6- to 8-week male C57BL/6 mice were obtained from the Model Animal Genetics Research Center of Nanjing University (Nanjing, China). Mice were grouped at SPF facility with controlled temperature (22 ± 2°C) and 12:12-h light–dark cycle. Animal welfare and experimental procedures were carried out strictly in accordance with the recommendation of Guide for the Care and Use of Laboratory Animals [Ministry of Science and Technology of China (11)], and the Nanjing University Animal Care and Use Committee (NJU-ACUC). The protocol was approved by the Nanjing University Animal Care and Use Committee (NJU-ACUC) and to minimize suffering and to reduce the number of mice used.

Procyanidin was purchased from Aladdin Co. Ltd. (Shanghai, China). Phorbolmyristate acetate (PMA), lipopolysaccharide (LPS), and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA). ATP was dissolved in ddH₂O (500 mM) and 0.1 M NaOH was used to adjust the pH value to 7.4 as stock solution. DSS was bought from MP Biomedical (Aurora, OH, USA). RPMI-1640 and fetal bowel serum were purchased from Life Technology (Carlsbad, CA, USA). Antibody for F4/80 was purchased from eBioscience (San Diego, CA, USA). Antibodies for NLRP3, phospho-p65, total p65, phospho-IKKα/β, total IKKα, anti-total IKKβ, phospho-IκBα, total IκBα and caspase-1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for apoptosis-associated speck-like protein containing a CARD (ASC) was purchased from Santa Cruz (Santa Cruz, CA, USA). Antibody for MMP9 was purchased from R&D System (Minneapolis, MN, USA). ELISA kits for human IL-1β were purchased from Dakewe Biotech Co. Ltd. (Beijing, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Human THP-1 cells were purchased from Shanghai Institute of Cell Biology (Shanghai, China) and maintained in RPMI 1640 medium, supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 10% fetal calf serum under a humidified 5% (v/v) CO₂ atmosphere at 37°C.

Cells (1 × 10⁶ per well) were cultured in a 6-well plate and treated with procyanidin in the presence or absence of LPS or ATP plus LPS for 6 h. Then, the cells were harvested and incubated with 2,7-dichlorofluorescein diacetate (DCFH-DA, Invitrogen, USA) at 37°C for 20 min and washed twice with cold PBS. DCF fluorescence distribution was detected by flow cytometry on a FACScan (Becton Dickinson, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Data were analyzed by Cell Quest software (Molecular Devices Corporation, CA, USA).

Real-time PCR was performed as described previously (12). Total RNA of colon tissue or THP-1 cells were reverse transcribed to cDNA and subjected to quantitative PCR, which was performed with the BioRad CFX96 Touch™ Real-Time PCR Detection System (BioRad, CA, USA) using iQTM SYBR^® GreenSupermix (BioRad, CA, USA), and threshold cycle numbers were obtained using BioRad CFX Manager software. The program for amplification was 1 cycle of 95°C for 2 min followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 95°C for 10 s. β-actin was used as an endogenous control to normalize for differences in the amount of total RNA in each sample, and the relative gene expression was calculated using comparative C_T method also referred to as 2^−ΔΔCT method, i.e., fold change = 2^−ΔΔCT = [(C_T gene of interest − C_T gene of actin control) sample A − (C_T gene of interest − C_T gene of actin control) sample B]. The primer sequences used in this study were as follows:

Human-TNF-α Forward 5′-TGGCCCAGGCAGTCAGA-3′;

Samples were collected and lysed in a lysis buffer containing protease inhibitor (protease inhibitor cocktail, Pierce). The proteins were fractionated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes. The membrane was blocked with 5% BSA for 2 h at room temperature. Different antibodies were incubated overnight at 4°C, and then incubated with secondary antibody. The software Quantity One (Bio-Rad Laboratories, Hercules, CA, USA) was used for densitometric analysis.

THP-1 cells (1 × 10⁶ per well) were cultured in a 6-well plate and treated with 500 nM PMA to differentiate to macrophage for 3 h. Supernatant was collected after LPS (100 ng/ml) incubation in THP-1 cells in the absence or presence of procyanidin. After adding loading buffer to each sample, same volume was loaded into the wells of gels (8% polyacrylamide gels containing 0.1% gelatin). After electrophoresis, each gel was incubated with 50 ml of developing buffer for 48 h (37°C) in shaking bath. Then the gels were stained with coomassie brilliant blue (1%, with 10% acetic acid, 10% isopropyl alcohol, diluted with water).

2.5% (wt/vol) DSS was used to induce acute colitis in mice. Mice were fed with DSS dissolved in drinking water continuously from day 0 to day 9, and then given normal drinking water for the next 2 days before sacrificed. Normal C57BL/6 mice received the same drinking water without DSS (n = 6–8 mice in each group). Procyanidin (10, 20, 40 mg/kg, i.g.) and mesalazine (200 mg/kg, i.g.) were dissolved in sodium salt of caboxy methyl cellulose (CMC-Na, 0.5%). Procyanidin and mesalazine were administered once a day from day 0 to day 11. The animals were observed once daily for weight, morbidity, and the presence of gross blood in feces and at the anus. The disease activity index (DAI) was calculated by assigning well-established and validated scores as previously described (13, 14). Briefly, the following parameters were used for calculation: (a) diarrhea (0 points = normal, 2 points = loose stools, 4 points = watery diarrhea); (b) hematochezia (0 points = no bleeding, 2 points = slight bleeding, 4 points = gross bleeding). At day 11 following induction with DSS, the animals were sacrificed, the entire colon was quickly removed for ex vivo study. Segments of the colon taken for histopathological essay were fixed in 10% normal buffered formalin, embedded in paraffin. Sections were stained with hematoxylin and eosin and histological score was evaluated (blinded) as follows: 0, no signs of inflammation; 1, low leukocyte infiltration; 2, moderate leukocyte infiltration; 3, high leukocyte infiltration, moderate fibrosis, high vascular density thickening of the colon wall, moderate goblet cell loss, and focal loss of crypts; and 4, transmural infiltrations, massive loss of goblet cell, extensive fibrosis, and diffuse loss of crypts.

F4/80 macrophage infiltration analysis was performed on paraffin-embedded colonic tissue sections (4–5 µm). Briefly, the sections were deparaffinized, rehydrated, and washed in 1% PBS-Tween20. Then, they were treated with 2% hydrogen peroxide, blocked with 3% BSA, and incubated for 2 h at room temperature with antibody for F4/80-FITC (1:100). The slides were then counter-stained with DAPI for 1 min. The reaction was stopped by thorough washing in PBS for 20 min. Images were acquired by confocal laser-scanning microscope (Olympus FV1000, Olympus, Japan). Settings for image acquisition were identical for control and experimental tissues.

Immunohistochemical analysis was performed on paraffin-embedded colon tissue sections (4–5 µm) to evaluate the p-p65 expression. Slides were deparaffinized, rehydrated, and blocked as described in Section “Gelatin Zymography,” and incubated with p-p65 antibody overnight at 4°C. Then, slides were incubated with streptavidin-HRP (Shanghai Gene Company, GK500705, Shanghai, China) for 40 min, then stained with DAB (Shanghai Gene Company, GK500705, Shanghai, China) substrate, and counter-stained with hematoxylin.

The amounts of IL-1β in the cultured cells were quantified by ELISA kits according to the manufacturer’s instructions.

Proteins from cells (1 × 10⁷ per well) were incubated with 2 µg of appropriate antibody and precipitated with protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoprecipitated proteins were separated by SDS-PAGE and western blot was performed with the indicated antibodies.

Results were expressed as mean ± SEM of three independent experiments and each experiment included triplicate sets. Data were statistically evaluated by one-way ANOVA followed by Dunnett’s test between control group and multiple dose groups. The level of significance was set at a P-value of 0.05.

Macrophages play a critical role in the initiation and progression of inflammation in IBD (15, 16), and ROS are predominantly generated from activated macrophages. We used THP-1 cells to examine the antioxidative effect of procyanidin. THP-1 cells were differentiated to macrophages by cultured with PMA (500 nM) for 3 h, and then single LPS (100 ng/ml) or LPS (100 ng/ml) combined with ATP (5 mM) stimulation—two classic methods of inducing activation of TLR4-NF-κB and the NLRP3 inflammasome, respectively—were applied to mimic the inflammatory condition in THP-1 cells. Under LPS challenge, ROS was generated by bind of TLR4 with NADPH oxidase 4 to produce superoxide (17) and according to former reference (14), ATP (5 mM) alone is also sufficient to induce ROS generation. In the present study, we found that the fold induction of ROS appeared to be the same with LPS and LPS + ATP, suggesting that the TLR pathway dominates the production of ROS. Since procyanidin has a powerful antioxidation effect, we investigated whether procyanidin also has an inhibitory effect on the production of ROS triggered by LPS or LPS plus ATP stimulation. As shown in Figures 1A,B, upon LPS stimulation or co-stimulation with LPS and ATP, increased amounts of ROS were generated in THP-1 cells, and procyanidin (10 µM) administration significantly suppressed the elevation of ROS.

Since LPS promotes the generation of ROS, which induce the activation of MMP9, we investigated whether procyanidin was able to inhibit MMP9. First, we examined the change in MMP9 expression by gelatin zymography during LPS stimulation for 0.5, 2, 6, 12, and 24 h. As shown in Figure 2A, elevated MMP9 was accompanied with LPS, especially in the 24th hour after LPS challenge. Next, we evaluated the inhibitory effect of procyanidin on MMP9 expression and found that procyanidin downregulated MMP9 from LPS administration for 24 h in a dose-dependent fashion (0.3, 1, 3, 10 µM) (Figure 2B).

The NLRP3 inflammasome is important in macrophage activation. Upon ATP stimulation, NLRP3 is co-localized with the apoptosis-associated speck-like protein containing a CARD (ASC), which has a caspase recruitment domain adaptor and pro-caspase 1, to form the inflammasome complex, resulting in the cleavage of pro-caspase 1 into its mature form. Therefore, the formation of the inflammasome complex is the core event in the activation of the NLRP3 inflammasome. In addition, IL-1β is an important proinflammatory cytokine in colitis and must be cleaved from its inactive form (pro-IL-1β) to the mature form by caspase 1. Here, we found that procyanidin (10 µM) significantly block the formation of the NLRP3 inflammasome induced by LPS and ATP (Figure 3A), resulting in decreased inflammasome activation and IL-1β release (Figures 3B–D). Together, these results suggested that procyanidin can interrupt the assembly of the NLRP3/ASC/caspase1 complex, thus inhibiting inflammasome activation and IL-1β release.

Commonly, TLR4, which belongs to the family of pattern recognition receptors in macrophages, is responsible for immune responses upon activation of pattern-associated molecular patterns, such as LPS. Following LPS stimulation, ROS can be produced by TLR4 binding to NADPH oxidase 4 to produce superoxide (17). However, ROS scavengers can block the NF-κB activation stimulated by LPS (18). As shown in Figure 4A, in THP-1 cells, the NF-κB signaling pathway was significantly elevated following LPS (100 ng/ml) stimulation, as evidenced by higher levels of p-p65, p-IKKα/β, and p-IκBα, which can be inhibited by procyanidin (0.3, 1, 3, 10 µM) in a dose-dependent manner. In addition, under LPS stimulation (100 ng/ml) for 20 min, p65 translocated from the cytoplasm into the nucleus, which was significantly blocked by procyanidin (10 µM) (Figure 4B). In the nucleus, p65 promoted the transcription of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α. As shown in Figures 4C–E, procyanidin (0.3, 1, 3, 10 µM) markedly decreased the mRNA level after 6 h stimulation with LPS in THP-1 cells.

To determine the ability of procyanidin to modulate inflammation, we investigated the therapeutic effect of procyanidin on DSS-induced experimental colitis in mice. Mice were orally treated with 2.5% DSS for nine consecutive days and then normal water for 2 days, inducing a model of acute colitis that was characterized by severe weight loss, evident diarrhea, rectal bleeding, and shortened colon length. Mesalazine, a cyclooxygenase (COX) inhibitor, which reduces the risk of recurrence of IBD, is by far the most frequently used drug in IBD treatment (19), and in this study, we used mesalazine as a positive control in mice (20, 21). As shown in Figure 5A, compared to the vehicle-treated group, oral treatment of procyanidin, especially at a dose of 40 mg/kg, markedly attenuated weight reduction. In addition, procyanidin also significantly prevented disease progression, as shown by the DAI and colon length (Figures 5B–D). Furthermore, the effect of procyanidin at the highest dosage (40 mg/kg) was equivalent to that of mesalazine.

Histology analysis implied that procyanidin exerted a better protective effect on DSS-induced inflammation in the colon (Figures 6A,B). Due to the critical role of macrophages in colitis, we next examined the effect of procyanidin on macrophage infiltration during colitis. As shown in Figures 6C,D, DSS induced obvious infiltration of F4/80 positive macrophages in the colon compared to vehicle-treated mice; by contrast, fewer infiltrating cells were observed in colonic samples from mice treated with procyanidin or mesalazine.

To clarify the in vitro results, mice were administered with 2.5% DSS for 0, 1, 3, 5, and 7 consecutive days, and the colons were collected. As shown in Figure 7A, MMP9 was significantly elevated with the development of DSS-induced colitis in colonic tissues. Therefore, MMP9 is markedly correlated with the severity of inflammation. However, the high expression of MMP9 was significantly decreased when mice were treated with procyanidin (10, 20, and 40 mg/kg), which was in line with the in vitro results (Figure 7B). In addition to protein level, procyanidin also decreased the elevated mRNA level of MMP9 in a dose-dependently way (10, 20, and 40 mg/kg); however, the effect of mesalazine on MMP9 expression was not significant (Figure 7C).

In accordance with the in vitro results, we also found that the NLRP3 inflammasome was markedly activated after DSS challenge in colonic tissue. However, procyanidin greatly inhibited the activation of the inflammasome, as evidenced by the decreased level of cleaved caspase-1 (Figures 8A,B).

As the NF-κB signaling pathway plays a crucial role in inflammation mediated by macrophages, we assessed the effects of procyanidin on the activation of the NF-κB signaling pathway, which is characterized by elevated levels of p-p65. Immunohistochemical staining (Figure 9A) revealed that the phosphorylation level of p65 was markedly increased in the colon in the DSS group, while procyanidin significantly inhibited the p-p65 level. In addition, the mRNA levels of IL-1β, TNF-α, and IL-6 were also decreased after procyanidin treatment compared to DSS treatment alone (Figures 9B–D).