Gastrointestinal digestion of food proteins and mechanisms of action of peptides on digestive health

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de lectura: 02-12-2016Esta tesis tiene embargado el acceso al texto completo hasta el 02-06-201

Inhibitory Effects of Peptide Lunasin in Colorectal Cancer HCT-116 Cells and Their Tumorsphere-Derived Subpopulation

The involvement of cancer stem-like cells (CSC) in the tumor pathogenesis has profound implications for cancer therapy and chemoprevention. Lunasin is a bioactive peptide from soybean and other vegetal sources with proven protective activities against cancer and other chronic diseases. The present study focused on the cytotoxic effect of peptide lunasin in colorectal cancer HCT-116 cells, both the bulk tumor and the CSC subpopulations. Lunasin inhibited the proliferation and the tumorsphere-forming capacity of HCT-116 cells. Flow cytometry results demonstrated that the inhibitory effects were related to apoptosis induction and cell cycle-arrest at G1 phase. Moreover, lunasin caused an increase in the sub-GO/G1 phase of bulk tumor cells, linked to the apoptotic events found. Immunoblotting analysis further showed that lunasin induced apoptosis through activation of caspase-3 and cleavage of PARP, and could modulate cell cycle progress through the cyclin-dependent kinase inhibitor p21. Together, these results provide new evidence on the chemopreventive activity of peptide lunasin on colorectal cancer by modulating both the parental and the tumorsphere-derived subsets of HCT-116 cells

Immunomodulatory effect of gut microbiota-derived bioactive peptides on human immune system from healthy controls and patients with inflammatory bowel disease

Bioactive peptides secreted by probiotic Bifidobacterium longum (peptide B7) and opportunistic pathogen Bacteroides fragilis (peptide B12) modulate the intestinal cytokine milieu in health. Here, we characterized their capacity to modulate both the mucosal cytokine production and the phenotype of circulating antigen presenting cells (APCs) in active inflammatory bowel disease (IBD). The IBD mucosa produced higher levels of pro-inflammatory cytokines referred to healthy controls (HCs). Peptides B7 and B12, however, did not ameliorate the mucosal cytokine milieu in IBD. Human circulating APCs (B-cells, monocytes, plasmacytoid dendritic cells (pDCs), and conventional dendritic cells (cDCs)) were characterized by flow cytometry in presence/absence of the peptides. Circulating B-cells, monocytes, and cDCs from IBD patients were more activated than those from HCs. Peptide B7, but not B12, decreased CCR2 expression on all APC subsets from HC, but not IBD patients. Moreover, both peptides tend to further increase their pro-inflammatory profile in IBD. In summary, IBD patients display mucosal and circulating APC pro-inflammatory properties. Peptide B7 immunomodulatory capacity elicited over circulating APCs from HC, but not IBD patients, suggests the presence of disrupted modulatory mechanisms for this peptide in IBD. Future studies should address the effect of bacteria-derived immunomodulatory peptides in non-inflamed (quiescent) IBD patientsThis work was supported by the Spanish Ministry of Economy (SAF2014-56642-JIN), the Spanish Ministry of Health (PIE13/00041), GETECCU (Grupo Español de Trabajo en Enfermedad Crohn y Colitis Ulcerosa), and the Community of Madrid (Consejería de Educación, Juventud y Deporte, Programa de Garantía Juvenil 2015 and 2016)

Milk Proteins, Peptides, and Oligosaccharides: Effects against the 21st Century Disorders

Milk is the most complete food for mammals, as it supplies all the energy and nutrients needed for the proper growth and development of the neonate. Milk is a source of many bioactive components, which not only help meeting the nutritional requirements of the consumers, but also play a relevant role in preventing various disorders. Milk-derived proteins and peptides have the potential to act as coadjuvants in conventional therapies, addressing cardiovascular diseases, metabolic disorders, intestinal health, and chemopreventive properties. In addition to being a source of proteins and peptides, milk contains complex oligosaccharides that possess important functions related to the newborn's development and health. Some of the health benefits attributed to milk oligosaccharides include prebiotic probifidogenic effects, antiadherence of pathogenic bacteria, and immunomodulation. This review focuses on recent findings demonstrating the biological activities of milk peptides, proteins, and oligosaccharides towards the prevention of diseases of the 21st century. Processing challenges hindering large-scale production and commercialization of those bioactive compounds have been also addressed

Differential effects of Anti-TNFα and Anti-α4β7 drugs on circulating dendritic cells migratory capacity in inflammatory bowel disease

Inflammatory bowel disease (IBD) is an idiopathic and chronic disorder that includes ulcerative colitis (UC) and Crohn’s disease (CD). Both diseases show an uncontrolled intestinal immune response that generates tissue inflammation. Dendritic cells (DCs) are antigen-presenting cells that play a key role in tolerance maintenance in the gastrointestinal mucosa. Although it has been reported that DC recruitment by the intestinal mucosa is more prominent in IBD patients, the specific mechanisms governing this migration are currently unknown. In this study, the expression of several homing markers and the migratory profile of circulating DC subsets towards intestinal chemo-attractants were evaluated and the effect of biological drugs with different mechanisms of action, such as anti-TNFα or anti-integrin α4β7 (vedolizumab), on this mechanism in healthy controls (HCs) and IBD patients was also assessed. Our results revealed that type 2 conventional DCs (cDC2) express differential homing marker profiles in UC and CD patients compared to HCs. Indeed, integrin β7 was differentially modulated by vedolizumab in CD and UC. Additionally, although CCL2 displayed a chemo-attractant effect over cDC2, while biological therapies did not modulate the expression of the homing markers, we paradoxically found that anti-TNF-treated cDC2 increased their migratory capacity towards CCL2 in HCs and IBD. Our results therefore suggest a key role for cDC2 migration towards the intestinal mucosa in IBD, something that could be explored in order to develop novel diagnostic biomarkers or to unravel new immunomodulatory targets in IBD.This study has been funded through the Instituto de Salud Carlos III (Sara Borrell fellowships, CD17/00014; CD21/00014), Asociación Española de Gastroenterología (Beca del Grupo Joven), Programa Estratégico Instituto de Biología y Genética Molecular (IBGM Junta de Castilla y León. Ref. CCVC8485), Plan Nacional (PID2019-104218RB-I00) from the Spanish Government, Janssen and MSD

Lunasin Peptide is a Modulator of the Immune Response in the Human Gastrointestinal Tract

[Introduction]: Lunasin is a soybean bioactive peptide with a variety of beneficial properties against chronic disorders. However, its effect in human primary intestinal cells remains unknown. Hence, this study aims to characterize its ex vivo biological activity in the human intestinal mucosa. [Methods and Results]: Human intestinal biopsies, obtained from healthy controls, are ex vivo conditioned with lunasin both in the presence/absence of lipopolysaccharide (LPS). Peptide maintains its stability during biopsy culture by HPLC-MS/MS analysis. Lunasin is bioactive in the human mucosa, as it induces IL-1β, TNF-α, IL-17A, CCL2, and PGE2/COX-2 gene expression together with an increased expression of tolerogenic IL-10 and TGFβ, while it also downregulates the expression of iNOS and subunit p65 from NF-κB. Indeed, lunasin also abrogates the LPS-induced pro-inflammatory response, downregulating IL-17A, IFNγ, and IL-8 expression, and inducing IL-10 and TGFβ expression. These results are also mirrored in the cell-free culture supernatants at the protein level by Multiplex. Moreover, lunasin further induces a regulatory phenotype and function on human intestinal conventional dendritic cell and macrophage subsets as assessed by flow cytometry. [Conclusions]: We hereby have characterized lunasin as an immunomodulatory peptide with potential capacity to prevent immune and inflammatory-mediated disorders in the human gastrointestinal tract.This work was supported by the Spanish Ministry of Economy (SAF2014-56642-JIN), the Spanish Ministry of Health (PIE13/00041), the Spanish Ministry of Science, Innovation and Universities (AGL2015-66886-R, PID2019-104218RB-I00), and the Community of Madrid (Consejería de Educación, Juventud y Deporte, Programa de Garantía Juvenil 2015 and 2016). S.F.T. is currently funded by the Instituto de Salud Carlos III (Sara Borrell fellowship CD17/00014). L.O.M. is funded by the Community of Madrid (BMD-5800). D.B. is funded by the Spanish Ministry of Science (RYC-2017-21606)

Long non-coding RNA signatures in the Ileum and Colon of Crohn’s disease patients and effect of Anti-TNF-α treatment on their modulation

Biological therapies only benefit one-third of patients with Crohn’s disease (CD). For this reason, a deeper understanding of the mechanisms by which biologics elicit their effect on intestinal mucosa is needed. Increasing evidence points toward the involvement of long noncoding RNAs (lncRNAs) in the pathogenesis of CD, although their role remains poorly studied. We aimed to characterize lncRNA profiles in the ileum and colon from CD patients and evaluate the effect of anti-TNF-α treatment on their transcription. Terminal ileum and left colon samples from 30 patients (active CD = 10, quiescent CD = 10, and healthy controls (HCs) = 10) were collected for RNA-seq. The patients were classified according to endoscopic activity. Furthermore, biopsies were cultured with infliximab, and their transcriptome was determined by Illumina gene expression array. A total of 678 differentially expressed lncRNAs between the terminal ileum and left colon were identified in HCs, 438 in patients with quiescent CD, and 468 in patients with active CD. Additionally, we identified three new lncRNAs in the ileum associated with CD activity. No differences were observed when comparing the effect of infliximab according to intestinal location, presence of disease (CD vs. HC), and activity (active vs. quiescent). The expression profiles of lncRNAs are associated with the location of intestinal tissue, being very different in the ileum and colon. The presence of CD and disease activity are associated with the differential expression of lncRNAs. No modulatory effect of infliximab has been observed in the lncRNA transcriptom

Effect of soybean peptide lunasin on the inflammatory and immune response of healthy human intestinal mucosa

Trabajo presentado al 2nd International Symposium on Bioactive Peptides, celebrado en Valencia (España) del 22 al 24 de mayo de 2019.Peer reviewe

Digestión gastrointestinal de proteínas alimentarias y mecanismos de acción de péptidos con efecto sobre la salud digestiva

In this Thesis several aspects related to the physiological effect of bioactive peptides on the digestive tract have been studied by a double approach. It has been investigated not only the modifications that food proteins and peptides undergo during the gastrointestinal digestion, but also the mechanisms of action involved in the biological function that bioactive peptides might exert due to their contact with digestive cells and receptors. Peptide lunasin, casein and whey proteins, and milk peptides were analyzed. Initially, the behavior of peptide lunasin under digestive conditions simulating the transit through the gastrointestinal tract in the absence or presence of soybean Bowman-Birk isoinhibitor 1 (IBB1), in both active and inactive states, was evaluated. IBB1 isoinhibitor exerted a protective effect on lunasin degradation during digestion. Protection against the action of pepsin was due to the presence of IBB1 and its higher size in comparison with that of peptide lunasin, independently of activity. However, an IBB1 dose-dependent protective effect was found at intestinal level, related to its inhibitory activity of pancreatic enzymes trypsin and chymotrypsin. The peptide profiles of gastric and gastrointestinal digests were characterized. It was demonstrated the notable resistance during the digestive process of some domains of peptide lunasin, especially regions 1SKWQHQQDSC10, 11RKQLQGVN18, 19LTPCEKHIME28 and 29KIQGRGDDDDDDDDD43. The transepithelial transport of these four fragments and the precursor lunasin was evaluated using Caco-2 cell monolayers. While some regions of these peptides were susceptible to epithelial brush-border peptidases, a marked resistance was found for others, particularly for fragments 1SKWQHQQDSC10 and 29KIQGRGDDDDDDDDD43. The transepithelial transport of lunasin and fragment 11RKQLQGVN18 was mediated through diffusion via the paracellular pathway. Additionally, lunasin, as well as its gastrointestinal digests in presence and absence of IBB1, and some lunasin-derived fragments identified in these digests were evaluated for their effect against the viability of gastric cancer AGS and colorectal cancer HT-29 and Caco-2 cells. It is highlighted that the fragment 1SKWQHQQDSC10 was the main responsible for the demonstrated inhibitory effect of lunasin on cellular viability, particularly in HT-29 cells. The chemopreventive mechanisms of action of lunasin against the proliferation of bulk colorectal cancer HCT-116 cells and the expansion of their tumorsphere-derived cancer stem-like subpopulation were evaluated. Peptide lunasin inhibited the viability of bulk tumor cells, as well as the tumorsphere-forming capacity of HCT-116 cells. The inhibitory activity was mediated by inducing apoptosis and arresting cell cycle at G1 phase. These effects were associated to a stimulatory effect on molecular marker caspase-3 linked to a cleavage on PARP signal, and a modest activation of p21 protein expression. Upon chemical-induced oxidative stress, lunasin-treated liver HepG2 cells showed an increased cellular viability. This protective effect was mediated through raising intracellular glutathione levels, and decreasing oxygen reactive species production and glutathione peroxidase and catalase activities. In addition, lunasin protected proteins from oxidative damage and inhibited caspase-3 mediated apoptosis.In the context of gastrointestinal mucus strengthening by food protein compounds, novel peptides derived from αs1-casein 144YFYPE148 and 144YFYPEL149 and, to a lower extent, 144YFY146 and 143AYFYPEL149 were found, for the first time, to exert an opioid agonistactivity. By molecular dynamics simulations of peptides binding with the μ-opioid receptor, it was proved that the carboxi-terminal proline residue from peptide 144YFYP147 affected to its interaction with opioid receptor and activity. On intestinal goblet cells, it was demonstrated that peptide 144YFYPEL149 was the minimum fragment able to stimulate expression of MUC5AC, the main secreted mucin gene in HT29-MTX cells. Later on, the protective effect of a peptic casein hydrolyzate containing these opioid peptides was studied in the rat intestinal mucus layer. This hydrolyzate stimulated the gene expression of mucins Muc2 and Muc3 in ileum and colon, and the fecal mucin secretion after its oral administration during two and eight weeks, enhancing in vivo the intestinal mucus barrier. Finally, protein degradation and peptide formation in digests obtained after in vivo and in vitro gastrointestinal digestion of casein and whey protein were analyzed.With this purpose, the peptide profile of jejunal digests of five human volunteers was characterized and compared with peptides generated during in vitro gastrointestinal digestion of milk proteins following a standardized and internationally harmonized static digestion protocol. Whereas protein degradation through the gastrointestinal tract was observed, some protein domains showed resistance against the action of digestive enzymes. In vivo and in vitro protein degradation and digests peptidome were similar. Spearman correlation coefficients between in vivo and in vitro digests were within the range of that obtained between the different human volunteers of the study. Therefore, this in vitro method represented a suitable model to physiologically simulate the gastrointestinal digestion, at least in the case of milk proteins. Altogether, these results demonstrated the key role that digestive tract can play on the bioactivity of ingested compounds. Moreover, the results have allowed increasing the knowledge on the mechanisms of action involved in the beneficial effects of peptide lunasin, milk proteins, and derived peptides on digestive health.Peer Reviewe

Modulatory Effects of a Lunasin-Enriched Soybean Extract on Immune Response and Oxidative Stress-Associated Biomarkers

The immune system, inflammatory, and oxidative processes are involved in the pathogenesis of a variety of chronic diseases. Soybean lunasin has emerged as one of the most promising food-derived peptides with a positive impact on health. A soybean extract enriched in lunasin (LES) was obtained and characterized, evaluating its behavior under simulated gastrointestinal conditions. Besides its in vitro radical scavenging capacity, the effects of LES on cell viability, phagocytic capacity, and levels of oxidative stress and inflammation-associated biomarkers were investigated in macrophages RAW 264.7. Different LES doses and treatment times were assayed. After extraction with an aqueous solvent, an enrichment with soluble proteins and small peptides, such as lunasin, was achieved. Lunasin was detected at the end of the simulated digestion, indicating its partial resistance to this process. LES showed radical scavenging activity. Moreover, at low doses, LES reduced reactive oxygen species (ROS) in macrophages, while an oxidizing effect was observed at the highest concentration. LES exerted an immunomodulatory action, increasing the production of nitric oxide (NO) in a dose- and time-dependent manner, and the phagocytic activity at short treatment times. LES also had a modulatory action on the secretion of pro-inflammatory (interleukin IL-6) and anti-inflammatory (IL-10) cytokines