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.
摘要：
晚期糖基化终产物（AGEs）是糖尿病代谢产物，可诱导巨噬细胞M1极化，这在糖尿病加剧的动脉粥样硬化中至关重要。苦参碱被证明具有抗动脉粥样硬化作用。本研究旨在探讨苦参碱对AGEs诱导的巨噬细胞M1极化的抑制作用及其分子机制。将原代小鼠巨噬细胞暴露于AGEs。AGEs受体(RAGE)和toll样受体4(TLR4)通过载体过表达。苦参碱用于治疗这些细胞。使用诱导型一氧化氮合酶（iNOS）表达和促炎细胞因子产生来评估巨噬细胞M1极化。通过细胞内活性氧（ROS）的产生来评估氧化应激，总抗氧化能力(TAC)和丙二醛(MDA)含量。通过实时PCR测定相对mRNA表达水平。蛋白质印迹用于评估蛋白质和蛋白质磷酸化水平。使用亚硫酸氢盐测序PCR(BSP)来评估DNA甲基化。苦参碱降低AGEs暴露-DNA甲基转移酶表达升高(DNAMTase，巨噬细胞中DNMT)3a和DNMT3b不受RAGE或TLR4过表达的影响。在苦参碱处理的AGEs暴露巨噬细胞中，GPX1启动子的DNA甲基化率从97.22％降低到66.67％。苦参碱上调GPX1表达，进一步抑制AGEs/RAGE介导的氧化应激。因此，苦参碱抑制下游TLR4/STAT1信号通路的激活,最终抑制AGEs诱导的巨噬细胞M1极化。然而,RAGE和TLR4过表达削弱了苦参碱的这些作用。这项研究的结果表明，苦参碱通过抑制RAGE诱导的氧化应激介导的TLR4/STAT1信号通路来抑制AGEs诱导的巨噬细胞M1极化。苦参碱通过抑制DNMT3a/b诱导的GPX1启动子DNA甲基化而增加GPX1表达，从而发挥抗氧化作用。
