| Background Macrophage-mediated inflammatory response plays an important role in the formation of atherosclerosis.Our previous cytological study found that ox-LDL promotes the M2 polarization of macrophages,enhances the phagocytosis of macrophages,and reduces the survival rate of macrophages,leading to an increase in IL-10 secretion and a decrease in TNFα secretion.The β-receptor agonist salbutamol promoted this effect,while theβ2-receptor antagonist ICI118551 inhibited it.Objective To compare the differences of atherosclerosis and macrophage polarization in apoe-/-mice fed with high-fat diet or normal diet,and investigate the effects of β2-adrenergic receptor inhibition of macrophage M2 polarization on atherosclerosis in apoe-/-mice.Methods Male apoe-/-mice with an average age of 5–6 weeks were randomly divided into four groups,and they were given a normal diet(Control group),a high-fat diet(Model group),a high-fat diet with β2 receptor stimulant salbutamol(SAL group),or a high-fat diet with β2 receptor antagonist ICI118551(ICI group)respectively after a week of adaptive feeding.They were sacrificed after feeding for 12 weeks.Blood samples were taken for determination of serum lipids,TNFα-α and IL-10.The mice were dissected and the large blood vessels were taken from the aortic root to the abdominal aortic bifurcation.The whole blood vessel was longitudinally cut open for oil red O staining,and the occurrence of AS plaques in each blood vessel segment of each group was observed according to the anatomical segments,and the plaque destruction rate was measured quantitatively.After transverse sections of the aortic roots were cut,HE staining was performed to compare the severity of AS plaques among different groups.The distribution and characteristics of M1/M2 cells in AS plaques corresponding to vascular segments in the shoulder of AS plaques and the vascular wall corresponding to AS plaques among different groups were compared by immunofluorescence staining.To compare the distribution characteristics and cell number ratio of M1/M2 cells in the samples with AS plaque rupture between the albuterol group and the ICI118551 group.Results(1)There was no significant difference in body weight of apoe-/-mice among different groups.Compared with the control group(fed with ordinary diet),the total cholesterol,low density lipoprotein cholesterol and triglyceride in the blood of mice in the model group(fed with high quality diet)were increased significantly,and there was no significant change in high density lipoprotein cholesterol.(2)The blood TNFα levels in the model group were significantly lower than those in the control group and ICI group,but significantly higher than those in the SAL group.IL-10 levels were significantly higher in the control group and the ICI group,but significantly lower than the SAL group.(3)Oil red O staining of longitudinal sections of aorta shows that the fatty plaque on the vascular wall of the typical sample was bright red,and almost colorless in other parts.The fat plaque area in the model group was significantly higher than that in the control group,while it was further increased in the SAL group and decreased in the ICI group as compared with that in the model group.Quantitative analysis showed that the plaque destruction rates in the control group and ICI group were lower than that in the model group,and that in the SAL group was higher than that in the model group.Moreover,there were significant differences in plaque destruction rate between each group and the model group.The plaque destruction rate in the ICI group was higher than that in the control group and there was a significant difference.(4)Qualitative analysis by HE staining of transverse sections of aortic roots showed that after 12 weeks of high-fat diet,the atherosclerotic plaques in the model group developed well.On the contrary,after 12 weeks of normal diet,no atherosclerotic plaques were found in the control group.Morphological analysis showed that the inner and outer layers of the vascular wall in the control group were normal.A large number of atherosclerotic plaques,cholesterol crystals and foam cells were observed in the model group.SAL group had more foam cells,disintegrants and cholesterol crystals,while ICI group had normal blood vessels and some foam cells.(5)Immunofluorescence staining of transverse sections of aortic roots showed that in the blood vessels at aortic roots of apoe-/-mice(control group)fed with ordinary diet for12 weeks,CD86+ cells(M1 type macrophages)were occasionally observed at the endovascular lumen,and CD163+ cells(M2 type macrophages)were observed at the adventitia.However,the total number was not large,and no double-positive co-localization of CD86/CD163 was observed.The model group found an adjacent lipid-striated plaque and an AS developing plaque on the same section.The M1 macrophage population was observed in the shoulder of the lipid-striated plaque,and CD163+ cells and the double positive co-localization of CD86/CD163 were observed in the translocation area of the partial lipid-striated core in the M1 macrophage population.More M1 type macrophage population could be seen in the shoulder of developing plaques,CD86/ CD163 double positive co-localization could be seen at the center of plaques above the shoulder,more CD163+macrophages could be seen near the lipid core,and no cells could be seen at the lipid core.In the albuterol group,multiple developing plaques and developed plaques were observed on the same section,and one relatively independent plaque was selected.A large number of M1 macrophages were observed at the shoulder of the plaque,and a large number of CD86/CD163 double-positive co-localization were observed at the M1 macrophages partial to the central side of the plaque.CD163+ cells were observed near the lipid core,and no cells were observed at the lipid core.No CD86-or CD163-positive stained cells was observed in the intima and CD86-positive macrophages in the adventitia of the ICI118551 group,and no CD163-positive stained cells was observed.(6)Quantitative analysis of immunofluorescence staining of transverse sections of aortic roots showed that compared with the control group,the total number of macrophages,the number of CD86 positive cells,the number of CD163 positive cells and the ratio of CD163/CD86 positive cells in the model group were all higher than those in the control group with significant differences.Compared with the model group,the above indicators in the SAL group were higher,while those in the ICI group were lower.Conclusion 1.Hyperlipidemia can induce M2-type differentiation of macrophages in atherosclerosis plaques,increase the number of macrophages under the intima,enhance the M2-type polarization of macrophages,and promote the progression of atherosclerosis.2.β2-adrenergic receptor antagonist can inhibit the increase in the number of macrophages in plaques induced by hyperlipidemia,and inhibit the macrophages from differentiating into M2 type,thus slowing the progression of atherosclerosis. |
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