| Part one Construction of Mouse Vascular Restenosis ModelObjective:To construct a mouse vascular intimal hyperplasia model,and discuss its related mechanisms that cause intimal hyperplasia and lumen stenosis.Methods:According to the method reported in the literature[1],unilateral femoral arteries were induced by nesting mice with silicone tubes to induce vascular intimal hyperplasia,sham operation was performed on the control side,no cuff was put around the femoral artery after isolation.Body weightwas continuously monitored after the operation.Blood vessels were taken for tissue morphology analysis and the changes in blood flow in the bilateral femoral arteries of the mice were detected with a small animal ultrasound instrument as an auxiliary diagnostic indicator of restenosis.Hematoxylin-eosin staining(HE staining)was performed on the femoral artery sections,and then analyze the neointimal area,medial area,and the ratio of intima to medial(I/M)with ImageJ software.Results:Compared with the sham operation side,the blood flow velocity[(126.2334±21.43199)vs(70.97854±4.646153),P=0.002]of the femoral artery operation side of the mouse was significantly accelerated compared with the sham operation side,that means the cross-sectional area of the vascular cavity on the surgical side was smaller than on the control side.The femoral arterial sections of mice was collected and HE staining was performed,and then analyze the neointimal area,medial area,and the ratio of intima to medial(I/M)with ImageJ software.The results showed that the intima area[(2563.67 ±398.31)vs(7974.67± 1491,69),P=0.004],medial area[(6939.67± 570.507)vs(12227.67± 1487.02),P=0.005]and the I/M ratio[(0.37± 0.04)vs(0.66 ± 0.13),P=0.019]on surgical side were higher than the control side(Figure 3D),the difference was statistically significant.Conclusion:A simple and practical model of intimal hyperplasia induced by vascular adventitia in mice was successfully constructed.This model is of great significance for the research on the occurrence,development and mechanism of vascular intimal hyperplasia and atherosclerotic diseases.Part two Effect of albendazole on smooth muscle cells in vitro.Objective:To evaluate the effect of Albendazole(ABZ)on the biological function of VSMCs by vitro experiments,and briefly explored its mechanism.Methods:Each experiment in vitro used the same volume of DMSO as the control of the experimental group,and the concentration of DMSO in the medium ≤0.2%.MTT assay was used to detect the effects of ABZ on the proliferation of VSMCs and endothelial cells(ECs).3-D cell migration experiments and Annexin V-PI staining were used to evaluate the effects of ABZ on the migration and apoptosis of VSMCs.Cell scratch experiments were used to evaluate the effects of ABZ on ECs migration.In addition,during the migration of VSMCs,the dynamic changes of the cytoskeleton structure of filamentous actin(F-actin)were explored by phalloidin staining.Western blot was used to detect the phosphorylation of myosin phosphatase 1(MYPT1)and myosin light chain 2(MLC).Results:In vitro experiments confirmed that 48 hours after ABZ treatment,the proliferation of VSMCs were significantly inhibited in 0.5 μM[(0.60±0.03)vs(0.50 ±0.04),P<0.001]and 1 μm[(0.60±0.03)vs(0.33± 0.03),P<0.001],and the minimum effective concentration was 0.5 μM.Compared with the control group,after 48 hours,ABZ in 0.5 μM[(118.55± 3.39)vs(81.00 ± 4.00),P<0.001]and μM[(118.55± 3.39)vs(78.875± 3.01),P<0.001]significantly inhibited cell migration through collagen.The results of Flow cytometer showed that after 48 hours of ABZ treatment,0.5 μM concentration had no significant effect on apoptosis[(18.29 ± 2.79)vs(20.02±2.23),P=0.420],but it could promote apoptosis at 1.0 μM[(18.29 ± 2.79)vs(25.13± 1.35),P=0.002].Previous studies in our laboratory have shown that ABZ blocks the cell cycle of VSMCs in the G2/M phase.The cell proliferation experiment of ECs using the MTT kit sho,wed that after 48 hours,the ABZ concentration in 0.5 μM[(0.34± 0.11)vs(0.42 ±0.09),P=0.146]and 1 μM[(0.34 ± 0.11)vs(0.40± 0.10),P=0.427]has no significant inhibition on the proliferation of ECs compared with the control group.Cell scratch experiments were used to estimate the effects of ABZ on wound healing healing area in vitro.ImageJ software was used to quantify the scratch area at 0h and 24h.ECs were treated with ABZ in 0.5μM and 1 μM for 24 hours,the wound healing area was the difference between the 0h and 24h values.The wound healing areas were significiently reduced by ABZ[(85423.89 ± 29521.03)vs(18211.33± 4985.28),P<0.01]and[(85423.89 ± 29521.03)vs(21810.00± 17750.62),P<0.001].Western blot results showed that after ABZ were applied for 24 hours,the levels of MYPT1 phosphorylation in 0.5 μM[(0.64 ± 0.12)vs(0.37 ± 0.12),p=0.04]and 1.0 μM[(0.64 ± 0.12)vs(0.24 ± 0.07),p=0.007]were significantly reduced.MLC phosphorylation levels in 0.5μm[(0.89±0.06)vs(0.56 ± 0.13),p=0.01]and 1.0μm[(0.89 ± 0.06)vs(0.34 ± 0.08),p<0.001]were also significantly reduced.Conclusion:In vitro experiments showed that ABZ inhibited the proliferation and migration of VSMCs,and showed less effect on apoptosis,but has no effect on the proliferation of ECs.The effect of ABZ on VSMCs migration was related to the disassembly of F-actin on the cytoskeleton,ABZ significantly inhibited the disassembly of F-actin,and also reduced the phosphorylation of MLC and MYPT1.Part three Effects of Albendazole on the Intimal Hyperplasia andRemodeling of the Femoral Artery in MiceObjective:A mouse vascular intimal hyperplasia model was used to evaluate the effect of ABZ in vivo on the formation of restenosis after mouse femoral artery injury,and to explore the possibility of ABZ as a potential preventive drug for vascular restenosis.Methods:10-week-old wild-type male C57BL/6 mice were used for experiment,and a cuff was used to nest the one side femoral artery of the mouse to induce a model of vascular intimal hyperplasia,and the other side was subjected to sham surgery.The other side was sham operated.Mice were randomly divided into an experimental group and a control group after operation,and the two groups of mice were fed with high-fat diet at the same time.During the experiment,ABZ was administered orally at a dose of 1.5 mg/day,and the control group was administered orally with the same volume of sesame oil.After 4 weeks,the severity of vascular intimal hyperplasia was evaluated with a small animal ultrasound system.The morphological changes of vascular tissues were observed after HE staining.The ImageJ software was used to quantify the intimal area,medial area,and ratio of intima to media(I/M).Immunohistochemical staining was used to evaluate the expression of smooth muscle cell phenotypic switching molecules(α-SMA),endothelial cell marker(CD31),macrophage marker(Mac-3),and smooth muscle cell nuclear proliferation antigen(PCNA).Results:Compared with the control group,the difference in blood flow velocity in the bilateral arteries of the experimental group was statistically reduced[(40.37± 2.88)vs(25.08±7.37),P=0.008],that means after ABZ treated,the cross-sectional area of the femoral artery lumen become larger than the control side,and the degree of stenosis was improved.The results of HE staining showed that ABZ significantly inhibited the intimal hyperplasia[(5605.79± 1246.96)vs(2988.66 ± 365.58),P=0.017]and the ratio of I/M[(1.12 ± 0.20)vs.(0.76 ± 0.15),P=0.005],there was no significant effect on the median area[(4114.46 ± 970.57)vs(3913.79 ± 528.51),P=0.661].The results of immunohistochemical staining showed that α-SMA positive cells were significantly reduced[(0.35±0.02)vs(0.21 ± 0.04),P=0.008].CD31 staining results indicate that ABZ has no effect on re-endothelialization.PCNA staining results showed that the number of PCNA-positive cells was significantly reduced after ABZ treatment[(0.32± 0.03)vs(0.20± 0.04),P=0.007].Compared with the control group,the number of positive macrophages also decreased[(0.08±0.02)vs(0.04± 0.01),P=0.03].Conclusion:ABZ effectively improved arterial intimal hyperplasia and vascular wall remodeling by acting on VSMCs and inflammatory cells,and has a satisfactory preventive effect on the formation of vascular restenosis after surgery. |
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