Matrine inhibits advanced glycation end products-induced macrophage M1 polarization by reducing DNMT3a/b-mediated DNA methylation of GPX1 promoter

Abstract

Advanced glycation end products (AGEs) are characterized diabetic metabolites inducing macrophage M1 polarization which is crucial in diabetes-exacerbated atherosclerosis. Matrine was proved anti-atherosclerotic. The current study was aimed to investigate the inhibitory effects of matrine on AGEs- induced macrophage M1 polarization and underlying molecular mechanisms. Primary mouse macrophages were exposed to AGEs. Receptor for AGEs (RAGE) and toll-like receptor 4 (TLR4) were over-expressed by vectors. Matrine was used to treat these cells. Inducible nitric oxide synthase (iNOS) expression and pro-inflammatory cytokine production were used to evaluate macrophage M1 polarization. Oxidative stress was assessed by intracellular reactive oxygen species (ROS) generation, total antioxidant capacity (TAC) and malondialdehyde (MDA) contents. Relative mRNA expression level was determined by real-time PCR. Western blotting was used to evaluate protein and protein phosphorylation levels. Bisulfite sequencing PCR (BSP) was used to evaluate DNA methylation. Matrine reduced AGEs exposure-elevated expressions of DNA methyltransferase (DNA MTase, DNMT)3a and DNMT3b in macrophages which were not affected by RAGE or TLR4 over expressions. DNA methylation rate of GPX1 promoter was reduced from 97.22% to 66.67% in AGEs- exposed macrophages treated by matrine. GPX1 expression was up-regulated by matrine, which further suppressed AGEs/RAGE-mediated oxidative stress. Thus, the activation of down-stream TLR4/STAT1 signaling pathway was inhibited by matrine treatment which eventually suppressed AGEs- induced macrophage M1 polarization. However, these effects of matrine were impaired by RAGE and TLR4 overexpression. Results from this study suggested that matrine inhibited AGEs- induced macrophage M1 polarization by suppressing RAGE-induced oxidative stress-mediated TLR4/STAT1 signaling pathway. Matrine exerted anti-oxidant effects via increasing GPX1 expression by inhibiting DNMT3a/b-induced GPX1 promoter DNA methylation.

Keywords: Advanced glycation end products; Macrophages; Matrine; Polarization.