Natural polysaccharides have emerged as an important class of bioactive compounds due their beneficial biological effects. Here we investigated the protective and healing effects of rhamnogalacturonan (RGal) isolated from Acmella oleracea (L.) R.K. Jansen leaves in an experimental model of intestinal inflammation in mice and in heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2). The findings demonstrated that RGal treatment for 7 days reduced the severity of DSS-induced colitis by protecting mice from weight loss, macroscopic damage and reduction of colon length. When compared to the DSS group, RGal also protected the colon epithelium and promoted the maintenance of mucosal enterocytes and mucus secreting goblet cells, in addition to conserving collagen homeostasis and increasing cell proliferation. In an in vitro barrier function assay, RGal reduced the cellular permeability after exposure to IL-1β, while decreasing IL-8 secretion and claudin-1 expression and preserving the distribution of occludin. Furthermore, we also observed that RGal accelerated the wound healing in Caco-2 epithelial cell line. In conclusion, RGal ameliorates intestinal barrier function in vivo and in vitro and may represent an attractive and promising molecule for the therapeutic management of ulcerative colitis.
Ulcerative colitis (UC) is a chronic relapsing and idiopathic disease characterized by a diffuse inflammation that affects the colonic mucosa with bloody diarrhea as a predominant symptom[
Taking into account, recent studies have shown that medicinal plant-derived extracts, herbs and dietary components such as flavonoids have anti-colitis activity, through controlling the levels of inflammatory mediators associated with the severity of active UC[
For instance, our research group demonstrated the gastroprotective properties of rhamnogalacturonan (RGal), a chemically-defined polysaccharide easily obtained in high yield from leaves of Acmella oleracea (L.) R.K. Jansen[
Considering this, the purpose of our study was to investigate the protective and healing effects of RGal in an acute experimental model of intestinal inflammation chemically induced by dextran sulfate sodium (DSS) in mice. Furthermore, to substantiate possible effects on barrier function, we employed in vitro assays using heterogeneous human epithelial colorectal adenocarcinoma (Caco-2) cells.
It is well known that the DSS is directly toxic to colonic epithelium leading to severe illness, characterized by shortening of the colon, bloody diarrhea and sustained weight loss, the main signal manifestations of UC[
When compared to healthy mice (naïve control group), animals that received DSS in drinking water and were treated with vehicle started to lose weight at day 5 (6.54%) and this extended up to the day 8 (27.30%), (Fig. 1A). The same holds for the DAI (Disease Activity Index), where the DSS group presented visible blood in the stool, as well as a colon length reduction (61%) when compared to the naïve control group (Fig. 1C,D). Moreover, the animals also presented occult blood in the feces (Supplementary Table S1).RGal protects mice against DSS-induced colitis. Effect of RGal treatment on (A) body weight change, (B) disease activity index, and (C) colon length. Mice were orally treated with vehicle (Control or DSS groups: water, 0.1 mL/kg) or RGal (
The anti-colitis effect promoted by RGal does not assume a dose-response relationship, both 3, 10 and 30 mg/kg achieved similar effects. Taking into account, we decided to work with a safer dose (10 mg/kg) in the subsequent experiments, considering the aggressiveness of the ulcerative colitis model induced by DSS. In the mice group submitted to DSS-induced colitis, RGal treatment (10 mg/kg) significantly reduced the body weight loss (day 8, 51%) and DAI from day 5 (28%) to day 8 (46%) when compared to the DSS group (Fig. 1A,B). RGal also prevented the reduction of colon length (RGal: 9.1 ± 0.2 cm) when compared to DSS group (DSS: 7.1 ± 0.2 cm) (Fig. 1C,D) and diminished the presence of occult blood in the feces when compared to DSS group (Supplementary Table S1).
Previous studies have shown that the colonic damage induced by DSS directly relate to cellular infiltration into the intestinal mucosa[
We also observed the maintenance of the total number of mucus secreting goblet cells with PAS and AB staining methods (Fig. 3A-I and M).RGal preserves the colonic goblet cells and the expression of MUC-1. Histochemical staining of colons for (A-C) neutral mucin-like glycoproteins (PAS, white arrows), (D-F) and (G-I) acid mucin [Alcian blue: pH 2.5 (black arrow) and pH 1.0 (black arrowhead)], ×400, bars = 50 μm. (J-L) Immunohistochemical staining for MUC-1 in colons, ×400, bars = 20 μm. (M,N) Quantification of mucus secreting goblet cells and MUC-1 expression, respectively. Mice were orally treated with vehicle (Control or DSS groups: water, 0.1 mL/kg) or RGal (10 mg/kg) for 7 days, once a day. Results are expressed as mean ± S.E.M or mean ± S.D. (n = 4-8) and analyzed using ANOVA followed by Bonferroni’s test.
To confirm previous data that showed an increase in the amount mucus goblet cells into the colonic tissue of RGal-treated animals, we also performed immunohistochemical analysis to determine the expression profile of MUC-1, a cell surface mucin involved in cellular signaling, adhesion, growth and immunological modulation[
Mice treated with DSS develop colitis that is characterized by transmural inflammation and fibrosis, which represents a serious complication of inflammatory bowel diseases (IBD). For this reason, we quantified the collagen deposition in the colonic tissue. The study of colonic sections stained with Sirius red demonstrated an extensive type III collagen deposits (70.28%) and reduction in type I collagen (55.88%) in DSS mice (Fig. 4D,E and H). On the other hand, the treatment with RGal normalized the proportions of collagens when compared to the DSS group (RGal collagen III: 1062.00 ± 87.52 μm, collagen I: 1036.00 ± 84.68 μm) (Fig. 4F,H).
Once we observed an improvement of the colonic tissue in the in vivo model of DSS-induced colitis we also determined whether the RGal is capable of accelerating Caco-2 cells healing. RGal (1000 µg/mL) greatly accelerated wound closure of scratched Caco-2 cell monolayers 24 and 48 h after wounding (Fig. 5A,B). The polysaccharide promoted lesion closure by 84 and 45% at 24 and 48 h, respectively, when compared to the control group (DMEM 24 h: 25.45 ± 2.82% and DMEM 48 h: 55.61 ± 4.00%).RGal accelerates the wound closure in Caco-2 cells. (A) Percentage of wound healing at 24 and 48 h after scratch (n = 3, in triplicate). (B) Representative images of scratched areas at 0, 24 and 48 h, ×10, bars = 1000 μm. Confluent cell monolayers were wounded with a pipette tip and incubated with medium alone (DMEM, control) or RGal (1000 μg/mL) for 48 h. Wound healing was photographed at 0, 24 and 48 h after scratch. Results are expressed as mean ± S.E.M. (n = 3) and analyzed using ANOVA followed by Bonferroni’s test. *P < 0.05 and **P < 0.01 compared to Control group.
The treatment of Caco-2 cell monolayer with IL-1β leads to disruption of intestinal barrier function in vitro and causes loss of barrier integrity[
After being exposed to IL-1β, the Caco-2 cells express and release many inflammatory mediators in the cell medium, such as IL-8. Under our experimental conditions, stimulation of Caco-2 cells with IL-1β (25 ng/mL) for 72 h increased the secretion of IL-8 in 949% when compared to the vehicle group (101.1 ± 0.8 pg/mL). On the other hand, RGal (1000 µg/mL) reduced in 24% the levels of IL-8, when compared to the IL-1β group (Fig. 6C).
Increased secretion of proinflammatory cytokines might cause paracellular permeability, due primarily to the disruption of tight junction proteins such as claudins and occludin[
Following 72 h exposure to IL-1β, fluorescent staining showed that intercellular junctions of cells markedly lost the well-defined bright green occludin outline, which suggested the disruption of the tight junction protein occludin. On the other hand, pre-stimulation of cells with RGal prevented this loss, showing uniform and continuous outlines (Fig. 6E).
Importantly, our results revealed that neither RGal nor IL-1β were cytotoxic for Caco-2 cells in all experimental sets (Supplementary Fig. S2).
The bioaccessible fraction is the amount of an ingested compound that is available for absorption in the body after digestion. In this context, we investigated the effect of simulated human digestive fluids on the chemical structure of RGal.
The development of new effective agents with minimal or without adverse effects is a constant search in the treatment of UC, that is a multifactorial disorder. Here, we describe a new role for RGal in decreasing the intestinal inflammatory process chemically induced by DSS in mice. We also demonstrated the reduction of epithelial permeability and cytokine secretion as well as the maintenance of the tight junction integrity in Caco-2 cells. The schematic summary of our results is shown in Fig. 8.Schematic summary of the results.
The DSS model is characterized by a general inflammatory process directly associated with body weight loss, bloody diarrhea and histopathologic changes that mimic some clinical aspects of UC in humans[
One of the most typical events underlying the pathogenesis of UC is the influx of inflammatory cells to the intestinal mucosa. The anti-inflammatory effects presented by RGal could be evidenced by the significantly reduction of intraepithelial lymphocytes infiltration. Additionally, we had previously demonstrated that RGal reduced the MPO and TNF-α levels in an experimental model of chronic ulcer induced by acetic acid[
Another important phenomenon involved in the pathogenesis of IBD due to the inflammatory process in the mucosa is the fibrosis. Complications of fibrosis can lead to the loss of tissue function and may contribute to the formation of intestinal stenosis[
In addition to the aforementioned characteristics, apoptosis has also been reported in IBD. It has already been proposed that the imbalance between apoptosis and proliferation of new cells causes relevant damage to the epithelial barrier. This hypothesis is supported by the finding that both increased apoptosis and decreased proliferation of new epithelial cells occurs in the acute phase of DSS-induced colitis[
To reinforce our preclinical data and confirm the protective effects of RGal, we initiated complementary in vitro experiments using Caco-2 cells. The intestinal epithelium is constantly exposed to an enormous diversity of stimuli, and the lesion of the intestinal epithelial cells is constant and almost unavoidable. In patients with UC, intestinal lesion is typical and during this process, the mucosal cells migrate to cover the injured area, regardless of the proliferation, trying to maintain the integrity of the intestinal barrier[
The repeated intestinal epithelial injury that occurs in the mucosa of UC patients leads to development increased paracellular permeability of intestinal epithelial cells and, consequently, epithelial barrier dysfunction. Accordingly, in vitro experiments conducted with human Caco-2 cells stimulated with IL-1β provides a good experimental model to evaluate it[
Accordingly, our measurements of FITC-dextran permeation clearly show that RGal markedly reduced the Caco-2 permeability after IL-1β stimulation, maintaining the cellular monolayer integrity. Also, we observed that RGal preserved the expression of claudin-1 and occludin, suggesting that the polysaccharide can prevent the abnormal altered permeability and consequently infections, and the establishment of inflammation in the gut. Additionally, the levels of pro-inflammatory chemokines, such as IL-8, have been found in the intestinal tissue of UC patients and, together with other cytokines, are responsible in initiating, mediating and perpetuating intestinal inflammation[
Although the dataset presented demonstrates the important biological effect of RGal related to the colonic protection and regeneration, the underlying mechanisms remain unclear. Interestingly, despite the pH changes of digestive fluids have caused de-esterification of RGal, the original main structure remains the same, showing that probably the polysaccharide is not digestible and may be available for fermentation in the large intestine. We can assume that RGal beneficial properties are product of indirect and direct ways of action. Indirectly, RGal acting as a substrate for bacterial fermentation in the large intestine and leading to the production of short-chain fatty acids (SCFAs) including acetate, propionate and butyrate. These SCFAs have been demonstrated in regulating intestinal immunity through the inhibition of histone deacetylases and the activation of G-protein coupled receptors such as GPR41, GPR43 and GPR109A[
On the other hand, RGal was also able to promote healing, reduced intestinal permeability, decreased secretion of IL-8, as well as maintaining the integrity of epithelial junction proteins in experiments in vitro in a simple culture system without the presence of bacteria. Despite these direct effects on epithelial cells are less explored, it is evident that RGal may be eliciting its effect through activation of a receptor, such as Toll-like receptors (TLRs). Indeed, some studies showed that the TLR signaling is involved in non-prebiotic effects of oligosaccharides[
Similar to our previous publication on the structure of rhamnogalacturonan (RGal), the polysaccharide was isolated from leaves of A. oleracea (L.) R.K. Jansen[
Experiments were performed using adult female Swiss mice (20-30 g), maintained in a controlled-temperature and luminosity environment (22 ± 2 °C, 12 h light/dark cycle). Mice were housed 10 per cage with wood shaving bedding with free access to pelleted food (Nuvilab CR-1, Quimtia S/A, Brazil) and tap water, and they were acclimated to the laboratory environmental conditions one week before the beginning of the experiments. After the experiments, the animals were euthanized by overdose of lidocaine [(4 mg/kg, intraperitoneal (i.p.)] and thiopental (100 mg/kg, i.p.). All protocols were performed upon approval by the Committee of Animal Experimentation of the Universidade Federal do Paraná (CEUA/BIO-UFPR, approval number 863) and were strictly performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (National Research Council, 2011)[
The experimental protocol used for the induction of ulcerative colitis was conducted as previously described[
Animals were divided into the following treatments groups: (i) naïve control group, that received only drinking water, (ii) DSS group treated with vehicle [C: water, 1 mL/kg, per oral (p.o.)] and (iii) DSS group treated with RGal (10 mg/kg, p.o.). Individual mice were monitored daily to determine the Disease Activity Index (DAI), according to changes of weight loss, stool consistency and occult blood[
The scores were described as follows: weight loss was graded as 0 if body weight increased or remained within 1% of the baseline; 1 for a 1-5% loss; 2 for a 5-10% loss; 3 for a 10-15% loss; or 4 for weight loss >15%. The stool consistency was graded as 0 for no diarrhea; 2 for loose stool that did not stick to the anus; and 4 for liquid stool that did stick to the anus. The presence of fecal blood received a value of 0 when assigned for none, 2 for moderate, and 4 for gross bleeding. At the end of experiment, the colons were collected, washed with saline (0.9%), the lengths were measured and then the tissues were immediately stored at −80 °C for further analysis.
For analysis of microscopic damage, the distal portion of each colon was excised and immediately fixed in Alfac solution (85% alcohol 80 °GL, 10% of formaldehyde at 40% and 5% glacial acetic acid) for 16 h and then transferred to 70% ethanol, embedded in paraffin wax and then sectioned at 7 µm thickness before being deparaffinized. Slides were stained using hematoxylin and eosin stain (H&E) to analyze the histopathological changes and to quantify the intraepithelial lymphocytes[
Immunohistochemistry for mucin 1 (MUC-1) and proliferating cell nuclear antigen (PCNA) was performed on 7 µm thickness paraffin-embedded sections from the colons of mice. Sections were blocked with 1% BSA for 30 min at room temperature and incubated overnight at 4 °C with primary antibodies anti-MUC-1 (rabbit polyclonal IgG, 1:100, Santa Cruz Biotechnology) and anti-PCNA (rabbit polyclonal IgG, 1:100, Santa Cruz Biotechnology). Subsequently, the sections were washed with 1% BSA/PBS and incubated with peroxidase conjugated secondary antibody (goat anti-rabbit, 1:100 Santa Cruz Biotechnology) at room temperature in a humid chamber for 1 h. Peroxidase binding sites were detected by staining using chromogen diaminobenzidine (DAB Substrate Kit, BD Pharmingen™). In all steps the sections were washed three times with PBS. At the last step, the slides were dehydrated and counterstained with Mayer’s hematoxylin. After, the sections were observed and photographed with a slide scanner from MetaSystems (MetaViewer
Human epithelial colorectal adenocarcinoma (Caco-2) cells line, purchase from ATCC (Manassas, VA), were grown to 100% confluence in 75 cm
To evaluate the intestinal mucosal healing, we used a classic in vitro wound healing assay, which consists in creating a “scratch” in a cell monolayer. Caco-2 cells were grown to confluence (3 days) on 6-well culture plates at a density of 2 × 10
To conduct the epithelial barrier integrity experiments, cells were seeded into polycarbonate filter membranes (0.4 μm pore size, 0.6 cm
Samples were collected from the basal compartment 72 h after IL-1β incubation and were centrifuged at 16000 × g for 10 min. The levels of IL-8 were evaluated using enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s recommendations (R&D Systems, Minneapolis, MN, USA). Absorbance was measured using a microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA) at 490 nm. Chemokine levels were extrapolated from the IL-8 standard curve (0-2000 pg/mL) and the results were expressed as pg/mL.
Cells were collected and homogenized in RIPA buffer containing Tris (1 M), NaCl (5 M), NP-40, EDTA (0.5 M), DTT (0.1 M), PMSF (0.1 M), and protease inhibitor cocktail (Roche Complete and Roche Phosstop). The homogenate was centrifuged at 3000 × g for 10 min at 4 °C, and the supernatants were stored at −70 °C for further analysis. DTT and bromophenol blue concentrations were adjusted to 100 mM and 0.1% w/v, before running on 12.5% denaturing polyacrylamide gels prior transfer. Membranes were probed sequentially with rabbit anti-human claudin-1 (1:1000, Invitrogen). Additionally, blots were probed with mouse anti-human β-actin (1:1000, Cell Signaling) as a loading control for epithelial proteins and this was used to enable normalization of all protein reactivities to ensure validity of quantitative blotting data. Membranes were incubated with their respective peroxidase conjugated anti-IgG secondary antibody (1:1000, Invitrogen) and then subsequently incubated with the enhanced chemiluminescence system (ECL Plus kit, Amersham GE Healthcare Life Sciences). Blots were carried out under identical conditions for each experiment/antibody, scanned (Bio-Rad
The cells were cultured in slides with 8-well chambers for 21 days and after the treatments the immunofluorescence technique was performed to verify the expression of occludin. The slides were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15-20 min at 4 °C. The cells were then washed again with PBS and incubated with blocking buffer containing 10% fetal bovine serum and 0.05% Tween for 1 h. After, 200 μl of the anti-occludin rabbit polyclonal antibody (1:1000, Invitrogen) was added in each well and incubated for 2 h at room temperature. The wells were washed with PBS and incubated for 1 h in the dark with 200 μl of the goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor
Cell viability was determined by MTS assay using CellTiter 96
The static in vitro digestion model to simulate the human gastrointestinal tract was performed according to a previously reported study with some modifications[
Deuterium oxide (D
All data were expressed as means ± S.E.M. Statistical analysis was performed using Kruskal-Wallis followed by Dunn’s test for non-parametric data, one-way or two-way ANOVA followed by Bonferroni´s test for parametric data. All analysis was conducted using GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA). Differences with p < 0.05 were considered statistically significant.
This work was supported by grants from the CNPq (National Council for Scientific and Technological Development, process number 425721/2016-7). D.M.F. and C.H.B. were recipients of CAPES (Coordination for the improvement of Higher Education Personnel) doctoral scholarship and post-doctoral fellowship, respectively. D.M.F and M.F.P.W. thank Capes-NUFFIC for providing scholarship support at Tytgat Institute for Liver and Intestinal Research (Amsterdam, The Netherlands).
D.M.F., M.F.P.W. and C.H.B. contributed to the concept and design of the study. D.M.F., A.M.N., A.P.S.F., P.S.W. and K.C.P.B. performed the experiments. T.R.C., D.M.G.S., F.B.L. and R.M.W. contributed to the analysis and interpretation of data. D.M.F., M.F.P.W. and C.H.B. wrote the manuscript. All authors contributed to the analysis and interpretation of data and critically reviewed and approved the final draft.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
The authors declare no competing interests.
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Supplementary information accompanies this paper at 10.1038/s41598-018-30526-2.
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PHOTO (COLOR): Dataset 1
By Daniele Maria-Ferreira; Adamara Machado Nascimento; Thales Ricardo Cipriani; Arquimedes Paixão Santana-Filho; Paulo da Silva Watanabe; Debora de Mello Gonçales Sant´Ana; Fernando Bittencourt Luciano; Karla Carolina Paiva Bocate; René M. van den Wijngaard; Maria Fernanda de Paula Werner and Cristiane Hatsuko Baggio