| Percutaneous transluminal coronary angioplasty is one of the most important methods for treating coronary heart disease in clinic, but restenosis after operation has reduced it's long effect. Many methords have been adopted to solve the problem, including high pressure balloon dilat, stent implanted, balloon cutting and laser plasty, but 10%-30% patients can be found the restenosis in stent or margin. Fingding the right drug to prevent and treat the stent restenosis is of great significance, which can reduce cardiovascular events in patients with coronary heart disease, improve quality of life, prolong life and alleviate the financial burden on individual countries. The intimal proliferation is the main pathological changes in restenosis, while the local inflammatory response is the main reason. The applications of anti-inflammatory may reduce intimal hyperplasia, The statins are lipid lowering drugs that effectively lower LDL cholesterol levels and reduce the risk of cardiovascular events in hyperlipidemic and normocholesterolemic patients.These clinical studies also suggest that statins exert vasculoprotective effects that cannot be fully explained by their cholesterol-lowering properties. Other studies indicate that atherosclerosis is not solely a lipid disorder but also an inflammatory disease. Statins have been found to exert antiinflammatory effects in vascular cells both in vitro and in vivo, indicating that they may exert important protective effects in the arterial wall. they can inhibite the smooth muscle cell proliferation, keep the plaque stable and improve the blood vessel endothelium function in atherosclerosis. We made the model of rabbits after balloon injury in abdominal aorta and culture the VSMC to evaluate the effect of rosuvastatin on neointimal hyperplasia, lumens stenosis and the effect on expression of TGF-β1, inorder to reveal its mechanism in restenosis.Part 1: Objective: Comparing different balloon's effect in making the balloon injuried artery models, finding the right balloon and methods to make the balloon injuried artery models.Methods :Methods 30 male new zealand rabbits were diveded into three groups, one is sham operated group, the others were made the balloon injuried artery madel by 2.5mm and 3.5mm balloons. The three groups were administrated the aspiran 25mg/d, and clopidogrel 25mg/d. After 4 weeks feeding,rabbits were killed by air embolism. 2cm segment of vascular tissue at the balloon injury were cut off, and we made 5μm thick sections and stained with HE, measured arterial intima and media thickness by the optical microscope, and calculated cross sections and Luminal diameter, neointimal smooth muscle cell layer and the middle layer of average thickness, and the average of three horizons, finally ratio of intima / media was calculated.Result :1. Comparison of the general state of the rabbits after the surgery: All rabbits were successfully operated, in 10mins-40mins, the time of the sham operated group was 15.5±3.28mins, which was shorter than the time of the 2.5mm balloon group 28.6±4.09mins, and shorter than the 3.5mm balloon group's time 34.9±6.06min, the operation time of the three groups obtained significant statistical difference, (P <0.05). 26 rabbits of the three groups began to drink water and food on the second day after the surgery, 3 rabbits on the third day. All returned to normal diet and exercise one week later. During 4 weeks feeding time, only one rabbit died of diarrhea 3 weeks later, the remaining 29 rabbits survived to the end of the experiment, without complications, the success rate was 95%. There was no significant difference in postoperative feeding and motility recovery.2. The observation of neointimal hyperplasia by optical microscope: HE staining disclosed that the vessel wall of the two groups balloon injury were not smooth, intima thickened, lumina narrowed, some of the internal elastic plate fractured under light microscope, the vascular smooth muscle cells near the internal elastic layer were arranged vertically, tending to grow to the direction of the endomembrane. Compared with the sham operated group, the average degree of arterial stenosis were 22.1%±3.5% and 32.5%±5.8%.The three groups were statistically significant, 3.5mm balloon group was more narrow than the 2.5mm group, which was more narrower than the sham group. The 3.5mm balloon group's intima thickness was 0.07±0.03 mm thicker than 2.5mm balloon group (0.03±0.02mm) and sham operated group (0.02±0.03mm), were significantly different (P <0.05). The middle thickness of 3.5mm balloon group was 0.16±0.03mm greater than the sham group's 0.12±0.02mm, were significantly different (P <0.05), but compared with the 2.5mm balloon group (0.14±0.02mm) , there is no statistically significant difference (P> 0.05). The ratio of Intima /middle in 3.5mm balloon group was 0.44±0.11 greater than 2.5mm balloon group's 0.21±0.05, and both balloon groups are higher than sham operation group's 0.17±0.06, three groups were statistically significant pairwise comparison (P> 0.05).Conclusion : The 3.5mm balloon cather was better than 2.5mm balloon cather in making the modle of artery balloon injury. The 3.5mm balloon cather was the ideal balloon catheter.Part 2:Objective: Observing the effect of rosuvastatin on progression of abdominal aorta intima-media thickness in rabbits injured by balloon.Method: 20 male new zealand rabbits made the model of abdominal aorta injured by balloon were divided in two groups: the rosuvastatin group and the control. Both of groups were administrated the aspiran 25mg/d, and clopidogrel 25mg/d, and the rosuvastatin group received rosuvastatin 1mg/(kg·d) in addition. 6 weeks later we collected the blood from the otomarginal vein of the rabbits, and measured the levels of total plasma cholesterol, triglyceride, high and low density lipoprotein-cholesterol,blood glucose, the homocysteic acid ,the function of liver and kidney by enzyme linked immunosorbent assay.and after we finished the abdominal aorta angiography, rabbits were killed by air embolism. 2cm segment of vascular tissue at the balloon injury were cut off, and we made 5μm thick sections and stained with HE, measured arterial intima and media thickness by the optical microscope, and calculated cross sections and Luminal diameter, neointimal smooth muscle cell layer and the middle layer of average thickness, and the average of three horizons, finally ratio of intima / media was got.Result :1. The blood biochemical of two groups:It shows that the liver and renal function and the level of homocysteine are no difference between two groups(P>0.05). But the Lipoprotein profiles are difference.The total plasma cholesterol(0.8±0.45 mmol/L), triglyceride(0.9±0.22 mmol/L) and low density lipoproteins(0.4±0.05 mmol/L) levels are lower in the rosuvastatin-treated group than that of (1.2±0.45mmol/L, 1.3±0.32 mmol/L, 0.6±0.11 mmol/L) in control. There are significances in statistics(P<0.05).2. The abdominal aorta angiography: We found that the distal of the aorta's intimas were rough from the result of abdominal aorta angiography, the lumen of blood vessels were stenosed about 30-50%, the smallest inner diameters were slightly larger in rosuvastatin-treated group(1.74±0.25) than that of the control(1.32±0.33), and the average stenosis degree was better in rosuvastatin-treated group (31.41%±7.08) than that of the control (55.25%±10.23). There are significances in statistics (P<0.05). But the inner diameters of reference vessel and the average length of stenosis are no significances (P>0.05).3. The result of hematoxylin-eosin staining: The inner surface of the vessel wall of the injured arteries were rough,the intimas were thickening, and the lumen of blood vessels were stenosed in both groups. The internal diameter of the cross section was 3.3±0.32 mm in rosuvastatin-treated group and 3.1±0.26 mm in control, nearly 7% was increased. And the thickness of intema and the ratio of the intima and the middle(0.04±0.02 mm,0.29±0.10 mm) were reduced 55% and 42% when compared with the control (0.08±0.03 mm, 0.50±0.11 mm) (P<0.05), but the thickness of the middle and the internal diameter of the cross section were not significantly different between two groups. Conclusion: Rosuvastatin can decrease the level of LDLC, TC, and TG. and it can inhibit the neointimal hyperplasia after balloon injury.Part 3:Objective: Observing the effect of rosuvastatin on expression of TGF-β1 in abdominal aorta of rabbits injured by balloon.Method: Using the section of artery balloon injury made before, Analyzing the level of TGF-β1 protein by standard immunohistochemistry, and the level of TGF-β1 mRNA by reverse transcription-polymerase chain reaction. Using UVP gel density scanner, detected the optical density and calculated the relative ratio.Result :1. The expression of TGF-β1 in abdominal aorta of rabbits by immunohistochemistry: The brown particle of TGF-β1 protein were found in both groups.they were analyzed by Imagepro plus 6.0 software, Comparison of the average absorbency of stained TGF-β1 in intima and the middle in both groups. In rosuvastatin-treated group, the average absorbency and the area of positive of TGF-β1 were dramatically lower than that in control group (0.413±0.13 vs 0.568±0.17, P < 0.05, 22.37±4.42% vs 40.58±7.45%, P < 0.05).2. The result of RT-PCR showed: there are 247bp's DNA amplification products in both groups's intima and middle. The quantity of DNA (25.32±5.13) was lower than that of the control(42.47±5.41), There were significant differences between the two groups, (P <0.05).Conclusion: Rosuvastatin can inhibit the expression of TGF-β1 in artery wall of rabbit after balloon injury.Part 4:Objective: Observing the effect of rosuvastatin on expression of TGF-β1 and SMAD3 in VSMC.Method: Primary cultured human umbilical vein smooth muscle cells, and passaged to 3-6, and stimulated with different concentrations of rosuvastatin calcium range from 10-7 to 10-3mol/L for 12 hours and 24 hours. And detected the expression of TGF-β1 and SMAD protein with Western-blot, and detected the expression of TGF-β1 and SMAD gene with RT-PCR。Result:1. Western blot analysis showed rosuvastatin calcium in concentration of 10-7-10-3mol/L can inhibit the expression of TGF-β1 protein. After stimulating 12 hours, TGF-β1 protein expression was gradually reduced dependant the concentration of rosuvastatin calcium increasing from 10-7-10-5mol/L, there were significant differences (P <0.05). Continue to increase rosuvastatin calcium concentration, the inhibiting effect was reduced, and the expression of TGF-β1 protein increased. After stimulating 24 hours, TGF-β1 protein expression was apparently discreased by rosuvastatin calcium in concentration of 10-7-10-3mol/L than the control, there were significant differences (P <0.05), but there was no significant differences between each of 10-7-10-3mol/L group(P >0.05).2. Effect of rosuvastatin on expression of TGF-β1 mRNA in VSMC: RT-PCR showed the expression of TGF-β1 mRNA in concentration of 10-5-10-3mol/L rosuvastatin calcium were lower than that of the control after simulating 3 hours. There were significant differences (P <0.05). The expression of TGF-β1 mRNA in concentration of 10-4 and 10-3mol/L rosuvastatin calcium group were lower than that of the 10-3mol/L group. There were significant differences (P <0.05). But after simulating 6 hours, The expression of TGF-β1 mRNA in all the rosuvastatin calcium group concentration from 10-7 to 10-3mol/L were lower than that of the control. There were significant differences (P <0.05). The quantity of TGF-β1 mRNA were discreasing with the rosuvastatin's concentration increasing. There is no significant differences between the 10-7mol/L group and 10-6 mol/L group, (P>0.05), but both of the groups and the 10-3mol/L group's expression were higher than that of the 10-5mol/L group, There were significant differences (P <0.05). and there is no significant differences between the 10-5mol/L group and 10-4mol/L group, (P>0.05).3. Effect of rosuvastatin on expression of SMAD3 protein in VSMC: The protein expression of SMAD3 were 84.652±14.5%, 70.651±8.71% and 78.245±5.21% of the control in 10-5-10-3mol/L groups after simulating 12 hours, there were significant differences (P <0.05). and they are 91.874±4.25% and 92.745±8.24% of the control in 10-7 mol/L and 10-6mol/L. There is no significant differences compared with the control. The protein expression of SMAD3 was lower only in 10-4 mol/L group compared with the 10-5 mol/L group, there were significant differences (P <0.05). After simulating 24 hours, The protein expression of SMAD3 were 85.547±8.47%, 75.698±7.24%, 68.354±7.65%, 57.568±9.32% and 80.536±6.35% of the control, there were significant differences (P <0.05). The expression were higher in 10-7mol/L and 10-3mol/L groups than in 10-5mol/L group, there were significant differences (P <0.05).Conclusion: Rosuvastatin can inhibit the expression of TGF-β1 in VSMC, it can inhibit the VSMC proliferation indirectly. |
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