| Atherosclerosis (AS) is a systemic disease, mainly involving large and medium arteries, the basic lesion is intimal lipid deposition, intimal lesions like fibrosis ,and proliferation of smooth muscle cells and connective tissue, causing the formation of thickening fibrosis of intimal lesions and atheromatous plaque, which hardens arterial wall, narrows lumens and induces secondary lesions, especially ischemic lesions of organs such as heart and ischemic lesions. For a long time, there are a number of theories on AS pathogenesis, including lipid infiltration of theory,macrophage receptor absence of theory,thrombotic of theory,haemodynamics of theory,middle layer accrementition of contractile fiber cells of theory,endothelial cell injury theory, injury and inflammatory response theory. However, none of them can explain the origin and development of AS to the full extent. Therefore, AS pathogenesis and therapy of the inquiry are the hot point in medical research.UⅡwas originally extracted from the tail spinal cord of fish pituitary, and shares its ubiquity in mollusks as well as mammals. GPR14, an isolated receptor and also a specific receptor of UⅡ, distributes mainly over the cardiovascular system. UⅡand GPR14 form UⅡ/UT system, triggering a series of biological effects, such as contraction of vascular function and promote mitogen effect. Lots of researches showed that UⅡplayed a important role in the homeostatic regulation of cardiovascular and the courses of the development of a disease. One of the main pathogenesis that bring about AS which may be the function of UⅡfunction in hemodynamics and the vascular wall and cells. Thus, the application of UⅡto preventing and curing AS which may has potential and important clinical value.Urantide, a peptide receptor antagonist derived from the human being of UⅡ, has been the best UⅡof receptor antagonist, famous for its effect, 50～100 times than other compounds. The function of Urantide can be highly selective, for example, it only confronts vasoconstrictor effect induced by UⅡ, but not affecting norepinephrine, ET-1 and other effects caused by spasm. Experimental in vitro study of rat aorta, Urantide can be competitively antagonized UⅡ on contraction of rat thoracic aorta, and significantly inhibited UⅡinduced human monocyte- derived macrophage cholesterol acyltransferase activity increases-1, thereby preventing the formation of foam cells. At present, the domestic Urantide studies have focused on myocardial ischemic injury and vascular remodeling, the Urantide role in AS has not been reported.In this study, morphology, biochemistry, immunocytochemistry, immunohistochemistry, flow cytometry, Western blot, RT-PCR and other techniques, through in vivo and in vitro methods confirmed the results, comparative the AS rat thoracic aorta and vascular smooth muscle cells (VSMC) in the UⅡand its receptor GPR14 changes, the relationship between UⅡand certain inflammatory mediators, and affected the extracellular matrix of the impact of the intervention to study under Urantide intervention , UⅡexisted the possible role and the mechanism of occurrance and development of AS in rats.The main results are as follows:(1) In vivo: AS group can be successfully replicated in rats, when VD3 70 U / kg was given by intraperitoneal injection,and fed with high fat diet during 4 W. The expression of Ca2 +, TG, TC, HDL and LDL in AS group were significantly higher than normal control group; and when we treated each group with Urantide,serum indicators were gradually decreased with the delivery time,catched or neared the level of positive control group. Control group were observed in rat thoracic aorta, the significant aortic intimal calcification, inflammatory cell infiltration, VSMC proliferation and foam-like cells, elastic fiber degeneration, fracture and collapse of the membrane atrophy, this was typical AS pathology; thoracic aorta UⅡand its receptor GPR14 gene and protein levels increased, the inflammatory mediators C-reactive protein (CRP) and monocyte chemoattractant protein-1(MCP-1) expression was increased, IL-6(IL-6) and transforming growth factor-β(TGF-β) expression was decreased; while Urantide treatment group increased with time, AS pathological changes gradually reduced, thoracic aorta pathology close to the positive group at 14 day in rats; reduction of UⅡand its receptor GPR14 gene and protein expression,will down regulated the levels of CRP and reduction of inflammatory mediators MCP-1 and up IL-6 and TGF-βexpression.(2) In vitro: In this experiment, cultured VSMC as the object of study by UⅡstimulated VSMC proliferation, and then observed in the UⅡreceptor antagonist Urantide intervention, UⅡand its receptor GPR14 in VSMC and its possible mechanism. The results showed that, 10-8 mol/L UⅡat each time point could promote VSMC proliferation, cell cycle, so that the percentage of Sphase cells, PI and SPF were increased significantly promoted VSMC UⅡ, GPR14 gene and a large number of protein expression. Concentration in the range of 10-10～10-6, Urantide inhibited VSMC proliferation, inhibition of VSMC UⅡ, GPR14 gene and protein expression, 10-6 mol/L Urantide group had the most significant effect. These results suggest that, UⅡcan promote the VSMC mitogen role of rats, so that increased UⅡand its receptor GPR14 UⅡexpression; Urantide could antagonize UⅡpromote the role of VSMC mitogen, down UⅡand its receptor expression GPR14 UⅡ. From this we infer that, UⅡand its receptor GPR14 UⅡcan promote the occurrence , development of AS, and Urantide can block the process of AS .The conclusions are as follows: 1. AS group can be successfully replicated in rats, when VD3 70 U / kg was given by intraperitoneal injection , and fed with high fat diet during 4 W;2. UⅡand its receptor GPR14 gene and protein expressed a large number in AS rat thoracic aorta, Urantide can be down regulated UⅡ,its receptor GPR14 gene and protein expression;3. UⅡcan promote inflammatory mediators CRP and MCP-1 expression, inhibited the expression of IL-6 and TGF-β; while inflammatory mediators CRP and MCP-1 expression are decreased treated by Urantide, increased the IL-6 and TGF-βexpression;4. UⅡcan promote the proliferation of rat VSMC, increases in UⅡand its receptor GPR14 gene and protein expression; Urantide then could inhibit VSMC proliferation and lower VSMC in the UⅡand its receptor GPR14 gene and protein expression;5. UⅡcan promote VSMC cultured supernatants of CRP, MCP-I, IL-6 and ColⅣexpression, Urantide can reduced CRP, MCP-I, IL-6 and ColⅣexpression;The results provides a comprehensive,new experimental evidence for the treatment of AS. Urantide have a protective effect on AS, can reduce the symptoms of AS, therefore, expected to become a new target for clinical treatment of AS.Innovation of this study: intraperitoneal injection of VD3 70 U/kg combined with high fat diet in 4 W, AS model can be successfully replicated in rats; the first time will be applied to rats Urantide AS study, certify that Urantide can down regulated the UⅡand its receptor GPR14 gene and protein expression of thoracic aorta and VSMC in rats, reducing inflammation, improving the AS lesions.
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