The Study On The Regulation Mechanism Of CCL20 And IL-17 In RAW264.7 And Their Correlations With Coronary Heart Disease

Background:Atherosclerosis(AS)is the main cause of coronary heart disease(CHD)and stroke,features inflammatory reaction,oxidative modification and lipid deposition.Macrophages and T cells can accelerate the pathological process of AS through activating and recruiting some inflammatory factors into plaque such as adhesion molecules,chemokines,and tumor necrosis factors.Chemokine act as a regulating factor of leukocyte transport in the process of immune surveillance and inflammation.Factors that affect the initial inflammatory response such as the generation of free radicals,the activation of the NF-?B pathway,the release of TNF-?and complement activation may induce the synthesis of chemokines.Chemokines CCL20 together with its sole receptor CCR6 is refered to as the called CCL20-CCR6 axis.It is important for regulating the atherosclerosis process through arresting mononuclear cells from bone marrow and recruiting into blood vessel walls.Inflammatory cytokine IL-17 is mainly secreted by Th17 cells and regulated by CCL20-CCR6 axis.However,the CCL20 and IL-17 expression in CHD especially in AMI and its regulation mechanisms in macrophages are still not clear.Objectives:To study the regulation mechanism of CCL20 and il-17 in macrophages and their relationship with coronary heart disease in mouse RAW264.7 cells and patients with coronary heart disease in China.Methods:Part one:1.OxLDL was used to stimulate RAW264.7 with different concentrations and time and the expression levels of CCL20 and IL-17 were tested in the model;2.Atorvastatin was used to interfere with the oxLDL induced model and the expression of CCL20 and IL-17 were tested;Part two:1.The activity of NF-?B was specifically inhibited in oxLDL induced model and Atorvastatin intervention model,the expression of CCL20 and IL-17 were tested;2.CCL20 overexpression plasmid was transfected into RAW264.7,the activity of NF-?B and the expression of IL-17 were tested;3.AKT-specific inhibitor and agonist were used to explore the relationship between AKT/NF-?B pathway and CCL20 and IL-17.Part three:1.Total of 206 patients were recruited and the CCR6 mRNA,CCL20 and IL-17expression were tested by RT-PCR and ELISA respectively;2.The correlations between CCR6 mRNA level,CCL20 and IL-17 concentration were analyzed,the correlations between these markers and severity of coronary artery stenosis were analyzed,and risk factors of CHD were analyzed by logistic regression.Results:1.OxLDL upregulated CCL20 and IL-17 expression in RAW264.7 in a concentration-dependent and time-dependent manner;2.Atorvastatin downregulated the expression of CCL20 and IL-17 in RAW264.7 induced by oxLDL,and in a concentration-dependent manner;3.NF-?B pathway participated in the regulation process of CCL20 and IL-17;4.The overexpression of CCL20 in RAW264.7 could activate NF-?B activity and regulate the secretion of IL-17;5.AKT may act as the upstream of the NF-?B signaling pathway to regulate the expression of CCL20 and IL-17;6.CCL20 and IL-17 concentration in plasma and the CCR6 mRNA level in PBMCs in AMI and SAP groups were significantly higher than control group;CCL20,CCR6 and IL-17 were positively correlated and were closely related to the severity of coronary artery stenosis;CCL20 and IL-17 expression were independent biomarkers of CHD.Conclusion:1.The AKT/NF-?B/CCL20-CCR6/IL-17 signaling pathway may play an important role in the development of atherosclerosis;2.OxLDL and atorvastatin can impact the activity of AKT/NF-?B signaling pathway in mouse macrophages and regulate the expression of CCL20 and IL-17;3.The overexpression of CCL20 can feedback to enhance NF-?B activity and strengthen pro-atherosclerotic function;4.The expression of CCL20 and IL-17 is related to the severity of coronary artery stenosis,may have good diagnostic value in CHD;5.CCL20 and IL-17 is an independent biomarker of CHD.