Comparison Of Intimal Hyperplasia In Different Apoe^-/- Mice After Carotid Artery Ligation And Study On The Intervention Of N-acetylcysteine（NAC）

Objective: Stroke is a leading cause of disability in Chinese adults,which seriously affects the patients’ ability to work and quality of life.Carotid atherosclerosis is one of the main cause of ischemic stroke,the short-term result of endovascular treatment for carotid artery stenosis or occlusion is fine.However,post-angioplastic restenosis and occlusion are still common problems in clinical treatment.Intimal hyperplasia is the main pathological process of post-angioplastic restenosis.Previous studies have shown that apolipoprotein E-knockout mice feeding with a Western diet,the lesions in the ligated carotid artery rapidly progress from a fatty streak consisting of macrophage-derived foam cells,to a fibrous plaque involving smooth muscle cells,to an advanced lesion containing neovessels.Macrophages,neutrophils and smooth muscle cells can be observed in the neointimal lesion.Previous studies have shown that inflammation and oxidative stress are the factors that affect the migration and proliferation of vascular smooth muscle cells(VSMC).The genetic susceptibility of C3H/He J(C3H)and C57BL6(B6)apolipoprotein E knockout mice to atherosclerosis was significantly different,analyzing the differences of cell components in the two strains are helpful to reveal mechanism of genetic susceptibility difference to atherosclerosis.In this study,we first compared the lesion size and lesion components of the two strains of mice under a Western diet,and analyzed the factors that may affect the formation of neointimal lesion.By intervention of macrophage and neutrophil infiltration and regulating the level of oxidative stress,the effect of these factors on the neointimal lesion was observed in B6 apolipoprotein E knockout mice which is susceptible to atherosclerosis,and analyze the possible mechanism.Then,after cultured vascular smooth muscle cells stimulated by ox-LDL,the relationship between N-acetylcysteine(NAC)and oxidative stress,proliferation and migration of vascular smooth muscle cells,and the effect of NAC on the secretion of inflammatory factors were analyzed.Methods: 1.In animal experiments,group A: B6-Apoe-/-and C3H-Apoe-/-of two genders started a Western diet at the age of 6 weeks,which lasted for 12 weeks,and bilateral carotid artery samples were obtained.Group B:4-8 week-old B6-Apoe-/-and C3H-Apoe-/-mice of two genders were used for carotid ligation,a Western diet was given 1 week before surgery and continued after ligation,mice were euthanized at 1 or 3 days or 1,2,or 4 weeks after surgery,the left and right common carotid arteries were obtained;plasma lipids level were measured by ELISA.In pathological examination,morphological measurement such as area of neointimal lesion,medial area were calculated.Cell components were analyzed by immunohistochemistry.In vivo intervention experiment,Group C: 8-week-old mice of two genders,mep1 α-/-/ Apoe-/-mice on a B6 genetic background were intraperitoneally injected with 250 μg of Ly6 G antibody per mouse every other day for 4 weeks,starting the day before carotid ligation.For mice that were not treated with the antibody,an equivalent amount of saline was administered.The mice were euthanized 4 weeks after surgery,and the heart and bilateral carotid tissues were obtained.Group D: 5-12 week-old mice of two genders,Apoe-/-mice on a B6 genetic background were intraperitoneally injected with 200 μg of anti CSF-1R antibody per mouse every 3 day for 2 weeks,starting the day before surgery.For mice that were not treated with the antibody,an equivalent amount of saline was administered.The mice were euthanized 2 weeks after operation,and the liver and bilateral carotid tissues were obtained.Group E: 8-week-old mice of two genders,left carotid ligation of Apoe-/-mice on a B6 genetic background were performed,a Western diet was given 1 week before ligation and continued after sugery,NAC(calculated according to 1.5g/ kg/day and daily drinking water of a 20 g mouse is about 4-6ml)was mixed in drinking water and fed from 1 week before operation,and lasted for 4 weeks after operation.Mice that were not treated with NAC were fed with normal water.The mice were euthanized 4 weeks after operation,and the bilateral carotid tissues were obtained.Plasma levels of inflammatory factors were measured by ELISA.In pathological examination,the area of intimal lesions were calculated.Cell components of neointimal lesion,aortic root lesion and specific cellular components of liver tissue were analyzed by immunohistochemistry.2.Human vascular smooth muscle cells were used in cell experiment,stimulation of vascular smooth muscle cells with 50 μg / ml ox-LDL,detection of proliferation ability of vascular smooth muscle cells by MTT,detection of migration ability of vascular smooth muscle cells by scratch test,measurement of ROS in vascular smooth muscle by flow cytometry.The levels of monocyte chemoattractant protein-1(MCP-1),tumor necrosis factor-α(TNF-α),interleukin-10(IL-10)and nitric oxide(NO)in the supernatant were detected by ELISA or spectrophotometry.Results: 1.After 12 weeks of Western diet,both B6-Apoe-/-and C3H-Apoe-/-mice developed severe hyperlipidemia.Compared with B6-Apoe-/-mice,the plasma lipid levels of C3H-Apoe-/-mice were higher.After 12 weeks of Western diet,B6-Apoe-/-mice developed progressive intimal lesion,while C3H-Apoe-/-mice developed no lesion.After ligation of carotid artery,at 1 week,2 weeks,4 weeks,B6-Apoe-/-mice showed greater intimal lesion than C3H-Apoe-/-mice.In terms of histopathology,C3H-Apoe-/-mice showed fatty streak lesions at 2 weeks after ligation,and by 4 weeks fibrous lesions had formed,although they were smaller than in B6-Apoe-/-mice,neovascularization and severe lumen stenosis occurred in B6-Apoe-/-mice at 4 weeks.The early neutrophil adhesion to endothelial cells and neutrophil infiltration in intimal lesions were observed in the carotid arteries of both strains of mice.In B6-Apoe-/-mice,macrophages infiltration of intimal lesions were found at 1 week,and macrophages infiltration were found at 2 weeks and 4 weeks in the two strains of mice.CD4 + T cells were observed in C3H-Apoe-/-mice,but not in B6-Apoe-/-mice.Smooth muscle cells staining were found in intimal lesion of two strains of mice,the immunoreactivity of α-smooth muscle actin in medial layer decreased.Compared with the control group,the neutrophil infiltration in the anti-Ly6 G antibody treatment group were significantly reduced,but the size of neointimal lesion was comparable with the control group.After anti-CSF-1R antibody treatment,the results of macrophage depletion were not stable,and the size of neointimal lesion in the treatment group was similar with that in the control group.The neointimal lesion size of antioxidant NAC treatment group was smaller than that of control group.2.After the vascular smooth muscle cells were stimulated with 50 μg/ml ox-LDL,it was found that 8000 cells per well group of smooth muscle cells could proliferate smoothly.NAC can significantly inhibit VSMC proliferation at 1m M and 2m M.In the scratch test,the migration area of vascular smooth muscle cells in 2m M NAC treatment group was smaller than that in the control group at 24 h and 48 h.Flow cytometry showed that 1m M NAC and 2m M NAC could inhibit ROS level of VSMC.The MCP-1,TNF-α,IL-10 and NO in the supernatant were detected by ELISA or spectrophotometry.It was found that NAC treatment after ox-LDL stimulated could inhibit the secretion of MCP-1 and TNF-α by vascular smooth cells(P < 0.05),and promote the secretion of IL-10 and NO by vascular smooth cells(P < 0.05).Conclusion: 1.The genetic background and arterial blood flow play an important role in the formation of carotid atherosclerosis in hyperlipidemic Apoe-/-mice.Neutrophils did not play a significant role in neointimal formation after carotid artery ligation in B6-Apoe-/-mice.NAC treatment inhibited intimal hyperplasia of B6-Apoe-/-mice after carotid artery ligation,partly because NAC can regulate the plasma levels of MCP-1,TNF-α and IL-10 in mice.2.NAC inhibited the migration and proliferation of vascular smooth muscle cells by inhibiting ROS.NAC can inhibit the proliferation and migration of VSMC by inhibiting the secretion of VSMC MCP-1 and TNF-α.NAC has the effect of promoting the secretion of VSMC IL-10 and NO.Antioxidant NAC is expected to be an effective drug to prevent arteriosclerosis occlusion and post-angioplastic restenosis.