The Effect And Molecular Mechanisms Of Advanced Glycation End Products(AGEs)on Macrophage Polarization

Objective: The present study aimed to explore the effect of AGEs on macrophage activation and its role in β-cell dysfunction.It mainly contained the following three parts: 1.To study the effect of AGEs on macrophage polarization in murine macrophage cell line Raw 264.7;2.To explore the signal pathway on macrophage polarization induced by AGEs;3.To study the role of AGEs induced macrophage phenotype reprograming in β-cell dysfunction.Method: Part 1: Raw 264.7 cells were cultured.AGEs was prepared and used to treat Raw 264.7.After that,the expression and secretion of proinflammatory cytokines,such as IL-1β,IL-6,and TNF-α,were examined,the M1 and M2 macrophageassociated markers expression were measured and the ROS levels were also detected.Part 2.We measured the phosphorylation of JNK,ERK p38 MAPK after incubating with AGEs.After adding certain concentration of JNK inhibitor SP600125,ERK inhibitor U0126 or p38 MAPK inhibitor SB203580 in Raw 264.7 culture medium with AGEs,the expression of pro-inflammatory cytokines,such as IL-1β,IL-6,and TNF-α,M1 and M2 macrophage-associated markers and intracellular ROS levels were detected.Part 3.MIN6 cells were cultured.MIN6 cells were cocultured with AGEs-pretreated macrophages,the expression of molecules involved in β-cell function and GSIS were measured.And the gene expression of monocyte chemotactic protein-1(MCP-1)was examined.Result:(1)AGEs treatment upregulated the expression of proinflammatory cytokines,such as IL-1β,TNF-α,and IL-6,at both the m RNA and secretion levels.The expression of M1 macrophage markers,such as i NOS,ROS and the surface marker CD11 c,was significantly upregulated,whereas the expression of M2 macrophage markers,such as Arg1 and CD206,was reciprocally downregulated upon AGEs stimulation.(2)The phosphorylation of JNK,ERK p38 MAPK was detectable as early as 15 min,and peaked at 30 min,followed by a slight drop after exposure to AGEs.Inhibition of MAPK activation by their respective inhibitors can significantly decreased the AGEs-induced production of pro-inflammatory cytokines,ROS,NO and M1-related markers.(3)Coculture with AGEs-pretreated macrophages significantly inhibited the expression of molecules involved in β-cell function and was accompanied by the impairment of glucose-stimulated insulin secretion(GSIS)in MIN6 cells.In addition,AGEs-pretreatment of Raw 264.7 cells induced MCP-1 expression in MIN6 cells.Conclusion: AGEs induce M1 macrophage phenotype polarization as well as inhibit M2 polarization and reduce IL-4-induced M2 skewing of macrophages.the AGE-induced polarization of macrophages to the proinflammatory phenotype might at least partially be linked to enhanced intracellular MAPK signaling.These data imply that AGEs-induced unfavorable M1/M2 polarization might be an indispensable potential mechanism responsible for β-cell dysfunction.The targeted inhibition or reversal of AGEs-induced macrophage M1 polarization may be a useful therapeutic intervention strategy for alleviating the progression of T2 DM.