Anti-Inflammatory Effect of an Apigenin-Maillard Reaction Product in Macrophages and Macrophage-Endothelial Cocultures

Abstract

Chronic inflammation is involved in the progression of various diseases, while dietary flavonoids are reported to possess antioxidative and anti-inflammatory properties against age-related diseases. Previously, an apigenin-Maillard reaction product, dimethylglyoxal apigenin (DMA), was identified by us and demonstrated to be antioxidative. In this study, we investigated the inhibitory effect of DMA on advanced glycation end product- (AGE-) induced inflammation in macrophages and macrophage-endothelial cocultures. Results showed that DMA remarkably inhibited the mRNA and protein expression of receptor for AGEs (RAGE), thereby inhibiting the production of ROS and proinflammatory cytokines, including tumor necrosis factor- (TNF-) α, interleukin (IL) 1, IL 6, and monocyte chemoattractant protein- (MCP-) 1 in RAW 264.7 cells. In the coculture system which was performed in the Boyden chamber, macrophage infiltration and adhesion to endothelial cells were significantly suppressed by DMA. Further study indicated that DMA decreased AGE-evoked IL 6 and MCP-1 secretion, which might be achieved through RAGE and its downstream-regulated transforming growth factor- (TGF-) β1 and intercellular adhesion molecule (ICAM) 1 expression in the coculture system. In conclusion, our study demonstrates that DMA, a thermally induced compound, has anti-inflammatory activity in both macrophages and macrophage-endothelial cocultures, offering a promising approach for slowing down the development of chronic diseases.

Figure 1

DMA suppresses AGE-induced oxidative stress in macrophages. (a) Chemical structures. (b) Cell viability of DMA and API assayed with CCK kit. The incubation time for DMA and API was 24 hrs. (c) ROS intensity by flow cytometry. RAW 264.7 macrophages were pretreated with 25 μM DMA or API for 24 hrs before stimulation with/without 200 μg/mL of AGEs for 24 hrs. ^∗∗∗ p < 0.001 vs. control; ^# p < 0.05 vs. AGE treatment.

Figure 2

DMA ameliorates AGE-induced inflammatory cytokine expression in macrophages. Relative mRNA levels by qPCR. RAW 264.7 macrophages were pretreated with 25 μM DMA or API for 24 hrs before stimulated with/without 200 μg/mL of AGEs for 24 hrs. ^∗∗ p < 0.01 vs. control, ^∗∗∗ p < 0.001 vs. control; ^# p < 0.05 vs. AGE treatment, ^## p < 0.01 vs. AGE treatment, ^### p < 0.001 vs. AGE treatment.

Figure 3

DMA ameliorates the AGE-induced RAGE expression in macrophages. RAW 264.7 macrophages were pretreated with 25 μM DMA or API for 24 hrs before stimulation with/without 200 μg/mL of AGEs for 24 hrs. (a) RAGE mRNA expression by qPCR. (b) RAGE protein expression by Western blot. Total cell lysates were obtained by lysis in RIPA buffer containing protease inhibitors. β-Actin served as a control. (c) RAGE protein fold changes were quantified with ImageJ software. ^∗∗ p < 0.01 vs. control, ^∗∗∗ p < 0.001 vs. control; ^## p < 0.01 vs. AGE treatment, ^### p < 0.001 vs. AGE treatment.

Figure 4

DMA protects AGE-induced macrophage infiltration. RAW 264.7 macrophages at the upper compartment of the Boyden chamber were incubated with medium (a), 500 μg/mL AGEs (b), 10 μM DMA + 500 μg/mL AGEs (c), 10 μM API + 500 μg/mL AGEs (d) for 24 hrs. Filtered macrophages in the microporous membrane were stained by 0.1% crystal violet, and representative photos were taken under a microscope. (e) Macrophage counting. The stain was washed off by 30% acetic acid and measured at a wavelength of 580 nm. ^∗∗∗ p < 0.001 vs. control; ^## p < 0.01 vs. AGE treatment.

Figure 5

DMA prevents AGE-induced macrophage adhesion to endothelial cells. HUVECs at the lower compartment of the Boyden chamber were incubated with medium (a), 500 μg/mL AGEs (b), 10 μM DMA + 500 μg/mL AGEs (c), or 10 μM API + 500 μg/mL AGEs (d) for 48 hrs. Macrophages adhered to HUVECs were stained by 0.1% crystal violet, and representative photos were taken under a microscope. (e) Macrophage counting. The stain was washed off by 30% acetic acid and measured at a wavelength of 580 nm. ^∗∗∗ p < 0.001 vs. control; ^### p < 0.001 vs. AGE treatment.

Figure 6

DMA ameliorates AGE-induced inflammatory cytokines in macrophage-endothelial cocultures. (a) Protein secretion of IL 6. (b) Protein secretion of MCP-1. After treatments, supernatants in the Boyden chamber were collected and assayed by ELISA kits. The incubation time with AGEs was 48 hrs. ^∗∗ p < 0.01 vs. control; ^## p < 0.01 vs. AGE treatment, ^### p < 0.001 vs. AGE treatment.

Figure 7

DMA decreases AGE-induced inflammation through RAGE in macrophage-endothelial cocultures. (a) Protein expression by Western blot. HUVECs at the lower compartment of the Boyden chamber were incubated with medium, 500 μg/mL AGEs, or 10 μM DMA + 500 μg/mL AGEs for 48 hrs. Total cell lysates were obtained by lysis in RIPA buffer containing protease inhibitors. β-Actin served as a control. (b) Fold changes were quantified with ImageJ software. ^∗∗∗ p < 0.001 vs. control; ^### p < 0.001 vs. AGE treatment.

Figure 8

Schematic diagram of DMA on AGE-induced inflammation in macrophages and macrophage-endothelial cocultures.