Buckwheat and buckwheat enriched products exert anti-inflammatory effect on myofibroblasts of colon CCD-18Co

Abstract

Buckwheat (BW) constitutes a good source of bioactive components that shows anti-inflammatory effects in vitro and in vivo. The use of functional foods in the prevention and treatment of inflammatory bowel diseases (IBDs) has aroused an increasing interest. This study investigates the effect of in vitro digested BW and BW-enriched products (BW-enriched wheat breads, roasted BW groats –fermented and non-fermented-, and BW sprouts) on colon myofibroblasts, cells involved in the regulation inflammatory response in the intestine. The cells were treated with the different digested-BW products, alone or together with TNF-α (20 ng/mL), and the effects on cell migration, mitochondrial membrane potential and cell cycle, processes altered during intestinal inflammation, were investigated. A significant reduction in TNF-α-induced migration (25.5%, p<0.05) and attenuation of TNF-α-altered cell cycle (p<0.05) was observed in myofibroblasts treated with BW-enriched white wheat bread. These results contribute to extend the beneficial effects derived from BW bioactive compounds, and suggest that BW consumption can exert beneficial effects on IBDs.

Introduction

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Chron’s disease, is a multifactorial disease of unknown etiology. Here, endogenous (gene and immune) as well as exogenous (lifestyle and diet) factors have been reported to significantly impact the pathogenesis of IBD ¹. Even though the relation between diet and IBD has been established, there is still an incomplete understanding of its role. Thus, some dietary habits (i.e., high animal fat consumption) can contribute to IBD development, whereas others, dietary ϖ−3 long-chain fatty acids as well as fiber rich fruit consumption can prevent or reduce the IBD pathological manifestations ².

BW is a pseudocereal that has received increasing attention as functional food ³. The interest of the food industry in the innovation and research of functional foods has launched numerous studies into the elaboration of an important variety of both BW grains and BW-containing foodstuff, including sprouts, groats, tea, bread, noodles, and beer ⁴. For example, BW-enriched cookies consumption has been proven to exert hypocholesterolemic activity in humans ⁵, while the inclusion of BW in honey and bread formulation increases the plasma antioxidant capacity in healthy volunteers ^{6, 7}.

Krkoskova and Mrazova⁸ reported that whole BW groats are composed of starch (55%), ash (2%), lipid (4%), protein (12%), carbohydrates (2%), dietary fiber (7%), and other compounds (18%) such as polyphenols. It is well known that BW is an important source of polyphenols intake. Flavone C-glucosides, including vitexin (Vi), isovitexin (IsoVi), orientin (Or) and isoorientin (IsoOr), as well as rutin (Ru) and quercetin (Q) (Figure 1) have been quantified to be in the range of 0.02 – 15.73 mg/g in BW grains and sprouts ^{9, 10}. In the gastrointestinal tract, Ru can reach the colon in its original form, where is metabolized by the microbiota, releasing Q ¹¹. Flavones C-glucosides have been documented to be poorly absorbed, remaining in the gastrointestinal tract up to 24 h ¹². The low absorption of these compounds and the concentrations achieved in the gut suggest that Ru, quercetin, and flavone C-glucosides ^{12, 13} stay at the intestinal level long enough to exert their beneficial effects via regulating cell physiology, promoting the proliferation of beneficial intestinal bacteria ¹⁴, and amelioration of intestinal inflammation ^15–19.

Figure 1.

Chemical structure of polyphenols present in the different buckwheat (BW) products investigated.

A major feature of the cytokine profile in IBD is an increased production of the TNF-α, a molecule that plays a crucial role in IBD onset ²⁰. Increased levels of TNF-α at the intestinal level leads to disruption of the physiological integrity of the intestinal barrier ²¹, allowing the translocation of the lumen content into the intestinal wall, where will contact with the cells of the sub-epithelial space. Here, myofibroblasts are found in the lamina propia. These cells interact with epithelial and immune cells to modulate the inflammatory response and regulate important processes such as fibrosis and tissue repair in the intestine²². The active role of myofibroblasts in the pathophysiology of IBD has resulted in an important number of in vitro studies investigating the potential anti-inflammatory effect of polyphenol-rich dietary foodstuff on intestinal fibroblasts ^{15, 23, 24}.

Even though there is compelling in vivo evidence about the contrasting role of dietary plant foods on the onset and progression of IBD ^{2, 25}, in vivo and in vitro studies have described that fruits and vegetables consumption can be considered a feasible therapy to ameliorate TNF-α effects on intestinal inflammation ^{26, 27}. Hence, further studies will help clarify to what extent and how far can the pathological manifestations of IBD be controlled by dietary plant-derived bioactive compounds. Hitherto, little is known about the effect of BW and BW-enriched products on TNF-α-inflamed intestinal cells. We hypothesized that BW consumption can exert anti-inflammatory effects by modulating processes altered during intestinal inflammation. Thus, this study aims at investigating the cellular responses of myofibroblasts (CCD-18Co cells) of colon treated with TNF-α and different BW products and/or the individual flavonoids. Specifically, we explored their effects on i) cellular migration, ii) cell cycle, and iii) mitochondrial membrane potential.

Material and methods

Materials.

α-amylase (A1031–5KU, 98.3 U/mg solid), pepsin (P7000, 92 U/mg solid), pancreatin (P7545, 8xUSP), bile salts extract (B8631), MTT [3-(4,5-dimethylthyazol-2-yl)-2,5-diphenyltetrazolium bromide], human recombinant TNF-α, rutin (quercetin-3-rutinoside), dihydrorhodamine 123 (DHR), propidium iodide (PI), RNAse A, and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vitexin (apigenin-8-C-glucoside), isovitexin (apigenin-6-C-glucoside), orientin (luteolin-8-C-glucoside), and isoorientin (luteolin-6-C-glucoside) were obtained from Extrasynthese (Genay, France). Dimethyl sulfoxide (DMSO) was purchased from MERK Millipore (Darmstadt, Germany). Ultrapure milliQ water was used for the elaboration of all solutions. All other chemicals were of analytical LC-MS grade.

BW products preparation.

The elaboration process of the different BW products is described in detail in supplementary material. Two types of BW-enriched wheat bread were prepared using dark wheat flour (BIO type 2000), white wheat flour (BIO type 500), and flour obtained by milling of roasted common buckwheat groats (BWg) (variety Kora) as described elsewhere ^28,29. The flour obtained from roasted groats was used to substitute dark wheat flour or white wheat flour (50% w/w) to produce BW-enriched dark wheat bread (_bBW) and BW-enriched white wheat bread (_b500BW), respectively. The BW-enhanced wheat breads formulation and baking conditions are shown in Supplementary Table 1. The bread powder was kept at −20 °C until analysis. Fermented roasted BW groats were elaborated as reported by Wronkowska et al ³⁰. The fermented product was freeze-dried and kept at −20 °C. BW sprouts (BWs) from common BW were provided by the Plant Breeding Station in Palikole (Poland) and were produced as described by Zielinska et al ³¹. Roasted BWg were obtained from a Polish local company (Olsztyn, Poland) and the material was freeze-dried, ground and kept at −20 °C ²⁸.

Flavonoids extraction.

50 mg of each freeze-dried BW products was extracted with 1 mL of 60% aqueous methanol containing 0,4% trifluroacetic acid. Each stage of extraction consisted of 30 s sonication (VC 750, Sonics & Materials, USA), followed by 30 s of vortexing and centrifugation at 13,200 × g for 10 min at 4°C in a centrifuge 5415R (Eppendorf, Germany). The supernatants were collected in 5 mL flask. The extraction process was performed in duplicate and repeated 5 times. The extracted samples were filtered (0.44 μm) and directly analyzed by HPLC coupled to SPD-M20A DAD detector (Shimadzu, Japan) to determine the flavonoid content in the different BW products (Table 1).

Table 1.

HPLC-DAD analysis of the concentration of flavonoids in the different buckwheat (BW) products

	Concentration (μg/g)				Concentration (μg/mg)
	bBW	b500BW	fBWg	roasted BWg	BWs
Orientin	-	-	-	-	3.51 ± 0.18
Isoorientin	-	-	-	-	7.88 ± 0.53
Vitexin	-	-	-	-	3.15 ± 0.11
Isovitexin	-	-	-	-	5.19 ± 0.14
Rutin	25.49 ± 2.61	35.45 ± 2.09	73.95 ± 6.00	59.45 ± 0.91	2.94 ± 0.07

Abbreviations: _bBW: Bread baked from “BIO” wheat flour and roasted BWF (50/50%);_b500BW: Bread baked from wheat flour type 500 and roasted BWF (50/50%);_fBWg: ‘Tempeh’ type product from dehulled roasted BW; BWg: Roasted BW groats; -: no detected.

In vitro digestion.

The lyophilized and milled BW products as well as a control sample (water) were subjected to an in vitro digestion as previously described ³² with some modifications. The procedure consisted of three stages: salival (pH 7.0), gastric (pH 2.0) and intestinal digestion (pH 7.5). 100 and 50 mg of BWs (BWs₁₀₀ and BWs₅₀, respectively) and 1 g of the rest of BW products were diluted in 10 mL of deionized water. 250 μL of α-amylase 98,3 U/mg solid diluted in 1 mM CaCl₂ solution (pH 7.0) was added to each sample and incubated under shaking in a water bath at 37°C for 5 min. In the gastric digestion the pH was reduced to 2.0 using HCl (6 N). Then, 0.025 gr pepsin (diluted in 0,1N HCl; 92 U/mg protein) was added per 0.5 g sample, and the mixture was incubated for 120 min. After the incubation, the pH was raised to 6,0 with 1M NaHCO₃ followed by the addition of 2.5 mL of a mixture containing pancreatin (activity 8x USP) and bile salts extract in 0,1M NaHCO₃. The pH was then adjusted to 7,5 with 1M NaHCO₃ and the samples incubated at 37°C for 120 min. The digestive enzymes were inactivated by heating (100 °C for 4 min), cooled and centrifuged at 13300 × g for 60 min at 4°C in a MPV-350R centrifuge (MPW Med. Instruments, Warsaw, Poland). The same digestion process was run in parallel of control samples containing water and a mixture of salt/enzymes. The supernatants were stored at – 20 °C until analysis and cellular assays were performed. For cell experiments, the digested products were sterilized (0.22 μm), diluted in DMEM and added to the cells.

Analysis of BW extracts by HPLC-diode array detector (DAD) analysis.

Prior to analysis, 250 μL ACN per 100 μL of digested solution was added followed by vortex and centrifugation at 15000 × rpm for 10 min (MPW 351R Centrifuge, Med. Instruments, Warsaw, Poland). The supernatant was evaporated in a vacuum concentrator plus (Eppendorf AG, Warsaw, Poland), and the samples diluted in 150 μL water:ACN (1:1 v/v). The samples were analyzed with a HPLC coupled to DAD (Shimadzu corp., Tokyo, Japan) to determine flavonoids composition. The separation was performed in a C18 column using water/ACN/formic acid (90:5:5) as mobile phase A, and water/ACN/formic acid (15:80:5) as phase B at 0.2 mL/min flow rate. The gradient started with 4% of solvent B. The concentration of B was increased up to 6% at 20 min, 11% at 35 min, 17% at 42 min and 80% at 45 min. This concentration was kept up to min 46, and then the initial conditions were re-established reaching 4% of B at 47 min. These conditions were maintained up to min 75. The flavonoids were identified according to their absorption spectra. The quantification was performed using pure external standards. This analysis was repeated 3 times (n = 3).

Cell line, culture conditions and cell treatment.

The myofibroblasts of colon CCD-18Co cell line was purchased from the American Type Culture Collection (ATTCC) (Rockville, MD, USA). Cells were routinely seeded at 6,000 cells cm⁻² and grown in Eagle’s minimum essential medium (EMEM) containing 10% v/v Fetal Bovine Serum (FBS) (Sigma, Saint Louis, USA), 2mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and sodium bicarbonate (1.5 g L⁻¹). The medium was also supplemented with penicillin (100 U mL⁻¹) and streptomycin (100 μg mL⁻¹). A pH between 7.2–7.4, 37 ºC of temperature and 5% CO₂/ 95% air atmosphere at constant humidity were the conditions to grow the cells. The myofibrobasts were subcultured using Trypsin-EDTA (0.25%–0.03%) after reaching 80% confluence. Population doubling levels (PDL) between 33 and 35 and passages ranging from 15 – 17 were used for all the experiments.

Cell viability assay.

Cell viability was evaluated by Trypan blue dye exclusion test. CCD-18Co cells were seeded in 6-well plates at 6,000 cells/cm² and were grown until confluence (5 – 6 days). Prior to the treatment of the cells, the digested BW products or control (salts + enzymes) were filtered (0.22 μm). The cells were treated with volumes ranging from 10 – 100 μL of the different digested BW products or control per 3 mL of medium in the presence or absence of TNF-α (20 ng/mL) for 48 h. Next, the cells were trypsinized and collected in 1 mL culture medium. 25 μL of the cell suspension was diluted in 75 μL of trypan blue, placed in a Neubauer hemocytometer (Fisher, Poznan, Poland) and cell viability determined. Viability results were expressed as percent of the control values. All experiments were repeated 3 times (n=3). Each experiment was performed in duplicate (2 wells per treatment).

Cell migration.

The migration assay was performed following a previously described method ³³. Confluent myofibroblasts were incubated in culture medium containing 0.1% (v/v) FBS for 24 h. Next, a horizontal scratch was made in the center of the well by scrapping the surface using a sterile ‘tip’, the cells were washed with PBS and treated as follows: i) first group: addition of 990 μL fresh medium (0.1% FBS v/v) and 10 μL digested _bBW, _b500BW, BWg, and _fBWg; ii) second group: addition of 950 μL fresh medium (0.1% FBS v/v) and 50 μL digested BWs (50 – 100 mg). Myofibroblasts treated with 10 or 50 μL of control digesta (salts + enzymes) were considered as controls of the first and second group, respectively.

In a second set of assays, the migration experiment was repeated treating the cells with the individual flavonoids present in the different BW products. The scratched cells were treated with three different concentrations (10, 1, and 0.1 μM) of rutin (Ru), vitexin (Vi), isovitexin (IsoVi), orientin (Or), and isoorientin (IsoOr). The cells were also treated with Q, at the same concentrations, since it is released from Ru in colon. Control cells were treated with an equivalent concentration of DMSO (0.5% v/v).

Different fields of the scratched area were photographed using a CKX41SF microscope (Olympus Corp., Tokyo, Japan) coupled to a DP25 digital color camera (Olympus, Tokyo, Japan). From 5 – 6 pictures from each well (2 wells per treatment) were taken of different sections of the scratched surface and the quantification was performed as previously described³⁴

Results are displayed as mean ± SD of 3 – 5 independent experiments (n = 3 – 5). Each experiment was performed in duplicate (2 wells per treatment).

Mitochondrial enzyme activity (MTT test).

In order to evaluate the impact of BW-enriched products on mitochondrial functionality, the maximum volumes of BW products and control (salt + enzymes) without cytotoxic effects were also tested by MTT. After the treatment with the digested-BW products, the medium was removed, the cells were washed with PBS and 200 μL MTT (1 mg/mL) added to the cells followed by incubation for 4 h at 37 °C. The MTT was removed and 100 μL DMSO added to dilute the formazan crystals. The formazan generated was determined at 570 and 690 nm (background subtraction) using a microplate reader (SynergyH1, Biotek, Warsaw, Poland). The results are shown as percentage (%) of control cells + SD. The experiments were repeated 3 times (n = 3) and each experiment was performed in triplicate (3 wells per treatment).

Cell cycle.

The cells were seeded at 6,000 cells/cm² in 6-wells plates, incubated for 2 days, and treated with the different digested BW products or control digesta as described before. After 48 h, cell culture medium was removed and added to a cytometer tube. The cells were rinsed with 1 mL PBS, which was mixed with the culture medium added previously to the cytometer tube. The cells were then trypsinized, added to the cytometer tube, and centrifuged at 125 × g for 7 min. The supernatant was discarded, the pellet re-suspended in 200 μL of PBS, and fixed in 2 mL ice-cold methanol (70:30, v/v) for 30 min. The pelleted cells (125 × g for 7 min) were diluted in 200 μL PBS containing 100 μg/mL of RNase and 40 μg/mL of propidium iodide (PI), and incubated 30 min at 37 °C. Cell cycle was analysed using a flow cytometer (BD LSR Fortessa Cell Analyzer; Erembodegem, Belgium) and FACS Diva Version 6.2 software (BD Bioscience, Warsaw, Poland). Cell cycle analysis of each treatment (2 plates per experiment) was repeated three times (n = 3). Results are displayed as mean ± SD.

Mitochondrial membrane potential (Δψ_m).

The analysis was performed using dihydrorhodamine (DHR, 20 mM in DMSO) and a PI solution (0.05 mg/ml PI; 1 mg/ml RNase A diluted in PBS) double labeling as described³⁵. After treatment with the digested-BW products, the cells were rinsed twice with PBS, trypsinized and incubated with 1 ml DHR solution (5 μM in PBS) for 30 min at 37 °C in darkness. Next, the DHR solution was aspirated, and the cells incubated with 500 μl PI staining solution for additional 15 min under the same conditions. DHR (λ_ex = 488 nm and λ_em = 525 nm) and PI (λexc = 488 nm and λem = 620 nm) fluorescent intensities were determined by flow cytometry (FACSAria II cell sorter). PI/DHR double negative cell ratio was used to determine Δψ_m changes. A minimum of 10,000 cells per treatment was analyzed in each replicate. The experiments were repeated 3 times (n = 3) and each experiment was performed in duplicate (2 wells per treatment).

Statistical analysis.

Results are displayed as mean ± standard deviation (SD). Prism 5 (GraphPad, La Jolla, CA, USA) was used for the analysis of significant differences. Kolmogorov Smirnov was used to determine the normal distribution of the data. ANOVA (1-way) followed by Dunnett post hoc analyses were performed to evaluate equality of variances and statistical differences between the treatments. Values p < 0.05 were indicated as statistically significant.

Results

Phenolic compounds in BW products before and after in vitro digestion.

The flavonoids composition determined in 50 mg of the different freeze-dried BW products investigated is shown in table 1. Despite flavones of C-glucosides were only detected in BWs, previous studies have described the presence of Vi (3.55 ± 0.16 μg/g) and IsoVi (1.60 ± 0.01 μg/g) in BWg ²⁸. The HPLC analysis of the in vitro-digested BW products showed no additional peaks formation compared to the chromatograms of non-digested samples. This observation may evidence that the compounds analyzed were stable to the proteolytic activity of gastrointestinal enzymes and harsh gastrointestinal conditions. Thus, unless microbial activity can degrade these compounds, our data suggest that the bioactive compounds from BW could reach the colon in their original form.

Cell viability.

Myofibroblasts treated for 48 h with TNF-α (20 ng/mL) together with _bBW, b₅₀₀BW, BWg, _fBWg or BWs exhibited cell viability values above 90% (Supplementary Figure S1). This value was similar to that observed for cells treated with equivalent volumes of the control digests (water + salts + enzymes) in the presence or absence of TNF-α. Based on these findings, for the rest of the experiments, cells were treated with the largest volume of _bBW (1.8 nM Ru), _b500BW (2 nM Ru), BWg (3 nM Ru), _fBWg (4.5 nM Ru) and BWs₁₀₀ (0.14 μM Or, 0.58 μM IsoOr; 0.28 μM Vi; 0.09 μM IsoVi; 0.71 μM Ru).

Effects on the migration of TNF-α-treated myofibroblasts.

We evaluated whether the treatment of the myofibroblasts with the digested-BW products affected their capacity to migrate in absence of TNF-α. As shown, cell migration was significantly (p<0.05) reduced by _bBW (50.3%) and BWs₁₀₀ (57.6%) (Figures 2A and B). In the presence of TNF-α (20 ng/mL) the colon fibroblasts migration was moderate, but significantly increased by 38.68% and 46.67% (p < 0.05) compared to the cells exposed to 10 and 50 μL digested control, respectively (Figures 2C and D). The TNF-α-induced migration was mainly ameliorated by _b500BW (25.5%; p<0.05) (Figure 2C). Similarly, BWs₁₀₀ reduced by 17.3% the migration in comparison with the TNF-α-treated cells (Figure 2D).

Figure 2.

Effect of the different buckwheat (BW) products investigated on colon myofibroblasts migration in absence (A,B) or presence (C,D) of the pro-inflammatory cytokine TNF-α (20 ng/mL) for 48 h of exposure. The histograms display the ability of the myofibroblasts to migrate as percent of scratched area covered. The cells corresponding to the histograms A and B were treated with 10 or 50 μL, respectively, of the digested-BW products and control digesta (Ct). The histograms C and D were also treated with 10 or 50 μL, respectively, of the digested-BW products and control digesta in the presence or absence (Ct) of TNF-α (20 ng/mL). The average ± SD from 3 – 5 different experiments are showed. * indicates statistically significant differences (p < 0.05) from Ct (A,B) and TNF-α-treated cells (C,D). Abbreviations: Ct: control digesta; _bBW: bread baked from “BIO” wheat flour and roasted BW flour (50/50%); _b500BW: bread baked from wheat flour type 500 and roasted BW flour (50/50%); BWg: roasted BW groats; _fBWg: ‘Tempeh’ type product from dehulled roasted BW; BWs₁₀₀: solution obtained from the digestion of 100 mg of BW sprouts; BWs₅₀: solution obtained from the digestion of 50 mg of BW sprouts.

Attempting to determine whether the flavonoids detected in the different BW products were responsible (at least in part) for the effects observed, the myofibroblasts of colon were co-treated with TNF-α (20 ng/mL) and Ru, Q, Vi, IsoVi, Or, and IsoOr at concentrations from 10 to 0.1 μM. Q showed a strong inhibitory effect on TNF-α-induced migration at 10 μM. Ru exerted a slightly non-significant reduction (16%) of cell migration at 10 μM, while the flavones C-glucosides had no significant effects on cell migration (Figure 3). These results indicate Ru and Q are not responsible for the effects observed, since the concentration needed to reduce TNF-α-induced migration is higher than that detected in the different BW products.

Figure 3.

Effect of isoorientin (IsOr), orientin (Or), isovitexin (IsVx), vitexin (Vx), quercetin (Q), and rutin (Ru) on TNF-α-treated myofibroblasts of colon (20 ng/mL) for 48 h. Control cells (Ct) were treated with equivalent volume of DMSO (0.5% v/v). The histograms display the ability of the myofibroblasts to migrate as a percent of scratched area covered after 48 h compared with time 0 (initial scratched area). The average ± SD from three different experiments (n = 3) are showed. Each experiment was performed in duplicate (2 wells per treatment). * (p < 0.05) and *** (p < 0.01) indicates statistically significant differences from TNF-α-treated cells.

Effects on cell cycle distribution.

We next investigated the effects of the digested-BW products on the cell cycle of TNF-α-treated cells (Figure 4). In this assay BWs₅₀ was discarded because it had no effect on TNF-α-induced migration. After the incubation of the myofibroblasts with TNF-α together with 10 or 50 μL of control digesta for 48 h there was quantified a decreased number of cells in phase G0/G1 (11.29% and 13.12%, respectively). This was accompanied by increased numbers of cells in phase S (76.3 and 122%, respectively) and G2/M (68 and 87.5%). The effects of TNF-α on cell cycle were slightly mitigated by _b500BW reducing the number of cells in phase S (15.24%, p<0.05) and G2/M.

Figure 4.

Analysis of the cell cycle in myofibroblasts CCD-18Co co-stimulated with TNF-α (20 ng/mL) and the different digested-buckwheat (BW) products for 48 h. Cells in exponential phase were treated with the different digested-BW products, at non-toxic concentrations, in the presence of TNF-α (20 ng/mL). Control cells were exposed to 10 and 50 μL of control digesta (Ct) (A and B, respectively). Values are expressed as fold-change (mean ± SD) of G0/G1, S, and G2 phases observed in control cells (set as 1.0). * (p < 0.05) and ** (p < 0.05) indicates statistically significant differences from TNF-α-treated cells. Abbreviations: Ct: control digesta; _bBW: bread baked from “BIO” wheat flour and roasted BW flour (50/50%); _b500BW: bread baked from wheat flour type 500 and roasted BW flour (50/50%); BWg: roasted BW groats; _fBWg: ‘Tempeh’ type product from dehulled roasted BW; BWs₁₀₀: solution obtained from the digestion of 100 mg of BW sprouts.

The influence of Ru and Q on myofibroblastś cell cycle was also investigated. In comparison to the TNF-α-treated cells, Ru at 10 μM reduced the number of cells in S (20.8%, p<0.05) and G2/M, while there was no effect on G0/G1 phase. Q at 1 μM had no effect on G0/G1 phase, but slightly decreased the number of cells in phase S and G2/M (Figure 5). Considering that the cells treated with the different BW products were exposed to Ru concentrations ranging from 1.80 nM to 0.71 μM, our results indicate that the effects exerted by the different BW products on cell cycle are not related to their Ru content. However, its effects might be mediated through Q (its hydrolysis product), since it modulated cell cycle to a similar extent than BW products at 1 μM (similar to the concentration of Ru detected in BW sprouts).

Figure 5.

Analysis of the cell cycle in myofibroblasts CCD-18Co co-stimulated with TNF-α (20 ng/mL) and rutin (Ru) or quercetin (Q) for 48 h. Cells in exponential phase were treated with the 10 μM Ru or 1 μM Q in the presence of TNF-α (20 ng/mL). Control cells (Ct) were treated in parallel with equivalent volume of DMSO (0.5% v/v). Values are expressed as fold-change (mean ± SD) of G0/G1, S, and G2 phases observed in control cells (set as 1.0). * (p < 0.05) indicates statistically significant differences from TNF-α-treated cells.

Effects on mitochondrial enzyme activity and mitochondrial membrane potential.

The effects on MTT transformation and Δψ_m in cells co-treated with TNF-α (20 ng/mL) and BW products for 48 h are displayed in Figures 6 and 7, respectively. TNF-α had no effect on mitochondrial activity (Figure 6A) or Δψ_m (Figure 7A) in the presence of 0.33% (v/v) digested control. A substantial reduction of 31.54% in MTT conversion (Figure 6B) along with an increase IP/DHR ratio (Figure 7B) was observed when the concentration of the digested control solution was 1.67% (v/v) in relation to control (Figure 6B). The treatment with BW-enhanced breads (_bBW and _b500BW) or BWg (fermented and non-fermented) also reduced the MTT conversion from 17 – 25% (Figure 6A), while increased the IP/DHR ratio (fold change: 1.37 – 2.18) compared to TNF-α (Figure 7A). BWs₁₀₀ caused a higher reduction in formazan formation (44.33%; p<0.05) in comparison to the other BW products (Figure 6), but no effect on Δψ_m compared to TNF-α-treated cells was observed (Figure 7B).

Figure 6.

MTT conversion in myofibroblasts of colon co-treated with TNF-α (20 ng/mL) alone or in combination with the different digested-buckwheat (BW) products for 48 h. (A) myofibroblasts co-treated with TNF-α and the BW-enhanced breads (_bBW and _b500BW) or BW groats (BWg, _fBWg) and (B) BW sprouts (BWs₁₀₀). The results are expressed as percent of the control cells (set as 100%). The experiments were obtained from three independent experiments (n = 3). Each experiment was performed in triplicates. * p < 0.05 compared to cells treated with digesta control. Abbreviations: Ct: control digesta; _bBW: bread baked from “BIO” wheat flour and roasted BW flour (50/50%); _b500BW: bread baked from wheat flour type 500 and roasted BW flour (50/50%); BWg: roasted BW groats; _fBWg: ‘Tempeh’ type product from dehulled roasted BW; BWs₁₀₀: solution obtained from the digestion of 100 mg of BW sprouts.

Figure 7.

Mitochondrial membrane potential (Δψ_m) in myofibroblasts of colon co-treated with TNF-α (20 ng/mL) alone or in combination with the different digested-buckwheat (BW) products for 48 h. (A) myofibroblasts co-treated with TNF-α and the BW-enhanced breads (_bBW and _b500BW) or BW groats (BWg, _fBWg) and (B) BW sprouts (BWs₁₀₀). The results are expressed as the average of the IP/DHR ratio obtained from three independent experiments (n = 3) and each assay analyzed in duplicate. Abbreviations: Ct: control digesta; _bBW: bread baked from “BIO” wheat flour and roasted BW flour (50/50%); _b500BW: bread baked from wheat flour type 500 and roasted BW flour (50/50%); BWg: roasted BW groats; _fBWg: ‘Tempeh’ type product from dehulled roasted BW; BWs₁₀₀: solution obtained from the digestion of 100 mg of BW sprouts; IP: propidium iodide; DHR: dihydrorhodamine.

Discussion

Currently, the treatment of IBD comprises the use of anti-inflammatory pharmacological agents, assuming side-effects and decreased quality of life of patients. An appropriate diet could bring great benefits decreasing side-effects in patients suffering of IBD in terms of reducing inflammation and medication ³⁶. BW has been proposed as a promising food to be included in the diet of patients experiencing intestinal inflammation³⁷. The anti-inflammatory effects at intestinal level of BW have been reported in animal models of intestinal inflammation by improving gut permeability, attenuating colonic mucosa inflammation, and modulating expression of tight junction proteins³⁸. Nevertheless, there is a lack of understanding in relation to i) the cellular mechanisms by which BW exerts its inflammatory activity, and ii) the molecule(s) responsible of the benefits observed.

In agreement with previous studies³⁹, our results showed that the phenolics present in BW (such as Ru and Q) are stable during in vitro digestion. These results indicate that the flavonoids, unless microbial activity modifies them, can reach the colon in their original physicochemical form and exert their beneficial effects.

Myofibroblasts migration is a core mechanism during intestinal inflammation that involves the movement of the intestinal fibroblasts to the inflamed area, contributing to tissue repair. During intestinal chronic inflammation, the fibroblasts are permanently activated synthesizing abnormal amounts of extracellular matrix, which can then lead to intestinal fibrosis, and express receptors for molecules involved in the regulation of the migration (i.e., TNF-α and its receptor)⁴⁰. The role of TNF-α on cell migration is very complex, and its effects vary depending on diverse factors such as tissue source, cell type, concentration, and exposure time ⁴¹. In this context, there have been described dissimilar effects of TNF-α regulating myofibroblasts function either reducing⁴² or promoting cell migration ³³. Under the conditions of the study, TNF-α (20 ng/mL; 48 h) increased the migration of the colon myofibroblasts compared with untreated cells. Regulation of intestinal fibroblasts migration by natural products has been proposed as a mechanism to attenuate intestinal inflammation⁴³. In this study, it has been shown the modulatory effect of different BW products on TNFα-stimulated myofibroblasts. The _b500BW product exerted a strong inhibitory effect on TNF-α-induced migration (Figure 3). According to studies where polyphenols reduced myofibroblasts migration in the absence of pro-inflammatory stimulus ⁴⁴, the BW products reduced fibroblasts migration in the absence of TNF-α. Overall, these results suggest that the modulatory effects of BW products involve additional signaling pathways other than the TNFα one. Previous research efforts have shown the potential of dietary bioactive compounds (such as polyphenols) to modulate the aryl hydrocarbon receptor (AhR) ⁴⁵. The AhR is a pleiotropic nuclear factor, expressed in intestinal fibroblasts, of critical importance in the inhibition of TNF-α-induced-collagen synthesis in the gut ⁴⁶. The hypothesized mechanism of polyphenols interaction with AhR could explain the positive effects of BW products through the reduction of TNF-α-induced fibroblasts migration. Additionally, it should not be ruled out that the effect of the different digested-BW products on TNF-α-induced fibroblasts migration might be exerted through innate immune receptors such as Toll-like receptor (TLR)-4 ⁴⁷, growth factors ⁴⁸, as well as COX-2 and PGE₂ ³³. These effects on cell migration might suppose an important immunonutritional contribution of BW-derived products against the undesirable effects (such as excessive fibrosis) resulting from the dysregulation of the intestinal homeostasis during chronic inflammation.

This study points out the importance of investigating the effects of whole products on the intestinal cells, BW and BW-enhanced products, containing mixtures of phenolic compounds with other nutrients. These complex mixtures exemplify a closer approximation to the content that can be found in the intestinal lumen after consuming BW products. Nevertheless, a study which parallels the whole products together with their individual phenolic compounds can help elucidate the molecules responsible of the biological effects exerted by the products. Like studies showing that Ru and Q reduce migration in intestinal cells ^{49, 50}, our results displayed a slight (16%) and strong (77.34%), respectively, inhibitory effect of these compounds on migration of TNF-α-stimulated cells at 10 μM (but not at 1 μM or lower). Despite 10 μM is a concentration that can be achieved in colon after consuming foodstuff containing Ru and Q ¹³, the results suggest that these compounds are not responsible for the effects observed in our in vitro study, since their concentrations are lower in the BW products investigated. Therefore, other compounds different than polyphenols such as fiber ⁵¹, and/or BW protein⁵², might be contributing to reduce the TNF-α effect.

Chronic inflammation disrupts the cell cycle of intestinal cells, thus altering intestinal homeostasis and inducing intestine impairment⁵³. Here, dietary interventions preventing cell biological alterations could have great benefits regulating intestinal homeostasis ⁵⁴. To date, numerous studies have investigated the effect of polyphenols and polyphenols-containing foods on cancer cells ^{55, 56}, while only a few studies have investigated the role of food products on cells cycle regulation in non-cancerous cell lines ⁵⁷. In line with studies describing the ability of TNF-α to alter fibroblasts’ cell cycle ⁵⁸, our results showed that the CCD-18Co myofibroblasts exposed to TNF-α (20 ng/mL) altered the physiological cellular distribution in the different cell cycle phases (Figure 4). An important mechanism to protect intestinal cells from the undesirable effect of oxidative agents is the prevention of cell cycle perturbation by natural products⁵⁹. The BW products reduced the effect of TNF-α on cell cycle progression. This effect was independent of their Ru content, since its concentration in the BW products is below 10 μM, which is the concentration required to reduce the TNF-α-induced cell cycle alteration. Q, a phenolic compound that comes from the hydrolysis of Ru, has been reported to be a potent regulator of cell cycle in numerous intestinal cell lines ⁶⁰. In this study, Q at 1 μM exhibited a modest capacity to decrease TNF-α effect on fibroblastś cell cycle by reducing about 10% the number of cells in phase S. Although this effect was rather moderate, it has to be considered that the continuous exposure of intestinal cells to Q due to the repeated consumption of BW products could help prevent/regulate cell cycle alteration during intestinal inflammation. A possible mechanism by which BW products could regulate the TNF-α-altered cell cycle checkpoint function is the modulation of cyclins ^{61, 62} as well as AhR^{63, 64}.

Besides fibroblasts migration and cell cycle, TNF-α also promotes inflammation through the generation of reactive oxygen species (ROS) in the mitochondria, reducing the mitochondrial membrane potential and causing cell death ^{65, 66}. In this study, TNF-α (20 ng/mL, 48 h) affected Δψ_m only in the presence of 1.67% (v/v) digested control. Co-treatment with the BW products increased cells sensitivity to TNF-α at the mitochondrial level. In agreement with our results, García-Nebot et al. (2011) reported a reduction in Δψ_m in Caco-2 cells exposed to digested caseinophosphopeptides⁵⁹. By contrast, a blackberry extract suppressed acrylamide-induced alteration of Δψ_m⁶⁷. These contradictory results could be explained by the generation of unidentified molecules generated during the in vitro digestion, exerting dissimilar effects on Δψ_m. We also have to consider that, despite the vast majority of studies has described BW as an antioxidant food ^{68, 69}, the pro-oxidant activity of methanolic BW extracts has also been reported ⁷⁰, what could partially explain the alteration of Δψ_m observed.

The alteration exerted on cell migration and cell cycle in the myofibroblasts by TNF-α is in concordance with the connection established between cell cycle and cell migration regulation reported in different cellular models^{71, 72}. The attenuation of the effect of the pro-inflammatory cytokine on cell cycle alteration exerted by some of the BW products investigated was accompanied by a reduction of cell migration in TNF-α-stimulated cells, indicating a link between these two processes. However, this effect was less clear in other products like _fBWg, where the effect of TNF-α was mitigated on cell migration but not on cell cycle. These results highlight the need of more studies in order to define the role of BW against chronic diseases such as IBDs, the molecule(s) involved in this response, and the mechanisms associated.

Conclusion

There is a growing number of in vitro studies investigating the biological activity of BW and/or BW-derived products. However, a limited number of investigations have looked at their anti-inflammatory activity. A PubMed search for “buckwheat and inflammation” will give less than 20 studies in the past 20 years. This indicates that the anti-inflammatory activity of BW is a field that needs to be explored. This study investigated for the first time the role of different digested BW products as well as their individual flavonoids in a TNF-α-treated myofibroblasts of colon cell model. In general, under the conditions of our study, the different BW products were able to modulate the response of the myofibroblasts in the presence of TNF-α. Our study also highlights the importance of testing in parallel bioactive rich nutritional products and their bioactive flavonoids to determine which compound(s) are responsible for the effects observed. However, the in vitro results obtained in this study cannot be extrapolated to in vivo models. Thus, it has to be considered that the concentration of polyphenols (such as Ru and Q) reached in the gastrointestinal tract after prolonged consumption of BW can be higher than that used in this study.

In conclusion, our results showed that the whole BW products reduced the effect of TNF-α on migration and cell cycle in the myofibroblasts, independently of their polyphenols content. Although the response observed in some cases was modest (expected for natural foodstuff), the effect exerted by BW products on the processes investigated might represent an alternative strategy against the detrimental effects caused during chronic intestinal inflammation. However, the study of mechanisms related to the inflammatory process (cytokines production, transcription factors, and/or pathways involved), as well as more human trials, and animal studies need to be performed to determine the benefits of BW against intestinal chronic diseases.