The Mechanism Of Transforming Growth Factor-β1 Promoting Polarization Of M2 Macrophages Through FoxO1 Pathway To Attenuate Atherosclerosis

Objective:The purpose of this study was to confirm that transforming growth factor-β1(TGF-β1)can promote the polarization of M2 macrophages in atherosclerotic plaques by up-regulating Fox O1,thereby reducing the occurrence and progression of atherosclerosis.To explore the molecular mechanism of TGF-β1’s anti-inflammatory effect in atherosclerotic plaques through Fox O1 signaling pathway.Methods:In this study,8-week-old Apo E-/-mice were fed high-fat diets to establish atherosclerotic model.C57BL/6J mice were used as the control group,and their diets was maintained for 12 weeks.The mice in the disease group were divided into high-fat diet group,high-fat diet +TGF-β1 group,high-fat diet +TGF-β1+ICG-001 group,high-fat diet +ICG-001 group,high-fat diet + anti-CD86 antibody group,high-fat diet+TGF-β1+ anti-CD86 antibody group.Mice in the disease group were fed high-fat diets for 12 weeks,and each intervention was given from the 10 th week of high-fat feeding to the end of the 12 th week.After 12 weeks,the serum,aorta and liver of each group were collected.Serum lipid levels were detected by biochemical method.Aortic tissues of each group were stained with gross oil red O,aortic valve HE staining,Masson staining and oil red O staining,respectively,to observe the progression of atherosclerotic plaques in each group.The expression of macrophages and M2 macrophages in each group was detected by immunofluorescence chemistry.Western blotting was used to detect the expression levels of markers such as macrophage,M2 macrophage and Fox O1pathway-related proteins in atherosclerotic plaques.SPSS 26.0 statistical software was used for analysis and Graphpad Prism 9.2 was used for mapping.Measurement data were expressed by Mean ± standard deviation(Mean ± SD).T-test and Oneway ANOVA were used for statistical analysis.P < 0.05 was considered statistically significant.Results:1.Mouse atherosclerosis(AS)model was established.Elevated levels of TG,CHO and LDL in peripheral blood of mice,gross oil red O staining,aortic root oil red O staining,HE staining and Masson staining showed the formation of atherosclerotic plaque and a large amount of lipid deposition in arterial wall.The average fluorescence intensity of F4/80 in AS was higher than that of the Control group by immunofluorescence chemistry,and the expression of CD68 in arterial tissues of AS model mice was increased by western blot assay(P < 0.01).2.TGF-β1 and TGF-β1 combined with ICG-001 intervention showed a decrease in blood lipid indexes among all groups,and oil red O staining,aortic root oil red O staining,HE staining and Masson staining showed a decrease in atherosclerotic plaque area and lipid deposition compared with HF group.The plaque area of TGF-β1 combined with ICG-001 was reduced compared with that of TGF-β1 group.Immunofluorescence chemistry showed that the positive staining area and fluorescence intensity of F4/80 in the HF+TI group were lower than those in the HF+T group(P < 0.001),and the positive staining area and fluorescence intensity of CD206 in the HF+TI group were higher than those in the HF+T group,but there was no statistical significance(P > 0.05).The relative protein expression of CD68 in HF+TI group was lower than that in HF+T group(P <0.001),and the relative protein expression of CD206 was higher than that in HF+T group(P < 0.01).Compared with HF group,Fox O1 protein expression was increased after TGF-β1 intervention,and Fox O1 protein expression was further increased after combined TGF-β1 and ICG-001 intervention(P < 0.001).TGF-β1 combined with ICG-001 reduced the expression of P-Fox O1(P < 0.01).3.TG,CHO and LDL decreased after anti-CD86 monoclonal antibody intervention(P=0.0141,P < 0.05;P = 0.0007,P < 0.001;P=0.0253,P < 0.05),the plaque area of gross oil-red O staining and aortic valve area decreased compared with HF group.Immunofluorescence staining showed that F4/80 positive staining area and average fluorescence intensity of anti-CD86 monoclonal antibody were lower than those of HF group(P < 0.0001).Conclusion:1.TGF-β1 attenuated atherosclerotic plaque in mouse AS model,and TGF-β1combined with ICG-001 showed no less anti-atherosclerosis effect than TGF-β1 alone;2.TGF-β1 combined with ICG-001 activated and upregulated Fox O1 pathway to promote the polarization of M2 macrophages in plaques of AS model mice,and reduce local infiltration of macrophages in plaques and alleviate atherosclerosis;3.Anti-cd86 monoclonal antibody can reduce the infiltration of macrophages in plaque and atherosclerosis in AS model mice.