muscle cell on the surface of endomembrane, the smooth muscle cell of medial were obviously hyperplasy, with punctiform and lamellar calcification; there are no accrementition in endomembrane and the medial inFusadil group and Valsartan group, which have few fatty substance deposition and foam cells forming and inflammatory cell infiltration between the endothelium cell and smooth muscle cell. Semiquantitative method of immunohistochemistry : The expression of AT1R and MCP-1 of arteriosclerosis group were significantly higher than others (P<0.001); The expression of AT1R in the Fasudil group were significantly higher than the normal control group and Valsartan group (P<0.05), but there were no difference between the normal control group and Valsartan group (P>0.05). The expression of MCP-1 was statistically higher in the Fasudil group and Valsartan group with comparison to the normal control group (P<0.05), while within these two groups there was no obvious differerce (P>0.05).conclusion: 1. The Rho/Rho kinase path has been activated in the forming process of arteriosclerosis and the inhibitor of Rho kinase has the role of inhibiting arteriosclerosis. 2. The activation of Rho/Rho kinase path has concerned with the activation of rennin- angiotensinsystem. 3. The action of Rho/Rho kinase path in arteriosclerosis may have some connection with promoting the more expression of MCP-1. The inhibitor of Rho kinase has the role of inhibiting arteriosclerosis, which may be a new drug for curing cardia and cerebrovascular disorder.