Cranberry fruit has been reported to have high antioxidant effectiveness that is potentially linked to its richness in diversified polyphenolic content. The aim of the present study was to determine the role of cranberry polyphenolic fractions in oxidative stress (OxS), inflammation and mitochondrial functions using intestinal Caco-2/15 cells. The combination of HPLC and UltraPerformance LC®-tandem quadrupole (UPLC-TQD) techniques allowed us to characterize the profile of low, medium and high molecular mass polyphenolic compounds in cranberry extracts. The medium molecular mass fraction was enriched with flavonoids and procyanidin dimers whereas procyanidin oligomers (DP > 4) were the dominant class of polyphenols in the high molecular mass fraction. Pre-incubation of Caco-2/15 cells with these cranberry extracts prevented iron/ascorbate-mediated lipid peroxidation and counteracted lipopolysaccharide-mediated inflammation as evidenced by the decrease in pro-inflammatory cytokines (TNF-α and interleukin-6), cyclo-oxygenase-2 and prostaglandin E2. Cranberry polyphenols (CP) fractions limited both nuclear factor κB activation and Nrf2 down-regulation. Consistently, cranberry procyanidins alleviated OxS-dependent mitochondrial dysfunctions as shown by the rise in ATP production and the up-regulation of Bcl-2, as well as the decline of protein expression of cytochrome c and apoptotic-inducing factor. These mitochondrial effects were associated with a significant stimulation of peroxisome-proliferator-activated receptor γ co-activator-1-α, a central inducing factor of mitochondrial biogenesis and transcriptional co-activator of numerous downstream mediators. Finally, cranberry procyanidins forestalled the effect of iron/ascorbate on the protein expression of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2). Our findings provide evidence for the capacity of CP to reduce intestinal OxS and inflammation while improving mitochondrial dysfunction.
Inflammation is the central component in inflammatory bowel diseases but it is accompanied by a persistent oxidative stress. The aim of our study consists in testing functional nutrients, endowed with anti-inflammatory and antioxidant properties, directly acting on the gut, and without evident side effects as observed with chemical agents.
The findings clearly show the potential of cranberry polyphenols to significantly alleviate intestinal oxidative stress and inflammation while improving mitochondrial dysfunctions via specific mechanisms.
Our work is critical in establishing novel functional aspects of polyphenols in the intestine, which may confer beneficial health impacts on inflammatory bowel diseases.
Polyphenols, a large class of plant-based organic compounds, are found in many fruits and vegetables, and thus serve as important dietary micronutrients. They are involved in plant defence against pathogens and ultraviolet damage  and are currently receiving much attention given their multiple related physiological and health effects, including antioxidative, anti-inflammatory and immune-regulatory actions [2,3]. Their potent free-radical scavenging activity and capacity to reduce inflammation  make them ideal candidate molecules not only for prevention, but also for complementary treatment strategies to improve health outcomes.
The dynamic cross-talk between intestinal epithelial cells and microflora represents one of the fundamental features of intestinal homoeostasis [5,6]. Given the unique environmental challenges, the gastrointestinal tract has become exquisitely sensitive to perturbations that affect its capacity to resolve oxidative stress (OxS). Accumulation of oxidative damage occurs when pro-oxidants overwhelm the endogenous antioxidant capacity, thereby leading to excessive production of reactive oxygen species (ROS). Free radical generation, which is intensively catalysed by redox-active metals such as iron owing to its catalysing role in Fenton chemistry, may be linked to gut-related diseases [7–9]. Undoubtedly, the intestine is highly vulnerable to oxidative damage due to its constant exposure to aerobic metabolism or luminal oxidants from ingested nutrients, local microbes or infections, ischaemia/reperfusion, gastric acid production and non-steroidal anti-inflammatory drugs [10–12]. Currently, OxS appears as an essential component of the signaling cascade of inflammation. It plays a significant role in inflammatory bowel diseases (IBD), chronic pathologies that are characterized by uncontrolled inflammation ultimately leading to mucosal disruption and ulceration .
Although polyphenols exhibit powerful antioxidant activity by acting as free radical scavengers, namely, hydrogen donating compounds, singlet oxygen quenchers and metal ion chelators [14–17], very few studies have evaluated their protective effects and mechanisms of actions in the intestinal mucosa. Moreover, only limited work has been carried out to explore their impact on the induction of cellular antioxidant defence via the modulation of gene and protein expressions. Such antioxidant defence may maintain intestinal epithelial cells in a reduced environment, thereby protecting them from OxS while preserving their innate function. Additionally, the influence of polyphenols on the interplay between mitochondria and OxS has not been thoroughly examined in the gut epithelium. Thus, in the present study, we have tested the hypothesis that polyphenols can attenuate OxS and inflammation in enterocytes. We investigated their inherent potential using Caco-2/15 cells, a recognized human intestinal model for the investigation of gut absorption and interactions, nutrition, toxicology, food microbiology, bioavailability tests and screening of drug permeability in discovery programmes [18–20]. To this end, we employed extracts containing polyphenolic compounds derived from cranberries whose polyphenols (CP) have a wide range of biological effects, including the ability to fight several bacterial strains through antimicrobial activity, to serve as antioxidants in conditions of OxS, to modulate activities of various enzymes and to regulate the expression of numerous genes [21,22]. Finally, we took advantage of the present study to analyse the impact of the degree of CP polymerization, which modulates CP uptake by the intestinal mucosa, bioavailability at both local and systemic levels and action on peripheral tissues.
MATERIALS AND METHODS
General chemicals and reagents
HPLC-grade acetonitrile, methanol, acetone and Optima grade water were from Fisher Scientific. Formic acid was purchased from Fluka. 3-(4,5-Dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was from Sigma. Chemicals and reagents for specific assays will be described in the respective sections. Fresh cranberries (Vaccinium macrocarpon Ait.) underwent chemical treatments for the extraction of fractions with different molecular masses, which were then lyophilized, milled and kept frozen at −20°C.
Extraction of polyphenolic fractions of different molecular masses
Freeze-dried cranberries (150 g) were extracted twice with 500 ml of an extraction solvent mixture containing acetone/water/acetic acid (70:29.5:0.5, by vol.). The extracts were combined and the acetone was evaporated at 50°C using a rotary evaporator under partial vacuum. The resultant slurry (approximately 300 ml) was extracted three times with 250 ml of hexane to remove lipid substances. The water slurry was evaporated again on a rotary evaporator to eliminate the residual hexane. The remaining aqueous fraction was diluted with an equal volume of 20% (v/v) methanol in water and applied to a Sephadex LH-20 column (30 mm×600 mm) that had previously been hydrated for more than 2 h in 20% (v/v) aqueous methanol. The samples were collected in three different fractions consisting of low, medium and high molecular mass CP (HC) compounds. Elution was performed with 500 ml of 60% (v/v) methanol/water for the low molecular mass CP (LC fraction) followed by 1 litre of 100% (v/v) methanol to obtain the medium molecular mass CP (MC fraction) and finally by 2 litres of 70% (v/v) acetone/water for elution of the high molecular mass CP compounds (HC fraction). A rotary evaporator operated at 50°C under partial vacuum was used to remove the organic solvent from each fraction. The residual concentrated water solutions were freeze-dried and generated three solid (powder) fractions.
Determination and characterization of cranberry polyphenol fractions
The total phenolic content of LC, MC and HC fractions was determined using the Folin-Ciocalteu method  with gallic acid as a standard. Briefly, 100 μl of Folin-Ciocalteu reagent (diluted 10-fold in ultrapure water) and 80 μl of sodium carbonate solution (7.5% in ultrapure water) were added to 20 μl of methanol in a 96-well plate. A blank sample and five calibration solutions of gallic acid (12.5 to 200 μg/ml) were analysed under the same conditions. After 1-h incubation at room temperature, the absorbance was measured at 765 nm using the Fisher Scientific Multiskan GO microplate reader. All determinations were carried out in triplicate, and results are expressed as g/100 g of extract mass.
Flavonoids were identified and quantified using an ACQUITY UltraPerformance LC® (UPLC) system coupled to a tandem quadrupole (TQD) mass spectrometer equipped with an ESI source, all from Waters. An Agilent Plus C18 column (2.1 mm×100 mm, 1.8 μm particle sizes), operated at 30°C, was employed. The flavonoids were separated using 0.2% acetic acid in ultrapure water and acetonitrile (solvent A and B, respectively) with a flow rate of 0.4 μl/min under the following gradient: 0–8 min, 5–50% B; 8–9.10 min, 50–90% B; 9.10–10 min, 90% B; 10–10.10 min, 90–5% B; 10.10–13 min, 5% B. Data were acquired with MassLynx V4.1 software and processed for quantification with QuanLynx V4.1 (Waters). The UPLC-TQD system was operated in negative ESI mode for flavonoids. Cone and collision gas (nitrogen) flow rates were 80 litres/h and 800 litres/h, respectively. The mass spectrometer parameters were first generated with Waters’ IntelliStart software (automatic tuning and calibration of the AQUITY-TQD), which were then manually optimized as following: capillary voltage 2.75 kV, source temperature 150°C and desolvation temperature 400°C. Quantification was performed in multiple reaction monitoring modes, tracking the transition of parent and product ions specific for each compound with the inclusion of an external calibration.
The procyanidin composition of cranberry fractions was analysed as previously described  by normal phase analytical HPLC using an Agilent 1260/1290 Infinity System. Samples (5 μl of 25 mg/ml solutions in acetone/ultrapure water/acetic acid, 70:29.5:0.5, by vol.) were injected into the HPLC system and the separation was performed at 35°C with a flow rate of 0.8 ml/min using a Develosil Diol column (250 mm×4.6 mm, 5 μm particle size) protected with a Cyano Security Guard column (Phenomenex). The elution was performed using a solvent system comprising solvents A (acetonitrile/acetic acid, 98:2, v/v) and B (methanol/water/acetic acid, 95:3:2, by vol.) in a linear gradient from 0 to 40% B in 35 min, 40 to 100% B in 40 min, 100% isocratic B up to 45 min and 100 to 0% B in 50 min. The column was re-equilibrated for 5 min between injections. Fluorescence of the procyanidins was monitored at excitation and emission wavelengths of 230 and 321 nm, respectively with the fluorescence detector set to low sensitivity with a gain of ×7 for the entire run. Individual procyanidins with degrees of polymerization (DP) ranging from DP1 to DP>10 were quantified using an external calibration curve of (−)-epicatechin, taking into account their relative response factors in fluorescence . The results are expressed as g/100 g of extract mass.
Intestinal Caco-2/15 cell culture
The human epithelial colorectal adenocarcinoma Caco-2/15 cell line, a stable clone of the parent Caco-2 cells (American Type Culture Collection), was employed. Intestinal Caco-2/15 cells were cultured as described previously [26–34]. Briefly, they were grown in MEM supplemented with 10% (v/v) decomplemented FBS, 1% (v/v) penicillin/streptomycin and 1% (v/v) non-essential amino acids (all reagents from Gibco-BRL) at 37°C, 95% humidity and 5% CO2 as described previously [26–34]. Caco-2/15 cells were maintained in T-75 cm2 flasks (Corning Glass Works) and were split (1:5) when they reached 90% confluence using 0.05% trypsin/0.5 mM EDTA (Gibco-BRL). For individual experiments, cells were plated at a density of 1×106 cells/well on six-well culture plates and were cultured for 10 days post-confluence, a period at which they are highly differentiated and appropriate for experimental treatments [26–34]. The medium was refreshed every second day.
Caco-2/15 cell integrity
After various treatments, cell integrity was estimated by viability, morphology and differentiation assays. Briefly, cell differentiation was assessed by determination of villin protein expression. Monolayer intactness and physical barrier function were tested by evaluating morphology, transepithelial electric resistance and occludin protein expression. Finally, cell viability was appraised using the MTT assay.
Induction of OxS and inflammation
Differentiated intestinal Caco-2/15 cells were used to explore the effects of the polyphenol fractions on OxS (induced by the mixture of 200 μM iron and 2 mM ascorbate, Fe/Asc) and inflammation [prompted by 200 μg/ml lipopolysaccharide (LPS)] as previously reported . CP fractions of different molecular masses (LC, MC and HC) at the concentration of 250 μg/ml were added to the apical compartment of Caco-2/15 cells for 24 h before incubation with Fe/Asc or LPS for 6 h at 37°C.
Measurement of lipid peroxidation
Estimation of lipid peroxidation was assessed by quantifying malondialdehyde (MDA) by HPLC following exposure of Caco-2/15 cells to Fe/Asc. Briefly, proteins were precipitated with 8% sodium tungstate (Na2WO4) (Aldrich). The protein-free supernatants were then reacted with an equivalent volume of 0.5% thiobarbituric acid solution (TBA; Sigma) at 95°C for 60 min. After cooling to room temperature, the pink chromophore [MDA-(TBA)2] was extracted with butan-1-ol and dried over a stream of nitrogen at 50°C for 3 h. The dry extract was then resuspended in 100% methanol before MDA determination by HPLC with a fluorescence detection (Jasco) set at 515 nm excitation and 550 nm emission.
Fatty acid (FA) analysis
Following differentiation, Caco-2/15 cells were incubated for 6 h at 37°C in the absence or presence of Fe/Asc (200 μM/2 mM) following pre-incubation with 250 μg/ml CP fractions. Cells were then homogenized in PBS containing 0.005% 2,6-di-tert-butyl-4-methylphenol (Sigma). Samples were subjected to trans-esterification and injected into a gas chromatograph using a 90 m×0.32 mm WCOT-fused silica capillary column VF-23ms coated with 0.25 μm film thickness (Varian) according to the method described previously .
Endogenous antioxidant enzyme activities
Differentiated Caco-2/15 cells were harvested in hypotonic lysis buffer (10 mM Hepes, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 0.2 mM PMSF). Total superoxide dismutase (SOD) activity was determined as described by McCord et al. . Briefly, superoxide radicals (O2−) were generated by the addition of xanthine and xanthine oxidase, and the oxidation of the SOD assay cocktail was followed using a spectrophotometer at 550 nm for 5 min. The same reaction was then repeated with the addition of the sample. The total SOD activity was then calculated. For glutathione peroxidase (GPx) activity, aliquots of cell homogenates were added to a PBS buffer containing 10 mM GSH, 0.1 U of glutathione reductase (GSH-Red) and 2 mM NADPH with 1.5% H2O2 to initiate the reaction. Absorbance was monitored every 30 s at 340 nm for 5 min . For GSH-Red activity, cell homogenates were added to a PBS buffer containing 2 mM NADPH and 10 mM GSSG to initiate the reaction. Absorbance was monitored every 30 s at 340 nm for 5 min . The assay for catalase (CAT) activity was adapted from the protocol reported by Jiang et al.  with measurement of Xylenol Orange oxidation at 560 nm in the presence of ferrous ions. Briefly, 100 μM H2O2 were added and the absorbance was monitored at 560 nm. Samples were then incubated on ice with the same H2O2 concentrations and allowed to react for 5 min. CAT activity can then be calculated using a standard curve.
Following incubation with various stimuli, differentiated Caco-2/15 cells were sonicated and the Bradford assay (Bio-Rad) was used to determine the protein concentration of each sample. Proteins were denatured in sample buffer containing SDS and 2-mercaptoethanol, separated SDS/PAGE (7.5%) and electroblotted on to Hybond nitrocellulose membranes (Amersham). Signals were detected with an enhanced chemiluminescence system for antigen–antibody complexes. Non-specific-binding sites of the membranes were blocked using defatted milk proteins followed by the addition of one of the following primary antibodies: 1:1000 polyclonal anti-villin (94 kDa; BD Biosciences); 1:1000 polyclonal anti-occludin (59 kDa; Abcam); 1:1000 polyclonal anti-cyclo-oxygenase-2 (COX-2) (70 kDa; Novus); 1:10000 polyclonal anti-nuclear factor κB (NF-κB) (65 kDa; Santa Cruz Biotechnology); 1:5000 polyclonal anti-inhibitor of NF-κB (IκB) (39 kDa; Cell Signaling); 1:5000 polyclonal anti-tumour necrosis factor (TNF)-α (26 kDa; R&D); 1:5000 monoclonal anti-interleukin (IL)-6 (25 kDa; R&D), 1:1000 polyclonal anti-nuclear factor erythroid-2-related factor 2 (Nrf2) (68 kDa; Abcam); 1:1000 polyclonal anti-peroxisome-proliferator-activated receptor γ co-activator-1 α (PGC-1α) (92 kDa; Abcam); 1:40 000 monoclonal anti-β-actin (42 kDa; Sigma; 1:1000 anti-mitochondrial transcription factorA (mtTFA) (25 kDa; Santa Cruz Biotechnology); 1:1000 polyclonal anti-mTFB1M (39 kDa; Novus Biologicals); 1:1000 polyclonal anti-mtTFB2M (40 kDa; Novus Biologicals); 1:1000 poly-clonal anti-mtRNA polymerase (135 kDa; Abcam); 1:1000 polyclonal anti-8-oxoguanine-DNA glycosylase (OGG1) (39 kDa; Novus Biologicals), 1:1000 monoclonal anti-cytochrome c (15 kDa; Novus Biologicals), 1:1000 anti-(apoptotic-inducing factor) (AIF) (67 kDa; Abcam) and 1:1000 anti-Bcl2 (26 kDa; Abcam).
The relative amount of primary antibody was detected with species-specific horseradish-peroxidase-conjugated secondary antibody (Jackson Laboratory). The β-actin protein expression was determined to confirm equal loading. Molecular size markers (Fermentas) were simultaneously loaded on gels. Blots were developed and the protein mass was quantitated by densitometry using an HP Scanjet scanner equipped with a transparency adapter and the UN-SCAN-IT gel 6.1 software.
Prostaglandin E2 determination
Cellular prostaglandin E2 (PGE2) was measured by ELISA (Arbor Assay). After a short incubation, the reaction was stopped and the intensity of the generated colour was detected in a microtitre plate reader (EnVision Multilabel Plate Readers, PerkinElmer) capable of measuring 450 nm wavelengths.
Mitochondrial and nucleus separation
Differentiated Caco-2/15 cells were washed twice with PBS and left on ice for 5 min in a buffer containing 250 mM sucrose, 3 mM EDTA and 1 mM DTT at pH 7.4. Cells were then scraped and homogenized with a glass pestle Dounce homogenizer. The homogenate was centrifuged at 1000 g for 10 min at 4°C to obtain the pellets containing nuclei. The supernatant was then collected and centrifuged at 10000 g for 10 min to obtain the pellets containing mitochondria. The pellet was resuspended in the above-mentioned buffer, then was quick-frozen and stored at −80°C. The nuclear samples were collected for protein expression analysis using Western blotting to evaluate NF-κB, Nrf2 and PGC-1α, whereas mitochondrial organelles were employed to assess their specific transcription factors [mtTFA, mtTFB1, mtTFB2 and mitochondrial RNA polymerase (POLRMT)], as well as OGG1, AIF, cytochrome c and Bcl-2.
Assessment of intracellular ATP
Intracellular ATP was measured using luciferase-driven bioluminescence using EnzyLight™ ADP/ATP Ratio Assay Kit (Bioassay systems) as reported previously . Values for mitochondria were then normalized further with regard to the protein content.
All values are expressed as means±S.E.M. Data were analysed by using a one-way ANOVA and the two-tailed Student's t test using the Prism 5.01 (GraphPad Software) and the differences between the means were assessed post-hoc using Tukey's test. Statistical significance was defined as P< 0.05.
Profile of phenolic compounds of cranberry fractions
Total contents of phenolic compounds in the different cranberry fractions were determined by the colorimetric Folin-Ciocalteu method (Table 1). A large variation was found in the composition of the different CP fractions ranging from 5.0±0.1 to 54.5±0.9 g of gallic acid equivalents/100 g of extract mass. The combination of UPLC coupled to a TDQ tandem quadrupole mass spectrometer revealed that the LC fraction was essentially constituted of small absorbable phenolic acids and some anthocyanins. The MC fraction was constituted of anthocyanin, flavonols and proanthocyanidin monomers and dimers. The HC fraction was devoid of phenolic acids and of anthocyanin but contained flavonols (35% of total) and procyanidins (65% of total). The latter were essentially composed of dimers (47%), oligomers (DP 3 to 5) (38%) and polymers (DP 5 to 10) (15%).
|.||Cranberry fraction .|
|Polyphenol compounds .||LC .||MC .||HC .|
|Oligomers (DP 3 and 4)||–||10.9±0.1|
|Polymers (DP 5 to > 10)||–||4.3±0.1|
|.||Cranberry fraction .|
|Polyphenol compounds .||LC .||MC .||HC .|
|Oligomers (DP 3 and 4)||–||10.9±0.1|
|Polymers (DP 5 to > 10)||–||4.3±0.1|
Cell integrity following various treatments
The effects of Fe/Asc and LPS on Caco-2/15 cells integrity were examined by morphological assessment, protein content quantification and MTT assay after a 6-h incubation period. The morphology and the protein content remained unchanged with the administration of Fe/Asc and LPS, as well as following treatment with the CP fractions (data not shown). Similarly, Caco-2/15 cell viability and monolayer membrane permeability were not affected by the addition of the different treatments (Supplementary Figure S1). On the other hand, an enhancement of cell viability and protein expression of occludin (a biomarker for tight junction and mucosal barrier function) was observed when Caco-2/15 cells were cultured in the presence of the MC fraction (Supplementary Figure S1). Thus, it can be concluded that cell integrity was not compromised by the experimental conditions.
Effects of CP fractions on lipid peroxidation
The extent of lipid peroxidation following the treatment of Caco-2/15 cells with Fe/Asc during 6 h was assessed by determining MDA levels. HPLC analyses indicated a 4-fold increase in MDA (P< 0.001) following the administration of the oxygen free radical generating system Fe/Asc as compared with controls (Figure 1A). The presence either of the LC, MC or HC cranberry fraction was found to counteract Fe/Asc-mediated lipid peroxidation, with a more favourable impact for HC (Figure 1A).
Effects of cranberry on lipid peroxidation and on regulatory endogenous antioxidant activities in Caco-2/15 cells
Since OxS may alter the composition and properties of the bilayer lipid environment, we next determined whether Fe/Asc was able to affect cellular FA profile. A significant decrease was observed in n-3 and n-6 polyunsaturated FAs (PUFAs) [eicosapentaenoic acid (EPA), 20:5n-3; docosahexaenoic acid (DHA), 22:6n-3; linoleic acid (LA), 18:2n-6] as well as in monounsaturated FAs (18:1n-9), which led to a reduction in the calculated total n-3, n-7 and n-9 PUFA contents compared with controls (Table 2). As n-3 PUFAs were more affected by OxS than n-6 PUFAs, an increase was observed in the n-6/n-3 ratio, which might promote an inflammatory state. On the other hand, pre-incubation with CP fractions restored the levels and composition of PUFAs. Once again, HC fraction polymers had a more favourable impact on bilayer lipid environment after the induction of OxS (Table 2).
|FAs .||Ctrl (%) .||Fe/Asc (%) .||LC + Fe/Asc (%) .||MC + Fe/Asc (%) .||HC + Fe/Asc (%) .|
|FAs .||Ctrl (%) .||Fe/Asc (%) .||LC + Fe/Asc (%) .||MC + Fe/Asc (%) .||HC + Fe/Asc (%) .|
Mechanisms for the action of CP fractions on OxS
As failure of antioxidant defence may favour the induction of OxS, we examined various endogenous antioxidant enzymes and we found that treatment of Caco-2/15 cells with Fe/Asc caused a significant augmentation in total SOD activity. In accordance with our previous results, pre-incubation with the CP fractions blunted the effects of OxS (Figure 1B). Under these same conditions, GPx activity was down-regulated by Fe/Asc and restored by treatment with the MC and HC fractions (Figure 1C). CAT activity was also affected by Fe/Asc and normalized by treatment with the CP fractions (Figure 1E). On the other hand, GSH-Red activity was not significantly affected by either the Fe/Asc or polyphenol treatments (Figure 1D).
Effects of CP fractions on inflammatory markers
Eicosanoids and cytokines are pro-inflammatory compounds produced by cells in response to injury. The formation of inflammatory eicosanoids such as PGE2 is synthesized from arachidonic acid by specific enzymes, especially COX-2. Our experiments showed that Fe/Asc and LPS elicited exaggerated synthesis of PGE2 and that pre-incubation with CP fractions completely blocked COX-2 induction by these pro-inflammatory treatments (Figures 2C and 2D). Noteworthy, we found distinct effects of the CP fractions on PGE2 production: although the HC fraction was the more efficient in lessening Fe/Asc-mediated PGE2 accumulation (Figure 2A), the LC fraction was more potent in lowering LPS-induced PGE2 accretion (Figure 2B). We then assessed the production of TNF-α and IL-6, two powerful inflammatory mediators, following the incubation of Caco-2/15 cells with Fe/Asc and LPS. Western blot analyses indicated that the CP fractions were able to significantly decrease and, in most cases, to completely abolish TNF-α and IL-6 production (Figures 3A–3D).
Regulatory effects of CP on oxidative stress and LPS-induced inflammation on PGE2 and COX-2 in Caco-2/15 cells
Effects of CP on oxidative stress or LPS-induced inflammation on pro-inflammatory cytokines in Caco-2/15 cells
Effects of CP fractions on key transcription factors
The NF-κB signalling pathway plays a crucial role in the initiation and amplification of inflammation via the modulation of multiple inflammatory mediators. Figure 4 shows that Caco-2/15 cells exposed to Fe/Asc or LPS displayed a high NF-κB/IκB protein ratio, which is indicative of elevated transcriptional activation of its target inflammatory genes (Figures 4A and 4D). Importantly, the CP fractions were found to blunt the activation of the NF-κB pathway in these cells. To further decipher the mechanisms of action of the CP fractions, we examined the transcription factors that are involved in the regulation of antioxidant genes expression and mitochondrial biogenesis. The protein mass of Nrf2 was down-regulated by Fe/Asc- or LPS-induced OxS and inflammation (Figures 4B and 4E). However, treatment with the CP fractions prevented the down-regulation of Nrf2 protein expression when the cells were pre-incubated with CP fractions before their exposure to Fe/Asc or LPS. We also assessed PGC-1α, a transcriptional co-activator known for up-regulating Nrf2. PGC-1α protein mass was reduced in response to both Fe/Asc and LPS treatments in Caco-2/15 cells (Figures 4C and 4F). However, pre-incubation with the CP fractions was able to prevent PGC-1α down-regulation by the inflammatory agents.
Effects of CP on oxidative stress or LPS-induced inflammation on key transcription factors in Caco-2/15 cells
Effect of cranberry procyanidin rich fraction (HC) on mitochondrial functions
The main function of the mitochondrion is the production of energy in the form of ATP via oxidative phosphorylation and oxygen consumption. We therefore assessed the amount of ATP levels in mitochondria extract from Caco-2/15 cells exposed to the Fe/Asc oxygen radical generating system. As observed in Figure 5(A), the administration of Fe/Asc led to a 6-fold reduction in ATP level compared with untreated cells. However, pre-incubation with the HC fraction almost fully prevented the reduction in ATP levels.
Effects of high molecular mass phenolic cranberry compounds on mitochondrial fonctions induced by OxS in Caco-2/15 cells
Protective effect of procyanidin rich fraction against OxS-induced apoptosis
In the process of programmed cell death, mediators of apoptosis are released from mitochondria through disruptions in the outer mitochondrial membrane and then participate in caspase activation and DNA degradation. In our study, we focused on one specific mediator of the outer mitochondrial membrane, the anti-apoptotic protein Bcl-2. Treatment of Caco-2/15 cells with Fe/Asc lowered Bcl-2 protein expression and pre-incubation with the HC fraction blunted this effect (Figure 5C). AIF is normally located in the inter-membrane space of mitochondria and is involved in initiating a caspase-independent pathway of apoptosis by causing DNA fragmentation and chromatin condensation. Furthermore, when cell death is triggered by an apoptotic stimulus, cytochrome c is released into the cytosol and contributes to the caspase-dependent pathway of apoptosis. Western blot analysis revealed an increase in the level of AIF (1.5-fold) and cytochrome c (2.5-fold) protein masses in the mitochondrial extract following the addition of Fe/Asc to Caco-2/15 cells (Figures 5B and 5D, respectively). Pre-incubation with the HC fraction prior to Fe/Asc administration prevented the rise in both AIF and cytochrome c protein mass.
Effect of procyanidin rich fraction on OGG1 repair enzyme level
The base excision repair pathway is primarily responsible for removing 8-OHdG from mitochondrial DNA . In humans, 8-OHdG is repaired by 8-oxoguanine DNA glycosylase (OGG1), an enzyme that recognizes and hydrolyses the aberrant base from the DNA backbone. As illustrated in Figure 6(A), treatment with Fe/Asc resulted in a significant (P< 0.01) reduction in OGG1 protein mass as compared with control cells. However, pre-incubation of Caco-2/15 cells with the HC fraction prevented the decline in OGG1 expression.
Effects of high molecular mass phenolic cranberry compounds on the mitochondrial protein expression of OGG1 and transcription factors by OxS in Caco-2/15 cells
Effects of procyanidin rich fraction on mitochondrial transcription factors
Human mitochondrial transcription requires bacteriophage-related RNA polymerase, POLRMT, mtDNA-binding protein, h-mtTFA/TFAM and two transcription factors/rRNA methyltransferases, h-mtTFB1 and h-mtTFB2. These crucial proteins define mitochondrial biogenesis and gene expression that together are believed to fine-tune mitochondrial functions [34,40,41]. Given the deleterious effects of Fe/Asc, it was mandatory to explore how OxS modulates the core protein components required for mitochondrial transcription. Western blot analyses showed a significant increase in POLRMT, mtTFA, mtTFB1 and mtTFB2 protein expression (Figures 6B–6E) in Caco-2/15 cells treated with Fe/Asc compared with control untreated cells. However, pre-incubation with the HC fraction was found to fully block the changes in these transcription factors.
Emerging data provide substantial evidence to classify numerous fruits as functional nutrients with several preventive and therapeutic health benefits [42–45]. Among them, cranberries have received attention as a result of their association with protection against urinary tract infections , glycaemic response improvement  and cardiovascular risk prevention [48,49], but only few reports have focused on gastrointestinal health. The present study was therefore conducted in order to test the potential effects of CP fractions on cell viability, membrane permeability, OxS, inflammation and mitochondrial functions in intestinal epithelial cells. We were particularly interested in determining which polyphenolic species was responsible for the presumed biological activity. We thus separated three fractions differing in their molecular size and intestinal absorption capacity. The LC fraction was composed of anthocyanins (1/3) and phenolic acids (2/3). The former of these molecules are relatively poorly absorbed and the latter have been shown to be readily absorbable by the enterocyte [50,51]. The MC fraction was composed of some poorly bioavailable anthocyanins, flavonols and small molecular mass procyanidins in approximately equal quantity. Importantly, the bioavailability of both the flavonols and the procyanidin monomers and dimers was previously evidenced . On the other hand, the HC fraction contained flavonols and procyanidin dimers but was particularly enriched in procyanidins, molecules with higher molecular mass and of which the oligomeric and polymeric forms are poorly absorbed . Hence, the distinctive difference between the MC and the HC fractions was the higher proportion of procyanidins that are not as well absorbed by the enterocyte.
The effects of polyphenols were investigated with Caco-2/15 cells that undergo a process of spontaneous differentiation leading to the formation of a monolayer of cells expressing several morphological and functional characteristics of mature human enterocytes. This remarkable intestinal model is regarded as the most appropriate for the investigation of gut absorption and interactions, nutrition, toxicology food microbiology, bioavailability tests and screening of drug permeability in discovery programmes. Multiple studies from our laboratory [54–61] have shown that Caco-2/15 monolayers are fully appropriate for the study of fat absorption, lipid/lipoprotein homoeostasis, OxS and inflammation. Importantly, when seeded on porous filters (Transwell), Caco-2/15 cells permit access to both sides of the bipolar intestinal epithelium: apical and basolateral compartments corresponding to intestinal lumen or serosal circulation, respectively. Therefore, the Caco-2/15 cell model has been proved to be a good alternative for human and animal studies and has emerged as one of the standard in vitro tools to predict in vivo intestinal absorption of various substances, such as polyphenols . For example, Caco-2 cells have been shown to produce sulfate and glucuronide conjugates of resveratrol  and to achieve sulfation that constitutes the primary route of intestinal metabolism of epicatechin, an antioxidant flavonoid in tea .
If one assumes a Western person consumes 1 g of polyphenols per day  and the volume of the intestine is about 6.25 litres, one would expect that the enterocytes are exposed to a concentration of about 235 μg/ml, which is very close to what we used in our experiments. Taking into account the volume of the colon (approximately 4.25 litres), we may obtain even higher values, which justifies the concentration (250 μg/ml) that was administered to Caco-2/15 cells. Therefore, the dose employed in our experiments is physiologically representative .
Our results showed that treatment of Caco-2/15 cells with CP fractions evoked no cell damage, but significantly increased occludin, a tight junction protein, suggesting an improved intestinal mucosal barrier that efficiently restricts paracellular permeability. The MC fraction caused the highest stimulation of occludin production, whereas HC improved the production of the protein under exposure to Fe/Asc stress. The high flavonol content of both the MC and HC fractions may explain this response since it was reported that quercetin enhances intestinal barrier by increasing the assembly of the tight junction protein occludin . This class of molecules, along with procyanidins, has been shown to improve the mucosal intestinal integrity  and kidney epithelial layer tightness .
Additionally, our experiments revealed the remarkable capacity of CP fractions to stimulate endogenous antioxidant mechanisms as they protected Caco-2/15 cells from Fe/Asc-induced OxS. CP prevented the increase in LPS-mediated pro-inflammatory cytokines, lowered PGE2 production via COX-2 inhibition and suppressed inflammation and NF-κB activation. Finally, procyanidin oligomers and polymers (HC fraction) restored mitochondrial function as suggested by the normalization of mitochondrial ATP production and apoptosis through modulation of local transcription factors and of PGC-1α, a key protein that controls mitochondrial biogenesis and respiration.
PGE2 production/biosynthesis during inflammatory processes is primarily modulated by the concerted activities of three enzymes: prostaglandin E synthase-1 (mPGES-1), COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The former two enzymes are involved in the synthesis of PGE2 and are inducible by a huge range of stimuli, which include pro-inflammatory IL-1β, TNF-α and IL-6 [70,71], whereas 15-PGDH is an enzyme degrading PGE2. The role of polyphenols in PGE2 pathway remains quite unclear. It is possible that different types of polyphenols may have distinct actions on the three enzymes, which may explain the discrepancy between COX-2 abundance and PGE2 concentrations.
ROS are highly reactive transient chemical molecules. In limited concentrations, they are indispensable in many normal cellular processes, but their generation under physiological condition is tightly controlled by a large number of antioxidant systems. Among the efficient antioxidant enzymes, the SOD catalysed the conversion of O2− into H2O2 and molecular oxygen, whereas the decomposition of H2O2 to non-toxic compounds is the main function of CAT and GPx . An imbalance between ROS and antioxidant defence causes OxS  that can elicit general damage to cells by promoting the oxidation of proteins, DNA and lipids  with a direct implication in the pathogenesis of IBD [75,76]. Limited efforts have been devoted to understand the influence of CP fractions on the gastrointestinal tract, despite being recognized for developing OxS in response to constant noxious luminal oxidant exposure. Our data clearly disclosed that the CP fractions acted as potent antioxidants that markedly reduced the extent of OxS. Apparently, one of the critical mechanisms for inactivating OxS by CP fractions was via the induction of endogenous antioxidant proteins (SOD, GPx and CAT). Also, our studies clearly pointed out that CP fractions positively modulated the antioxidant defence by up-regulating Nrf2, a central transcription factor that initiates the transcription of cytoprotective genes following binding to specific DNA sites termed antioxidant response elements (AREs) .
Caco-2/15 cells have the ability to activate the transcription factor Nrf2, a master regulator of cellular defences against OxS. Under basal conditions, Nrf2 is bound to its cytosolic inhibitor Keap1, which functions as an adaptor protein in the cullin 3 (Cul3)-based E3 ligase complex that ubiquitinates Nrf2 resulting in proteasomal degradation . In the presence of oxidative or electrophilic stress, ubiquitination of Nrf2 is disturbed allowing the accumulation of Nrf2 in the nucleus where Nrf2 binds to the ARE  in the regulatory regions of the target genes and drives their expression. In our studies, Nrf2 was assessed in the nucleus, which reflects its full potential to play its cytoprotective regulatory role.
In the present work, CP fractions not only targeted antioxidant components, but also reinforced anti-inflammatory mechanisms. Indeed, the incubation of Caco-2/15 cells with LPS in presence of CP fractions led to the prevention of NF-κB activation and to the reduced generation of the pro-inflammatory mediators TNF-α and IL-6. The CP fractions also targeted the COX-2 pathway further amplifying the protection against inflammation. The inactivation of NF-κB probably led to the down-regulation of COX-2, a pro-inflammatory enzyme responsible for the elevated levels of PGE2 prostanoids . Overall, our data demonstrated that the different CP fractions are able to suppress LPS-induced pro-inflammatory cytokines and COX-2 expression via the control of NF-κB signal transduction pathway. We therefore propose that maintaining antioxidant and anti-inflammatory homoeostasis of intestinal tissue by CP may help prevent and/or provide added benefit to treatments of intestinal diseases leading to obvious health benefits.
Mitochondrial processes are of great importance to cells of multiple organs and include energy metabolism, generation of free radicals, calcium homoeostasis and initiation of apoptosis via release of the respiratory protein cytochrome c. Their membranes are potential targets of OxS, but mitochondria are also a source of pro-oxidants and critical regulators of survival and death, thereby contributing to complex diseases [81–83]. Based on the facts that grape seed procyanidins (containing a mixture of monomers, dimers, oligomers and polymers) modulate energy metabolism in skeletal muscle mitochondria  and that quercetin flavonols preferentially accumulate in mitochondria and prevent damage by ROS , we specifically studied the impact of the HC fraction on mitochondrial activity and integrity, assuming that this fraction contained a high quantity of both flavonols and procyanidins. Our data point out that OxS activated apoptotic signalling pathway as indicated by a significant increase in the pro-apoptic protein cytochrome c and a down-regulation of the anti-apoptotic protein Bcl-2. It is believed that the accumulation of OxS by-products in mitochondria probably constitutes the upstream cascade trigger that leads to apoptosis . Accordingly, we showed the anti-apoptotic effects of cranberry flavonols and procyanidins given the increment of mitochondrial Bcl-2 and the reduced expression of cytochrome c and AIF in their presence. Since Bcl-2 is a key anti-apoptotic regulator of mitochondrial outer membrane permeabilization and a prerequisite for cytochrome c release from mitochondria to cytosol, the positive modulation of this pathway by cranberry flavonols and procyanidins definitely suggests an inhibition of apoptosis. According to available studies, the flavonol quercetin altered the intramitochondrial Ca2+ mobilization, cytotoxicity and apoptosis induced by indomethacin and thus appears to prevent mitochondrial dysfunction by regulating intracellular Ca2+ homoeostasis and preventing apoptosis .
Mitochondrial biogenesis is dependent on the cross-talk between the nuclear and mitochondrial genomes orchestrated by PGC-1α . PGC-1α also mediates mitochondrial DNA transcription and replication through two nuclear-encoded genes, including mtFA and mtFB . Our studies support the notion of a protective effect of cranberry flavonols and procyanidins on mitochondrial functioning perturbations by restoring ATP synthesis via the increase in PGC-1α protein expression. Additional studies are necessary to delineate whether the stimulation of ATP production by CP is accomplished via mitochondrial biogenesis and/or bio-energetically mitochondrion efficiency. The former action is favoured since it is already known that quercetin induces muscle mitochondrial biogenesis and contributes to improving exercise recuperation by increasing PGC-1α and SIRT1 expression . Finally, as suggested by our findings, cranberry flavonols and procyanidins were able to raise OGG1, the DNA repair enzyme, which eliminated DNA damage caused by OxS, thereby preventing the initiation of the vicious cycle of ROS.
The polymers of the HC fraction are not absorbed by intestinal epithelial cells, but could provide a physical barrier for free radicals and antioxidant protection . In addition, they may interact with the lipid bilayer and proteins of the membrane, which may alter its chemico-physical properties, thereby generating signals that constitute input for the enterocytes to adjust their metabolism and to influence a number of physiological processes, including glucose uptake via alterations of glucose transporter genes [92,93] and insulin sensitivity  through enhancing insulin signalling inflammatory, cholesterol and lipogenic pathways in intestinal enterocytes . Additional studies are evidently necessary to uncover the mechanisms of action of HC polyphenols to modulate mitochondrial adaptations.
In conclusion, our data support the importance of examining the roles of CP in regulating cellular processes related to OxS and inflammation, as well as the cross-talk between cellular compartments, which increases our understanding of the mode of actions of this unique blend of polyphenols. Such advances may help determine whether CP constitute novel therapeutic approaches to better treat IBD.
antioxidant response element
degrees of polymerization
high molecular mass CP
inflammatory bowel diseases
inhibitor of nuclear factor κB
low molecular mass CP
medium molecular mass CP
mitochondrial transcription factor A
mitochondrial transcription factor B
nuclear factor κB
nuclear factor erythroid 2-related factor 2
peroxisome-proliferator-activated receptor γ co-activator-1-α
mitochondrial RNA polymerase
polyunsaturated fatty acid
reactive oxygen species
tumour necrosis factor-α
UltraPerformance LC®-tandem quadrupole
Marie-Claude Denis, Alexandra Furtos and Emile Levy participated in the design of the study. Marie-Claude Denis, Stéphanie Dudonné, Carole Garofalo and Alain Montoudis conducted the experiments. Marie-Claude Denis, Yves Desjardins, Edgar Delvin and Emile Levy analysed and interpreted the data. Marie-Claude Denis, Valérie Marcil, Yves Desjardins, André Marette and Emile Levy contributed to the writing of the paper.
We thank Schohraya Spahis for her excellent technical assistance.
The present study was supported by the J. A. DeSève Research Chair in Nutrition, the Canadian Foundation of Innovation (to E.L.), Leahy Orchards & Appleboost Products Inc. and scholarship award from the Fonds de recherche du Québec-Nature et technologies (to M.C.D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.