RESUMO
BACKGROUND: The Mediterranean diet (MD), known to prevent obesity, overweight and the related non communicable diseases (NCD), is based on typical dishes, foods and on a common cultural milieu. Although MD is the basis of dietary guidelines, the prevalence of obesity, overweight and NCD, is increasing both in Western regions, and even more in Middle Eastern regions (MER). This study aimed to analyze (i) the impact of different levels of adherence to the MD, in Italy and MER, on body mass index (BMI) (ii) the bromatological composition of a simulated 7-days food plan (7-DFP) based on Italian or MER typical meals, following MD criteria and the Italian or MER food base dietary guideline; (iii) the optimization of nutrients impacting on NCD. METHODS: The 7-DFPs were implemented using a dietary software. The association between adherence to MD and BMI was evaluated by pooled estimated ORs (with 95% confidence intervals and p-values). Pooled measures were obtained by the methods appropriate for meta-analysis. The different food-based guidelines have been compared. RESULTS: The pooled ORs of obese status comparing medium vs. high adherence to MD were: 1.19 (95% C.I.: 0.99; 1.42, p-value = 0.062) and 1.12 (95% C.I.: 0.90; 1.38, p-value = 0.311) for MER and Italy respectively. For the comparison of low vs. high adherence, the pooled ORs were 1.05 (95% C.I.: 0.88; 1.24, p-value = 0.598) for MER, and 1.20 (95% C.I.: 1.02; 1.41, p-value = 0.031) for Italy when outliers are removed. High adherence to the MD resulted as potential protective factor against obesity. In MER 7-DFP: total fats is higher (34.5 E%) vs. Italian 7-DFP (29.4 E%); EPA (20 mg) and DHA (40 mg) are lower than recommended (200 mg each); sugars (12.6 E%) are higher than recommended (< 10 E%). Calcium, Zinc, and vitamin D do not reach target values in both 7-DFPs. CONCLUSION: This study highlights that, even when 7-DFPs follow MD and refer to nutrient needs, it is necessary to verify nutrient excesses or deficits impacting on NCD. High MD adherence is protective toward NCDs. MD principles, and energy balance should be communicated according to socioeconomic and educational levels.
Assuntos
Índice de Massa Corporal , Dieta Mediterrânea , Doenças não Transmissíveis , Humanos , Itália , Oriente Médio , Doenças não Transmissíveis/prevenção & controle , Doenças não Transmissíveis/epidemiologia , Feminino , Masculino , Obesidade/prevenção & controle , Obesidade/epidemiologia , Adulto , Pessoa de Meia-Idade , Estado NutricionalRESUMO
Tendon disorders are common medical conditions that could lead to significant disability, pain, healthcare costs, and a loss of productivity. Traditional approaches require long periods of treatment, and they largely fail due to the tissues weakening and the postoperative alterations of the normal joint mechanics. To overcome these limitations, innovative strategies for the treatment of these injuries need to be explored. The aim of the present work was the design of nano-fibrous scaffolds based on poly(butyl cyanoacrylate) (PBCA), a well-known biodegradable and biocompatible synthetic polymer, doped with copper oxide nanoparticles and caseinphosphopeptides (CPP), able to mimic the hierarchical structure of the tendon and to improve the tissue healing potential. These were developed as implants to be sutured to reconstruct the tendons and the ligaments during surgery. PBCA was synthetized, and then electrospun to produce aligned nanofibers. The obtained scaffolds were characterized for their structure and physico-chemical and mechanical properties, highlighting that CuO and CPP loading, and the aligned conformation determined an increase in the scaffold mechanical performance. Furthermore, the scaffolds loaded with CuO showed antioxidant and anti-inflammatory properties. Moreover, human tenocytes adhesion and proliferation to the scaffolds were assessed in vitro. Finally, the antibacterial activity of the scaffolds was evaluated using Escherichia coli and Staphylococcus aureus as representative of Gram-negative and Gram-positive bacteria, respectively, demonstrating that the CuO-doped scaffolds possessed a significant antimicrobial effect against E. coli. In conclusion, scaffolds based on PBCA and doped with CuO and CPP deserve particular attention as enhancers of the tendon tissue regeneration and able to avoid bacterial adhesion. Further investigation on the scaffold efficacy in vivo will assess their capability for enhancing the tendon ECM restoration in view of accelerating their translation to the clinic.
Assuntos
Embucrilato , Nanofibras , Humanos , Engenharia Tecidual , Alicerces Teciduais/química , Escherichia coli , Tendões , Nanofibras/química , Poliésteres/químicaRESUMO
BACKGROUND: Betaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it also shows an antioxidant activity. Moreover, BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). The processes of myogenesis and osteogenesis involve common mechanisms with skeletal muscle cells and osteoblasts sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation. METHODS: The effect of BET was tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible effects of BET on hObs differentiation were evaluated by real time PCR, western blot and immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes. RESULTS: Real time PCR results showed that BET stimulated significantly the expression of RUNX2, osterix, bone sialoprotein and osteopontin. Western blot and immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. BET induced a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels and CaMKII signaling activation. A significant rise in IGF-I mRNA at 3 and 6 h and a significant increase of IGF-I protein at 6 and 24 h after BET stimulus was detected. Furthermore, BET was able to increase significantly both SOD2 gene expression and protein content. CONCLUSIONS: Our study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are enhanced by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. Taken together, these results suggest that BET could be a promising nutraceutical therapeutic agent in the strategy to counteract the concomitant and interacting impact of sarcopenia and osteoporosis, i.e. the major determinants of senile frailty and related mortality.
Assuntos
Betaína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/citologia , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Modelos Biológicos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismoRESUMO
OBJECTIVE: To evaluate general knowledge about nutrition in an Italian population of children, pre-adolescents and adolescents. DESIGN: Knowledge about nutrition-related items such as healthy eating, breakfast, snacks, fast food, beverages, fruits and vegetables, cereals and tubers, meat/fish/legumes/eggs, milk and dairy products, fats and dressings, and sweets was analysed by means of a self-administered questionnaire (QuesCA IT) containing thirty-one questions, that was translated and adapted from a Swiss version (QuesCA) previously used in Geneva and Vaud. SETTING: North of Italy (Bergamo, Milan). SUBJECTS: Students (n 614) belonging to two different age groups: 9-11 years (GR1) and 12-16 years (GR2). RESULTS: Data analysis showed that nutritional knowledge varied in relation to the age of the participants, increasing in particular in the older group, although this difference was not statistically significant for all the considered items. Nutritional knowledge also varied in relation to the gender of the participants, with females in particular seeming to possess better cognition. For each age group there was poor knowledge about the items healthy diet, snacks, milk and dairy products, meat/fish/legumes/eggs, and fats and dressings. Moreover, the percentage of participants who declared own knowledge as insufficient was higher in GR2 compared with GR1. CONCLUSIONS: The present research demonstrates a lack of knowledge about the main concepts of healthy nutrition both in the youngest and oldest participants of the survey. This evidence, together with the presence of higher self-consciousness in GR2, should be taken into account in specific educational interventions during the school period.
Assuntos
Ciências da Nutrição Infantil , Dieta , Comportamento Alimentar/psicologia , Conhecimentos, Atitudes e Prática em Saúde , Adolescente , Fatores Etários , Criança , Laticínios , Feminino , Educação em Saúde/normas , Humanos , Itália , Masculino , Avaliação Nutricional , Valor Nutritivo , Lanches , Inquéritos e QuestionáriosRESUMO
Autophagy is a catabolic pathway involved both in tissue homeostasis and in cellular response to stress. The precise role of autophagy in cancer is still undefined and seems to depend on the tumor stage, appearing tumor-suppressive in physiological conditions and helpful to tumor progression in the established tumor. Here we analyzed by immunohistochemistry Beclin-1, p62, and LC3B, autophagic markers, in human specimens of normal breast, bone metastasis together with pair-matched invasive breast carcinoma of no special type (IBC-NST) as well as non-metastatic breast carcinoma, to disclose the possibility that they could be early prognostic indicators of the evolution of the disease toward the worst outcome. Different regions of metastatic carcinomas, i.e., areas adjacent to the tumor without signs of neoplastic growth, dysplastic lesions, and areas with invasive growth were considered. The pattern of autophagic parameters showed differences among the stages of breast carcinoma progression with a trend that indicated the activation of autophagic process in normal breast (Beclin-1 more elevated than p62), a pattern that was maintained in non-metastatic carcinoma. As the neoplasia proceeds with malignancy, the modification of the pattern of expression of autophagic markers (low ratio between Beclin-1 and p62) in areas of invasive growth of carcinomas suggested inhibition of the process. Of note, the parameters showed a different pattern in bone metastasis with respect to bone metastatic (bm)-IBC-NST, suggesting the reactivation of the autophagic process in the new growth site, helpful to the colonization. The course of autophagy markers during tumor progression could have a prognostic value towards bone metastasis and reveal different roles of the process in different phases of neoplastic growth. The understanding of the role of autophagy in bone metastasis could disclose new therapeutic targets to improve the conditions of patients.
Assuntos
Proteínas Reguladoras de Apoptose , Autofagia , Proteína Beclina-1 , Biomarcadores Tumorais , Neoplasias Ósseas , Neoplasias da Mama , Progressão da Doença , Imuno-Histoquímica , Proteínas de Membrana , Proteínas Associadas aos Microtúbulos , Proteína Sequestossoma-1 , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Proteína Beclina-1/metabolismo , Neoplasias Ósseas/secundário , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Autofagia/fisiologia , Proteína Sequestossoma-1/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Pessoa de Meia-Idade , Adulto , Prognóstico , IdosoRESUMO
Tendon disorders are common injuries, which can be greatly debilitating as they are often accompanied by great pain and inflammation. Moreover, several problems are also related to the laceration of the tendon-to-bone interface (TBI), a specific region subjected to great mechanical stresses. The techniques used nowadays for the treatment of tendon and TBI injuries often involve surgery. However, one critical aspect of this procedure involves the elevated risk of fail due to the tissues weakening and the postoperative alterations of the normal joint mechanics. Synthetic polymers, such as thermoplastic polyurethane, are of special interest in the tissue engineering field as they allow the production of scaffolds with tunable elastic and mechanical properties, that could guarantee an effective support during the new tissue formation. Based on these premises, the aim of this work was the design and the development of highly porous 3D scaffolds based on thermoplastic polyurethane, and doped with chondroitin sulfate and caseinophosphopeptides, able to mimic the structural, biomechanical, and biochemical functions of the TBI. The obtained scaffolds were characterized by a homogeneous microporous structure, and by a porosity optimal for cell nutrition and migration. They were also characterized by remarkable mechanical properties, reaching values comparable to the ones of the native tendons. The scaffolds promoted the tenocyte adhesion and proliferation when caseinophosphopetides and chondroitin sulfate are present in the 3D structure. In particular, caseinophosphopeptides' optimal concentration for cell proliferation resulted 2.4 mg/mL. Finally, the systems evaluation in vivo demonstrated the scaffolds' safety, since they did not cause any inflammatory effect nor foreign body response, representing interesting platforms for the regeneration of injured TBI.
Assuntos
Sulfatos de Condroitina , Alicerces Teciduais , Alicerces Teciduais/química , Porosidade , Sulfatos de Condroitina/química , Poliuretanos/química , Engenharia Tecidual/métodos , Regeneração Óssea , TendõesRESUMO
Paraprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes' formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.
RESUMO
In this paper, we show that the pH optimum for the plasma membrane (PM)-associated activity of four glycohydrolases (conduritol B epoxide sensitive ß-glucosidase, ß-glucosidase GBA2, ß-hexosaminidase and ß-galactosidase) measured on intact cells is acidic. Moreover, we show that drugs able to modify the efflux of protons across the PM, thus locally affecting the extracellular proton concentration close to the PM, are able to modulate the activities of these enzymes. These data strongly suggest that pH-dependent modulation of PM-associated glycohydrolases activities could be an effective way to locally modulate the cell surface glycoconjugate composition.
Assuntos
Membrana Celular/enzimologia , Concentração de Íons de Hidrogênio , beta-Galactosidase/metabolismo , beta-Glucosidase/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acetazolamida/farmacologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Ácido Egtázico/farmacologia , Ativação Enzimática , Fibroblastos/enzimologia , Glioma/enzimologia , Humanos , Neuroblastoma/enzimologia , Omeprazol/farmacologia , PrótonsRESUMO
Bone metastases represent the main problem related to the progression of breast cancer, as they are the main cause of death for these patients. Unfortunately, to date, bone metastases are incurable and represent the main challenge for the researcher. Chemokines and cytokines affect different stages of the metastatic process, and in bone metastases, interleukin (IL) -6, IL-8, IL-1ß, and IL-11 participate in the interaction between cancer cells and bone cells. This review focuses on IL-11, a pleiotropic cytokine that, in addition to its well-known effects on several tissues, also mediates certain signals in cancer cells. In particular, as IL-11 works on bone remodeling, it plays a relevant role in the osteolytic vicious cycle of bone resorption and tumour growth, which characterizes bone metastasis. IL-11 appears as a candidate for anti-metastatic therapy. Even if different therapeutic approaches have considered IL-11 and the downstream-activated gp130 signaling pathways activated downstream of gp130, further studies are needed to decipher the contribution of the different cytokines and their mechanisms of action in breast cancer progression to define therapeutic strategies.
RESUMO
Bone metastasis is a serious and often lethal complication of particularly frequent carcinomas, such as breast and prostate cancers, which not only reduces survival but also worsens the patients' quality of life. Therefore, it is important to find new and/or additional therapeutic possibilities that can counteract the colonization of bone tissue. High adherence to the Mediterranean diet (MD) is effective in the prevention of cancer and improves cancer patients' health, thus, here, we considered its impact on bone metastasis. We highlighted some molecular events relevant for the development of a metastatic phenotype in cancer cells and the alterations of physiological bone remodeling, which occur during skeleton colonization. We then considered those natural compounds present in MD foods with a recognized role to inhibit or reverse the metastatic process both in in vivo and in vitro systems, and we reported the identified mechanisms of action. The knowledge of this bioactivity by the dietary components of the MD, together with its wide access to all people, could help not only to maintain healthy status but also to improve the quality of life of patients with bone metastases.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/terapia , Quimioterapia Adjuvante , Dieta Mediterrânea , Progressão da Doença , Alimentos , Neoplasias da Próstata/terapia , Neoplasias da Mama/patologia , Feminino , Humanos , Masculino , Neoplasias da Próstata/patologiaRESUMO
Infant biscuits (IBs) are part of complementary feeding from weaning up to the age of five years. They normally contain bovine milk proteins, which can influence bone development. This potential effect was investigated using experimental baked IBs, which were prepared from doughs containing different type of dairy proteins: milk protein concentrate (IB1), whey protein isolate (IB2), and skimmed milk powder (IB3). Dairy protein-free (IB0) and gluten-free (IB4) biscuits were also formulated. The in vitro gastrointestinal digests of IBs (IBDs) were tested on a co-culture of Caco-2/HT-29 70/30 cells as an in vitro model of human small intestine. None of the IBDs influenced cell viability and monolayer integrity, while IBD0 and IBD4 increased Peptide-YY production. The basolateral contents of Transwell plates seeded with Caco-2/HT-29 70/30 co-culture, mimicking metabolized IBDs (MIBDs), were tested on Saos-2 cells, an in vitro model of human osteoblast-like cells. After incubation, MIBD0, lacking dairy proteins, decreased the cell viability, while MIBD2, containing whey protein isolate, increased both the viability and the number of cells. MIBD2 and MIBD4, the latter containing both casein and whey proteins, increased alkaline phosphatase activity, a bone differentiation marker. These results highlight that IBs containing dairy proteins positively affect bone development.
RESUMO
OBJECTIVES: The intestinal cell function can be modulated by the type and quantity of nutrients. The aim of this study was to evaluate the effects of an excess of nutrients on intestinal morphofunctional features and a possible association of inflammation in a 70/30 Caco2/HT-29 intestinal in vitro co-culture. METHODS: An excess of nutrients (EX) was obtained by progressively increasing the medium change frequency with respect to standard cell growth conditions (ST) from confluence (T0) to 15 d after confluence (T15). RESULTS: In comparison with the ST group, the EX group revealed a maintenance in the number of microvilli, an increase in follicle like-structures and mucus production, and a decrease in the number of tight junction. The specific activity of markers of intestinal differentiation, alkaline phosphatase and aminopeptidase N, and of the enterocyte differentiation specific marker, dipeptidyl peptidase-IV, were progressively raised. The transepithelial electrical resistance, indicative of the co-culture barrier properties, decreased, whereas Lucifer yellow Papp evaluation, an index of the paracellular permeability to large molecules, showed an increase. Reactive oxygen species and nitric oxide production, indicative of an oxidative status, together with interleukin-6, interleukin-8, indicative of a low-grade inflammation, and peptide YY secretion were higher in the EX group than in the ST group. The differences between ST and EX were particularly evident at T15. CONCLUSION: These data support the suitability of our in vitro gut model for obesity studies at the molecular level and the necessity to standardize the medium frequency change in intestinal culture.
Assuntos
Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Nutrientes/metabolismo , Transporte Biológico , Células CACO-2 , Técnicas de Cocultura , Células HT29 , Humanos , Técnicas In Vitro , Permeabilidade , Junções Íntimas/metabolismoRESUMO
Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance.
Assuntos
Matriz Óssea/efeitos dos fármacos , Carnitina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Antioxidantes/metabolismo , Matriz Óssea/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Humanos , Sialoproteína de Ligação à Integrina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteopontina/metabolismo , Oxirredução , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição Sp7/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Health benefits are routinely attributed to whey proteins, their hydrolysates and peptides based on in vitro chemical and cellular assays. The objective of this study was to track the fate of whey proteins through the upper gastrointestinal tract, their uptake across the intestinal barrier and then assess the physiological impact to downstream target cells. Simulated gastrointestinal digestion (SGID) released a selection of whey peptides some of which were transported across a Caco-2/HT-29 intestinal barrier, inhibited free radical formation in muscle and liver cells. In addition, SGID of ß-lactoglobulin resulted in the highest concentration of free amino acids (176â¯nM) arriving on the basolateral side of the co-culture with notable levels of branched chain and sulphur-containing amino acids. In vitro results indicate that consumption of whey proteins will deliver bioactive peptides to target cells.
Assuntos
Estresse Oxidativo/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas do Soro do Leite/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Células CACO-2 , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Trato Gastrointestinal/metabolismo , Células HT29 , Humanos , Lactoglobulinas/metabolismo , Leite/metabolismo , Peptídeos/isolamento & purificação , Peptídeos/metabolismoRESUMO
Carotenoids, tocols, phenolic acids and antioxidant capacity were studied during in vitro digestion of water biscuits (WB) from bread wheat, einkorn and einkorn-pseudocereals. Antioxidant and cytotoxic activities of digestates were also measured using a 70% Caco2/30% HT-29 human intestinal co-culture layer. Antioxidant profiles differed among WB formulations. Only hydrophilic molecules were solubilised by gastric digestion. After intestinal digestion, 77% carotenoids and 67% tocols were released. Soluble-conjugated phenolic acids increased (30%) and insoluble-bound forms decreased (17%), suggesting partial conversion from bound to conjugated form. After intestinal digestion, antioxidant capacity increased regardless of type and amount of antioxidants in undigested or digested WB. All WB, especially those with quinoa flour, reduced the AAPH pro-oxidant capacity in co-culture cells. These results highlight the potential health benefits of underutilized crops and the need for in vitro or in vivo models to better address potential bioactivity at intestinal and target organs level.
Assuntos
Antioxidantes/análise , Células CACO-2 , Carotenoides , Farinha , Humanos , FenóisRESUMO
An intestinal 70/30 Caco2/HT-29 co-culture was set up starting from the parental populations of differentiated cells to mimic the human intestinal epithelium. Co-culture was harvested at confluence 0 (T0) and at 3, 6, 10, and 14 days post confluence after plating (T3, T6, T10, and T14, respectively) for morphological and functional analysis. Transmission electron microscopy revealed different features from T0 to T14: microvilli and a complete junctional apparatus from T6, mucus granules from T3, as also confirmed by PAS/Alcian Blue staining. The specific activity of alkaline phosphatase (ALP), aminopeptidase N (APN), and dipeptidyl peptidase IV (DPPIV) progressively increased after T0, indicating the acquirement of a differentiated and digestive phenotype. Transepithelial electrical resistance (TEER), indicative of the barrier properties of the monolayer, increased from T0 up to T6 reaching values very similar to the human small intestine. The apparent permeability coefficient for Lucifer Yellow (LY), along with morphological analysis, reveals a good status of the tight junctions. At T14, HT-29 cells reduced to 18.4% and formed domes, indicative of transepithelial transport of nutrients. This Caco2/HT-29 co-culture could be considered a versatile and suitable in vitro model of human intestinal epithelium for the presence of more than one prevalent intestinal cell type, by means of a minimum of 6 to a maximum of 14 post-confluence days obtained without the need of particular inducers of subclones and growth support to reach an intestinal differentiated phenotype.
Assuntos
Antígenos de Diferenciação/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Modelos Biológicos , Transporte Biológico Ativo , Células CACO-2 , Técnicas de Cocultura , Humanos , PermeabilidadeRESUMO
Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT-29 cells that have been shown to be differentiated in culture. The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs beta-CN(1-25)4P and alpha(s1)-CN(59-79)5P, the commercial mixture CPP DMV, the 'cluster sequence' pentapeptide, typical of CPPs, and dephosphorylated beta-CN(1-25)4P, [beta-CN(1-25)0P]. The biological effect was found to be: (a) maximal with beta-CN(1-25)4P and null with the 'cluster sequence'; (b) independent of the presence of inorganic phosphate; and (c) maximal at 4 mmol.L(-1) Ca2+. The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by beta-CN(1-25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+-free Krebs/Ringer/Hepes; (e) formation of bigger aggregates (150 nm radius) with beta-CN(1-25)0P. With both beta-CN(1-25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at 4 mmol.L(-1) Ca2+.
Assuntos
Cálcio/metabolismo , Caseínas/metabolismo , Fosfopeptídeos/fisiologia , Sequência de Aminoácidos , Células HT29 , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development.
Assuntos
Células CACO-2/patologia , Diferenciação Celular/fisiologia , Células CACO-2/enzimologia , Células CACO-2/ultraestrutura , Contagem de Células , Técnicas de Cultura de Células/métodos , Processos de Crescimento Celular/fisiologia , Humanos , FenótipoRESUMO
In the present work, Grana Padano (GP) and Trentingrana (TN) cheeses at different ripening time were in vitro digested. To study calcium uptake and utilization, the intact digestates (selected doses that do not alter cell viability and Transepithelial Electrical Resistance) were administered to Caco2/HT-29 70/30 cells, cultured on a semipermeable membrane in transwells, as a model of human intestinal epithelium. Intact digestates as well as the whole basolateral solutions (mimicking the passage of digestates through intestinal cells before reaching the blood flow and bone) in parallel were further administered to human osteoblast-like cells SaOS-2 to study the extracellular bone matrix formation. In vitro digestates deriving from GP and TN promoted calcium uptake and extracellular bone matrix formation independently of both the cheese type and its ripening period (13, 19 or 26months). The present study reports the ability of whole digestates of GP and TN cheeses to improve intestinal calcium absorption and bone matrix formation in vitro. Once fully explored at bone level, this finding could better support the role of cheese in ameliorating calcium deficiencies and associated diseases in vivo.