| Objective:The pathogenesis of Atherosclerosis(AS)involves such characteristic chronic inflammatory lesions AS activation of inflammatory cells,abnormal lipid deposition and plaque formation in the artery intima,thus AS has previously been defined as a chronic inflammatory disease.However,an increasing amount of evidence suggests that immune regulation is involved in and affects the occurrence and development of AS.CD4+T cells play an auxiliary role in cellular immunity,while helper cell 17(Th17)and Regulatory T cells(Treg)cells have been confirmed to play a regulatory role in AS progression,and their specific mechanisms need further study.Glucagon-like peptide-1(GLP-1)has been clinically observed to be beneficial to patients with AS.Currently,studies have shown that GLP-1 has lipid-lowering and immunomodulatory effects,but the mechanism of cardiovascular benefits brought by GLP-1 is not very clear.To some extent,this has hindered the wide clinical application of GLP-1.At present,there is little evidence on whether GLP-1 influences the progression of atherosclerosis by regulating immune imbalance,and whether this process is related to CD4+T cell differentiation.The purpose of this study was to explore whether GLP-1 intervention in CD4+T cells caused functional changes of macrophages co-cultured with T cells,and to further explore the mechanism related to GLP-1 regulation of Treg/Th17 ratio and delay of AS in vitro.Methods:Abdominal macrophages and spleen CD4+T cells were isolated from C57 male mice aged 12 weeks.CD4+T cells were divided into five groups: blank control group,GLP-1 10nmol/L,GLP-1 100nmol/L,GLP-1 inhibitor Avexitide 100nmol/L,Avexitide100nmol/L+GLP-1 100nmol/L.After 24 h of intervention,the contents of IL-10 and IL-17 in the superneant were determined by enzyme-linked immunoassay.Treg cells were isolated for western and PCR analysis.The related indexes of Treg cells were Foxp3,IL-10,and RORγt,IL-17.The above five groups of CD4+T cells were transwell co-cultured with abdominal macrophages.CD4+T cells were removed 24 h later,and co-cultured macrophages were Oxidized Low Density Lipoprotein(Ox-LDL).Oil red O staining was performed to observe and calculate the degree of macrophages phagocytosis of lipids.The cholesterol effluence indicators of macrophages,such as ABCA1 and ABCG1,and the key indicators of the pathway involved,such as PI3 K,AKT and m TOR,were determined by western and PCR.Results:1.mRNA and protein expression levels of Foxp3 and RORγt in CD4+T cellsCompared with blank control group,Foxp3 mRNA and protein expression levels increased in GLP-1 intervention group,while RORγt mRNA and protein expression levels decreased in Avexitide intervention group,Foxp3 mRNA and protein expression levels decreased while RORγt mRNA and protein expression levels increased in Avexitide intervention group.The differences were statistically significant(p<0.05).Compared with Avexitide intervention group,the mRNA and protein expressions of Foxp3 in Avexitide+GLP-1 intervention group were increased,while the mRNA and protein expressions of RORγt were decreased,with statistical significance(p<0.05).2.mRNA,protein expression and cytokine secretion levels of IL-10 and IL-17 in CD4+T cellsCompared with the blank control group,the mRNA level,protein expression level and secretion level of IL-10 in GLP-1 intervention group were increased,while the mRNA,protein expression level and secretion level of IL-17 were decreased,and the mRNA,protein expression level and secretion level of IL-10 in Avexitide intervention group were decreased.The mRNA,protein expression and secretion levels of IL-17 were increased,and the differences were statistically significant(p<0.05).Compared with Avexitide intervention group,mRNA,protein expression and secretion levels of IL-10 were increased in Avexitide+GLP-1 intervention group,while mRNA levels of IL-17 were decreased,with statistical significance(p<0.05).3.Ox-LDL induced macrophage foam and oil red O stainingCompared with blank control group,the degree of cell foam in GLP-1 intervention group was decreased,and that in Avexitide+GLP-1 intervention group was decreased,the difference was statistically significant(p<0.05).4.mRNA and protein expression levels of ABCA1 and ABCG1 in CD4+T cocultured macrophagesCompared with blank control group,mRNA and protein expressions of ABCA1 and ABCG1 in GLP-1 intervention group were increased,while those in Avexitide intervention group were decreased,with statistical significance(p<0.05).Compared with Avexitide intervention group,mRNA expression levels of ABCA1 and ABCG1 in Avexitide+GLP-1 intervention group were increased,with statistical significance(p<0.05).5.Explore the mechanism of GLP-1 and its antagonists regulating the proportion and function of CD4+T cells differentiationThe protein-protein interaction network between GLP-1 and CD4+T cells predicted by String database showed that the PI3K/AKT/m TOR pathway was a node connecting key proteins related to GLP-1/GLP-1R and Treg/Th17 cells.On this basis,PCR verification showed that compared with blank control group,mRNA expressions of PI3 K,AKT and m TOR in GLP-1 intervention group were decreased,while mRNA expressions of PI3 K,AKT and m TOR in Avexitide intervention group were increased,with statistical significance(p<0.05).Compared with Avexitide intervention group,mRNA expressions of PI3 K,AKT and m TOR in Avexitide+GLP-1 intervention group were decreased,and the differences were statistically significant(p<0.05).Conclusion:The proportion of CD4+T cells differentiated into Treg/Th17 cells regulated by GLP-1 and its receptor antagonist Avexitide was related to PI3K/AKT/m TOR pathway.The increase of Treg/Th17 cells reduced macrophage foam by affecting the cholesterol effluence pathway.GLP-1 up-regulates the proportion of CD4+T cells that differentiate into Treg/Th17 cells and reduces macrophage foaming in atherosclerosis by affecting cholesterol effluence. |
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