The Antioxidant Procyanidin Reduces Reactive Oxygen Species Signaling in Macrophages and Ameliorates Experimental Colitis in Mice

Abstract

Management of inflammatory bowel disease (IBD) is a real clinical challenge. Despite intense investigation, the mechanisms of IBD remain substantially unidentified. Some inflammatory conditions, such as matrix metalloproteinases (MMPs) and the nuclear factor-κB (NF-κB) and NOD-like receptor protein 3 (NLRP3) inflammasome signaling pathways, are reported to contribute to the development and maintenance of IBD. Regulation of their common upstream signaling, that is, reactive oxygen species (ROS), may be important to control the progression of IBD. In the present study, we found that procyanidin, a powerful antioxidation flavonoid, has a significant effect on ROS clearance on THP-1 macrophages after lipopolysaccharide (LPS) or LPS-combined adenosine triphosphate stimulation, thus downregulating MMP9 expression, suppressing NF-κB signaling, and interrupting the formation of the NLRP3 inflammasome. Moreover, our in vivo data showed that procyanidin attenuated Dextran sulfate sodium-induced experimental colitis in a dose-dependent fashion by suppressing the expression of MMP9, NF-κB, and NLRP3 inflammasome signaling in colonic tissues in mice. Overall, our results suggested that targeting ROS could be a potential therapeutic choice for colonic inflammation.

Keywords: MMP9; NF-κB; NLRP3 inflammasome; inflammatory bowel disease; procyanidin; reactive oxygen species signaling.

Figure 1

Procyanidin inhibited the generation of reactive oxygen species (ROS) in THP-1 cells. (A) THP-1 cells were pretreated with 500 nM phorbolmyristate acetate (PMA) for 3 h and were then cultured with 100 ng/ml lipopolysaccharide (LPS) for 2 h in the absence or presence of procyanidin (10 µM). (B) THP-1 cells were preincubated with 500 nM PMA for 3 h, followed by a 3 h treatment with procyanidin (10 µM) and 100 ng/ml LPS as well as a culturing with 5 mM adenosine triphosphate (ATP) for 1 h. ROS was stained with DCFH and measured by flow cytometry. ^##P < 0.01 vs. vehicle group, **P < 0.01 vs. LPS alone or LPS + ATP-treated group.

Figure 2

MMP9 expression was consistently upregulated following lipopolysaccharide (LPS) stimulation and was inhibited by procyanidin treatment in THP-1 cells. (A) THP-1 cells were preincubated with 500 nM phorbolmyristate acetate (PMA) for 3 h, followed by 100 ng/ml LPS for different times (0, 0.5, 2, 6, 12, and 24 h); supernatants were collected to measure MMP9 expression by gelatin zymography. (B) THP-1 cells were preincubated with 500 nM PMA for 3 h, followed by 24 h treatment with 100 ng/ml LPS in the presence or absence of procyanidin (0.3, 1, 3, and 10 µM); MMP9 expression was examined by gelatin zymography. ^##P < 0.01 vs. vehicle group, *P < 0.05, **P < 0.01 vs. LPS alone.

Figure 3

Procyanidin suppressed the activation of NLRP3 inflammasome in THP-1 cells. THP-1 cells were pretreated with 500 nM phorbolmyristate acetate for 3 h and cultured with 100 ng/ml lipopolysaccharide (LPS) in the absence or presence of procyanidin (10 µM) for 3 h, followed by a 1 h incubation with 5 mM adenosine triphosphate (ATP). (A) Proteins were isolated and immunoprecipitated with an antibody against ASC. (B,C) Protein levels of pro-caspase 1, cleaved caspase 1, ASC, and NLRP3 were determined by western blot. (D) Released interleukin (IL)-1β in the supernatant was analyzed by ELISA. ^##P < 0.01 vs. vehicle group, *P < 0.05, **P < 0.01 vs. LPS + ATP-treated group.

Figure 4

Procyanidin suppressed the NF-κB signaling pathway in THP-1 cells. THP-1 cells were treated with different concentrations of procyanidin (0.3, 1, 3, 10 µM) in the presence or absence of lipopolysaccharide (LPS) (100 ng/ml) for 30 min. (A) Protein levels of p-p65, p65, p-IKKα/β, IKKα, IKKβ, p-IκBα, and IκBα were determined by western blot. (B) Subcellular localization of p-p65 was examined by immunofluorescence by a confocal microscope. (C–E) mRNA levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were measured 6 h after LPS stimulation by real-time PCR. ^##P < 0.01 vs. vehicle group, *P < 0.05, **P < 0.01 vs. LPS group.

Figure 5

Procyanidin administration ameliorated Dextran sulfate sodium (DSS)-induced experimental colitis in mice. Mice were given 2.5% DSS in drinking water for nine consecutive days and then given normal drinking water for the next 2 days before being sacrificed. Procyanidin were orally administered daily (10, 20, and 40 mg/kg) from day 1 to day 11. (A) Body weight changes during the disease process. (B) Disease activity index was calculated. (C,D) Macroscopic images and length of the colon from each group were measured. Data are presented as the means ± SEM (n = 6–8 per group). ^##P < 0.01 vs. normal, *P < 0.05, **P < 0.01 vs. DSS-treated group.

Figure 6

Procyanidin treatment prevented dextran sulfate sodium (DSS)-induced colon damage and macrophage infiltration in mice. (A) Serial sections of colon tissues were stained with H&E. (B) Histopathological scores of each group were determined. (C) Sections of colonic tissue were immunostained with antibody for F4/80-FITC (green) and DAPI (blue) and were observed using a confocal laser-scanning microscope. Magnification: 200×. (D) Fluorescence intensity of each group was determined. ^##P < 0.01 vs. normal, **P < 0.01 vs. DSS-treated group.

Figure 7

MMP9 expression was elevated after dextran sulfate sodium (DSS) administration and was inhibited by procyanidin treatment in mice. (A) Mice were orally administered 2.5% DSS for various times (0, 1, 3, 5, and 7 days). Paraffin-embedded colon tissue sections were stained for MMP9. (B) Paraffin-embedded colon tissue sections from vehicle and procyanidin-treated group were stained for MMP9. (C) The mRNA expression of MMP9 was determined by real-time PCR. ^##P < 0.01 vs. normal, *P < 0.05, **P < 0.01 vs. DSS-treated group.

Figure 8

Procyanidin treatment inhibited dextran sulfate sodium (DSS)-induced activation of NLRP3 inflammasome in mice. (A,B) Colon tissue protein was extracted from mice, and the protein levels of pro-caspase 1 and cleaved caspase 1 were examined by western blot. ^##P < 0.01 vs. normal, *P < 0.05, **P < 0.01 vs. DSS-treated group.

Figure 9

Procyanidin suppressed the activation of the NF-κB signaling pathway in mice. (A) Paraffin-embedded colon tissue sections from vehicle or procyanidin-treated group were stained for p-p65. (B–D) mRNA levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were determined by real-time PCR. ^##P < 0.01 vs. normal, *P < 0.05, **P < 0.01 vs. dextran sulfate sodium (DSS)-treated group.