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1.
Artigo em Inglês | MEDLINE | ID: mdl-32384765

RESUMO

(1) Background: The gastrointestinal tract (GI) tract is one of the main organs exposed to particulate matter (PM) directly through ingestion of contaminated food or indirectly through inhalation. Previous studies have investigated the effects of chronic PM exposure on intestinal epithelia in vitro using Caco-2 cells and in vivo using mice. In this study, we hypothesized that chronic PM exposure would increase epithelial permeability and decrease barrier function due to altered redox homeostasis, which alters levels and/or localization of barrier-associated proteins in human three-dimensional (3D) intestinal tissues. (2) Methods: Transepithelial electrical resistance (TEER) in tissues exposed to 50, 100, 150, 250, and 500 µg/cm2 of PM for 1 week and 2 weeks was analyzed. Levels and localization of tight junction proteins zonula occludens protein 1 (ZO-1) and claudin-1 and desmosome-associated desmocollin were analyzed using immunofluorescence. As a marker of oxidative stress, levels of 4-hydroxy-nonenal (4HNE) adducts were measured. (3) Results: No differences in TEER measurements were observed between exposed and un-exposed tissues. However, increased levels of 4HNE adducts in exposed tissues were observed. Additionally, decreased levels of ZO-1, claudin-1, and desmocollin were demonstrated. (4) Conclusion: These data suggest that chronic PM exposure results in an increase of oxidative stress; modified levels of barrier-associated proteins could possibly link to GI tract inflammatory conditions.


Assuntos
Células CACO-2 , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Mucosa Intestinal/metabolismo , Material Particulado/farmacologia , Junções Íntimas/metabolismo , Animais , Células CACO-2/efeitos dos fármacos , Células CACO-2/fisiologia , Humanos , Intestinos/fisiopatologia , Proteínas de Membrana/metabolismo , Camundongos , Oxirredução , Material Particulado/administração & dosagem , Proteínas de Junções Íntimas
2.
Sci Rep ; 10(1): 2793, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066787

RESUMO

Carbon-based nanomaterials are being increasingly used, demanding strong information to support their safety in terms of human health. As ingestion is one of the most important exposure routes in humans, we have determined their potential risk by using an in vitro model simulating the human intestinal barrier and evaluated the effects of both graphene oxide (GO) and graphene nanoplatelets (GNPs). A coculture of differentiated Caco-2/HT29 cells presenting inherent intestinal epithelium characteristics (i.e. mucus secretion, brush border, tight junctions, etc.) were treated with GO or GNPs for 24 h. Different endpoints such as viability, membrane integrity, NPs localization, cytokines secretion, and genotoxic damage were evaluated to have a wide view of their potentially harmful effects. No cytotoxic effects were observed in the cells that constitute the barrier model. In the same way, no adverse effects were detected neither in the integrity of the barrier (TEER) nor in its permeability (LY). Nevertheless, a different bio-adhesion and biodistribution behavior was observed for GO and GNPs by confocal microscopy analysis, with a more relevant uptake of GNPs. No oxidative damage induction was detected, either by the DCFH-DA assay or the FPG enzyme in the comet assay. Conversely, both GO and GNPs were able to induce DNA breaks, as observed in the comet assay. Finally, low levels of anti-inflammatory cytokines were detected, suggesting a weak anti-inflammatory response. Our results show the moderate/severe risk posed by GO/GNPs exposures, given the observed genotoxic effects, suggesting that more extensive genotoxic evaluations must be done to properly assess the genotoxic hazard of these nanomaterials.


Assuntos
Dano ao DNA/efeitos dos fármacos , Grafite/farmacologia , Intestinos/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Células CACO-2/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Grafite/química , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Nanopartículas/química , Nanoestruturas/química , Junções Íntimas/efeitos dos fármacos
3.
Sci Rep ; 9(1): 16647, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719636

RESUMO

The present state of cancer chemotherapy is unsatisfactory. New anticancer drugs that marginally improve the survival of patients continue to be developed at an unsustainably high cost. The study aimed to elucidate the effects of insulin (INS), an inexpensive drug with a convincing safety profile, on the susceptibility of colon cancer to chemotherapeutic agents: 5-fluorouracil (FU), oxaliplatin (OXA), irinotecan (IRI), cyclophosphamide (CPA) and docetaxel (DOC). To examine the effects of insulin on cell viability and apoptosis, we performed an in vitro analysis on colon cancer cell lines Caco-2 and SW480. To verify the results, we performed in vivo analysis on mice bearing MC38 colon tumors. To assess the underlying mechanism of the therapy, we examined the mRNA expression of pathways related to the signaling downstream of insulin receptors (INSR). Moreover, we performed Western blotting to confirm expression patterns derived from the genetic analysis. For the quantification of circulating tumor cells in the peripheral blood, we used the maintrac method. The results of our study show that insulin-pretreated colon cancer cells are significantly more susceptible to commonly used chemotherapeutics. The apoptosis ratio was also enhanced when INS was administered complementary to the examined drugs. The in vivo study showed that the combination of INS and FU resulted in significant inhibition of tumor growth and reduction of the number of circulating tumor cells. This combination caused a significant downregulation of the key signaling substrates downstream of INSR. The results indicate that the downregulation of PIK3CA (phosphatidylinositol 3-kinase catalytic subunit alpha), which plays a critical role in cell signaling and GRB2 (growth factor receptor-bound protein 2), a regulator of cell proliferation and differentiation may be responsible for the sensitizing effect of INS. These findings were confirmed at protein levels by Western blotting. In conclusion, these results suggest that INS might be potentially applied to clinical use to enhance the therapeutic effectiveness of chemotherapeutic drugs. The findings may become a platform for the future development of new and inexpensive strategies for the clinical chemotherapy of tumors.


Assuntos
Antineoplásicos/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Proteína Adaptadora GRB2/antagonistas & inibidores , Insulina/farmacologia , Animais , Western Blotting , Células CACO-2/efeitos dos fármacos , Células CACO-2/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Colorretais/metabolismo , Ciclofosfamida/uso terapêutico , Docetaxel/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Fluoruracila/uso terapêutico , Proteína Adaptadora GRB2/metabolismo , Humanos , Irinotecano/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/metabolismo , Oxaliplatina/uso terapêutico
4.
Food Funct ; 10(10): 6936-6944, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31591629

RESUMO

Probiotic starters for Chinese pickle fermentation were screened from three anti-Candida lactic acid bacteria (LAB). Similar to commercial starters, the candidates Lactobacillus plantarum AT4 and L. plantarum AT28 could acidize the pickle within 10 days. The isolates AT4 and AT28 maintained 6.0-7.0 log cfu ml-1 in the pickle products and their inhibition rates against Candida increased to 83.7% and 92.9% after fermentation, respectively. Only L. plantarum AT4 satisfied the essential probiotic requirements such as tolerance to the gastrointestinal environment (pH 2.0, 0.5% bile salts and 0.5% phenol) and adhesion to Caco-2 cells. The pickle product fermented with L. plantarum AT4 presented comparable levels of lactic and acetic acids, volatile profile and sensory value to the commercial one. The results suggested that L. plantarum AT4 can be used as a probiotic starter for industrial fermentation of Chinese pickle, and the pickled product may be utilized as a promising anti-Candida probiotic.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Lactobacillus plantarum/fisiologia , Probióticos/química , Probióticos/isolamento & purificação , Probióticos/farmacologia , Grupo com Ancestrais do Continente Asiático , Aderência Bacteriana , Células CACO-2/efeitos dos fármacos , Fermentação , Alimentos e Bebidas Fermentados , Microbiologia de Alimentos , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Microbiota , Paladar
5.
Artigo em Inglês | MEDLINE | ID: mdl-31481446

RESUMO

P-glycoprotein (ABCB1), an ATP-binding-cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions (DDIs). ABCB1 has been suggested to interact with many antivirals used to treat HIV and/or chronic hepatitis C virus (HCV) infections. Using bidirectional transport experiments in Caco-2 cells and a recently established ex vivo model of accumulation in precision-cut intestinal slices (PCIS) prepared from rat ileum or human jejunum, we evaluated the potential of anti-HIV and anti-HCV antivirals to inhibit intestinal ABCB1. Lopinavir, ritonavir, saquinavir, atazanavir, maraviroc, ledipasvir, and daclatasvir inhibited the efflux of a model ABCB1 substrate, rhodamine 123 (RHD123), in Caco-2 cells and rat-derived PCIS. Lopinavir, ritonavir, saquinavir, and atazanavir also significantly inhibited RHD123 efflux in human-derived PCIS, while possible interindividual variability was observed in the inhibition of intestinal ABCB1 by maraviroc, ledipasvir, and daclatasvir. Abacavir, zidovudine, tenofovir disoproxil fumarate, etravirine, and rilpivirine did not inhibit intestinal ABCB1. In conclusion, using recently established ex vivo methods for measuring drug accumulation in rat- and human-derived PCIS, we have demonstrated that some antivirals have a high potential for DDIs on intestinal ABCB1. Our data help clarify the molecular mechanisms responsible for reported increases in the bioavailability of ABCB1 substrates, including antivirals and drugs prescribed to treat comorbidity. These results could help guide the selection of combination pharmacotherapies and/or suitable dosing schemes for patients infected with HIV and/or HCV.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Fármacos Anti-HIV/farmacologia , Antivirais/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Idoso , Animais , Sulfato de Atazanavir/farmacologia , Benzimidazóis/farmacologia , Células CACO-2/efeitos dos fármacos , Células CACO-2/metabolismo , Interações Medicamentosas , Feminino , Fluorenos/farmacologia , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Hepatite C/complicações , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Imidazóis/farmacologia , Intestinos/efeitos dos fármacos , Lopinavir/farmacologia , Masculino , Maraviroc/farmacologia , Pessoa de Meia-Idade , Ratos , Ratos Wistar , Ritonavir/farmacologia , Saquinavir/farmacologia , Zidovudina/farmacologia
6.
Mutat Res ; 845: 402980, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31561898

RESUMO

TiO2 particles are widely used in products for everyday consumption, such as cosmetics and food; their possible adverse effects on human health must therefore be investigated. The aim of this study was to document in vitro impact of the food additive E171, i.e. TiO2, and of TiO2 nanoparticles, on a co-culture of Caco-2 and HT29-MTX cells, which is an in vitro model for human intestine. Cells were exposed to TiO2 particles three days after seeding, i.e. while they were not fully differentiated. Cell viability, reactive oxygen species (ROS) levels and DNA integrity were assessed, by MTT assay, DCFH-DA assay, alkaline and Fpg-modified comet assay and 8-oxo-dGuo measurement by HPLC-MS/MS. The mRNA expression of genes involved in ROS regulation, DNA repair via base-excision repair, and endoplasmic reticulum stress was assessed by RT-qPCR. Exposure to TiO2 particles resulted in increased intracellular ROS levels, but did not impair cell viability and did not cause any oxidative damage to DNA. Only minor changes in mRNA expression were detected. Altogether, this shows that E171 food additive and TiO2 nanoparticles only produce minor effects to this in vitro intestinal cell model.


Assuntos
Células CACO-2/efeitos dos fármacos , Aditivos Alimentares/toxicidade , Células HT29/efeitos dos fármacos , Titânio/toxicidade , 8-Hidroxi-2'-Desoxiguanosina/análise , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Aditivos Alimentares/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estresse Oxidativo , Tamanho da Partícula , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
7.
Planta Med ; 85(11-12): 987-996, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31350736

RESUMO

The rise of diabetes incidence in Nigeria enhances the use of popular remedies that may interact with conventional therapies. The aqueous extracts of 27 popular Nigerian "antidiabetic" plants were tested for their in vitro effects on glutathione levels within HepG2 cells, P-glycoprotein (P-gp)-mediated Rh-123 efflux activity in Caco-2 vincristine-resistant cells, and modulation of glibenclamide transport in Caco-2 monolayers. The extract from Ximenia americana significantly depleted intracellular glutathione at 100 µg/mL similarly to the reference buthionine sulphoximine (p < 0.05). Other 10 extracts raised glutathione levels. Eight extracts inhibiting P-gp efflux in a concentration-dependent manner (p < 0.01) were selected for further evaluation in a bi-directional transport model across Caco-2 monolayers: Annona senegalensis, Bridellia ferruginea, Cassytha filiformis, Daniellia ogea, Khaya ivorensis, Syzygium guineense, Terminalia avicennioides, and X. americana. When interferences in paracellular transport were discarded, only 3 of them may be modulating the efflux ratio of glibenclamide (efflux ratio: 2.65 ± 0.13) in the same manner the reference drug verapamil (efflux ratio: 1.14 ± 0.25, p < 0.01) does: Syzygium guineense (efflux ratio: 1.70 ± 0.23, p < 0.01), Terminalia avicennioides (efflux ratio: 1.80 ± 0.25, p < 0.05), and X. americana (efflux ratio: 1.66 ± 0.10, p < 0.01). HPLC-UV analyses for P-gp inhibitors in these extracts revealed several phenolic compounds such as rutin, gallic acid, and ellagic acid reported to decrease P-gp expression and/or directly modify its function. In conclusion, some popular herbal medicines used by Nigerian diabetic patients are here shown to potentially affect glibenclamide absorption at concentrations that could be reached in the intestinal tract.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Glibureto/metabolismo , Hipoglicemiantes/farmacologia , Medicina Tradicional Africana , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Células CACO-2/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Interações Ervas-Drogas , Humanos
8.
Sci Rep ; 9(1): 10609, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337851

RESUMO

4-thiazolidinones, which are privileged structures in medicinal chemistry, comprise the well-known class of heterocycles and are a source of new drug-like compounds. Undoubtedly, the 5-bulky-substituted-2,4-thiazolidinediones - a class of antihyperglycemic glitazones, which are peroxisome proliferator-activated receptor gamma (PPARγ) agonists, are the most described group among them. As there are various chemically distinct 4-thiazolidinones, different subtypes have been selected for studies; however, their main pharmacological profiles are similar. The aim of this study was to evaluate the anticancer activity of 5Z-(4-fluorobenzylidene)-2-(4-hydroxyphenylamino)-thiazol-4-one (Les-236) in four human cancer cell lines, A549, SCC-15, SH-SY5Y, and CACO-2, and investigate its impact on the production of reactive oxygen species (ROS) and the apoptotic process as well as cytotoxicity and metabolism in these cell lines. The cell lines were exposed to increasing concentrations (1 nM to 100 µM) of the studied compound for 6, 24, and 48 h, and later, ROS production, cell viability, caspase-3 activity, and cell metabolism were examined. The obtained results showed that the studied compound decreased the production of ROS, increased the release of lactate dehydrogenase, and decreased cell metabolism/proliferation in all the five cell lines at micromolar concentrations. Interestingly, over a wide range of concentrations (from 1 nM to 100 µM), Les-236 was able to increase the activity of caspase-3 in BJ (after 6 h of exposure), A549, CACO-2, and SCC-15 (after 48 h of exposure) cell lines which could be an effect of the activation of PPARγ-dependent pathways.


Assuntos
Antineoplásicos/farmacologia , Tiazóis/farmacologia , Células A549/efeitos dos fármacos , Células A549/metabolismo , Apoptose/efeitos dos fármacos , Células CACO-2/efeitos dos fármacos , Células CACO-2/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Relação Dose-Resposta a Droga , Humanos , L-Lactato Desidrogenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Chemosphere ; 228: 139-148, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31029959

RESUMO

Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filtering shellfish and can cause human intoxications. Humans can be exposed to combinations of several phycotoxins. The toxicological effects of phycotoxin mixtures on human health are largely unknown. Published data on phycotoxin co-exposure show that okadaic acid (OA) is simultaneously found with pectenetoxin-2 (PTX-2), 13-desmethylspirolide C (also known as SPX-1), or yessotoxin (YTX). Therefore, the aim of this study was to examine the effects of three binary mixtures, OA/PTX-2, OA/SPX-1 and OA/YTX on human intestinal Caco-2 cells. A multi-parametric approach for cytotoxicity determination was applied using a high-content analysis platform, including markers for cell viability, oxidative stress, inflammation, and DNA damage. Mixtures effects were analyzed using two additivity mathematical models. Our assays revealed that OA induced cytotoxicity, DNA strand breaks and interleukin 8 release. PTX-2 slightly induced DNA strand breaks, whereas SPX-1 and YTX did not affect the investigated endpoints. The combination of OA with another toxin resulted in reduced toxicity at low concentrations, suggesting antagonistic effects, but in increased effects at higher concentrations, suggesting additive or synergistic effects. Taken together, our results demonstrated that the cytotoxic effects of binary mixtures of lipophilic phycotoxins could not be predicted by additivity mathematical models. In conclusion, the present data suggest that combined effects of phycotoxins may occur which might have the potential to impact on risk assessment of these compounds.


Assuntos
Células CACO-2/efeitos dos fármacos , Combinação de Medicamentos , Interações Medicamentosas , Toxinas Marinhas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Furanos/farmacologia , Humanos , Inflamação , Intestinos/citologia , Macrolídeos , Toxinas Marinhas/análise , Ácido Okadáico/análise , Ácido Okadáico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Oxocinas/farmacologia , Piranos/farmacologia , Frutos do Mar/análise , Frutos do Mar/toxicidade , Compostos de Espiro/farmacologia
10.
Food Res Int ; 118: 101-107, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30898345

RESUMO

This study aimed to evaluate the potential anti-inflammatory role of the most produced form of lactoferrin expressed in various expression systems (Fe-saturated recombinant human Lf, rhLf) and its hydrolysate in concentrations resembles that found in mature human milk. Co-culture model consisted of CaCo-2 and RAW 246.7 cell lines was used to evaluate the potential anti-inflammatory activity of rhLf and its hydrolysate. During this experiment, CaCo-2 monolayer permeability and integrity was assayed through the measurement of transepithelial electrical resistance (TEER values). Also, the production of reactive oxygen species (ROS), nitric oxide (NO) and different cytokines (IL-8, IL-1ß, IL-6, IL-10, IL-12p70, and TNF-α) were measured. The treatment with rhLf and its hydrolysate protected the monolayer integrity against LPS effect and reduced IL-8 and ROS production. This effect was dependent on the dose and 2mgmL-1 of rhLf hydrolysate was more effective. The addition of rhLf and its hydrolysate to infant formula is a prominent step towards improving both infant formula functionality and newborn health. Thus, these functional ingredients could be incorporated in infant foods. In this context, ongoing researches are conducted to clarify this effect whether by using synthetic peptides or by using LPS-sepsis animal.


Assuntos
Anti-Inflamatórios/farmacologia , Lactoferrina/farmacologia , Lipopolissacarídeos/efeitos adversos , Proteínas Recombinantes/farmacologia , Animais , Células CACO-2/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Fórmulas Infantis , Recém-Nascido , Lactoferrina/metabolismo , Camundongos , Leite Humano , Óxido Nítrico/metabolismo , Células RAW 264.7/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo
11.
Food Res Int ; 119: 634-642, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30884698

RESUMO

Titanium dioxide (TiO2) is enclosed in many consumer products including pharmaceuticals, cosmetics, and foods. TiO2 (E171) is daily ingested as mixed nano- and submicron-sized particles since it is approved as a white colorant in Europe in a wide variety of food products, Noteworthy, the relevant risk assessment has never been satisfactorily concluded and growing alarms for human hazards deriving from TiO2 exposure are incrementally reported. The objective of the present study was to establish conceivable mechanisms by which nano-sized TiO2 particles affect physiological function of the intestinal epithelium layer. The well-established Caco-2 cell line differentiated for 21 days on permeable supports was used as a predictive model of the human intestinal mucosa to identify the biological response triggered by TiO2 particles. Exposure to 42 µg/mL TiO2 nanoparticles disrupted the tight junctions-permeability barrier with a prompt effect detectable after 4 h incubation time and wide effects on barrier integrity at 24 h. Transport and ultrastructural localization of TiO2 nanoparticles were determined by ICP-OES, TEM and ESI/EELS analysis, respectively. Nano-sized particles were efficiently internalized and preferentially entrapped by Caco-2 monolayers. Storage of TiO2 nanoparticles inside the cells affected enterocytes viability and triggered the production of pro-inflammatory cytokines, including TNF-α and IL-8. Taken together these data indicate that nano-sized TiO2 particles exert detrimental effects on the intestinal epithelium layer.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Nanopartículas/química , Titânio/efeitos adversos , Células CACO-2/efeitos dos fármacos , Citocinas/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Aditivos Alimentares/química , Humanos , Interleucina-8 , Nanopartículas/toxicidade , Tamanho da Partícula , RNA Mensageiro , Fator de Necrose Tumoral alfa/metabolismo
12.
Nutrients ; 11(3)2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841512

RESUMO

The aim of this study was to determine the anti-inflammatory potential of pomegranate peel extracts (PPE) prepared from waste material of pomegranate juice production both in vitro on Caco-2 cells and ex vivo using porcine colonic tissue explants. Caco-2 cells were stimulated in vitro by TNF and colonic tissue explants were stimulated ex vivo with lipopolysaccharide (LPS). Both tissues were co-treated with PPE at 0, 1.0, 2.5, 5.0, 10 and 25 µg/mL. The secretion of CXCL8 in the supernatant of both experiments was determined by enzyme linked immunosorbent assay (ELISA) and the relative expression of inflammatory cytokines were evaluated in the colonic tissue by quantitative polymerase chain reaction (QPCR). The 2.5 to 25 µg/mL of PPE suppressed CXCL8 (p < 0.001) in the Caco-2 cells, whereas CXCL8 production was suppressed by only 5 and 25 µg/mL (p < 0.01) of PPE in the colonic explants. The 5 µg/mL of PPE also suppressed the expression of IL1A (p < 0.05), IL6 (p < 0.01) and CXCL8 (p < 0.05) in LPS challenged colonic tissues compared to controls. In conclusion, the 5 µg/mL of PPE consistently elicits strong anti-inflammatory activity. These results support the potential of bioactive compounds from the waste peel of pomegranate in terms of their anti-inflammatory activity in cells and tissues of the gastrointestinal tract.


Assuntos
Anti-Inflamatórios/farmacologia , Frutas/química , Lythraceae , Extratos Vegetais/farmacologia , Animais , Células CACO-2/efeitos dos fármacos , Colo/efeitos dos fármacos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos , Reação em Cadeia da Polimerase , Suínos
13.
Phytomedicine ; 55: 269-281, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668439

RESUMO

BACKGROUND: The degree of intracellular drug accumulation by specific membrane transporters, i.e., MDR1, BCRP, and MRP, and the degree of detoxification by intracellular metabolic enzymes, i.e., CYP3A4 and GST, provide control for cancer chemotherapy through diminishing the propensity of cancer cells to undergo apoptosis which in turn modulates the unresolved and complex phenomenon of multidrug resistance (MDR) for the cancer cells. HYPOTHESIS/PURPOSE: This study dwells into the interaction details involving ABC-transporters, CYP3A4, GST and cytotoxic effects of resveratrol on different cell lines. METHODS: Resveratrol was evaluated for its ability modulating the expression and efflux functions of P-gp /MDR1, MRP1, and BCRP in the multidrug-resistant human colon carcinoma cell line, Caco-2, and CEM/ADR5000 cells through flow cytometry and RTPCR technique. RESULTS: The resveratrol influenced P-gp and MRP1 efflux functions whereby it increased rhodamine 123 with calcein accumulation in concentration-dependent manner (1 - 500 µM) in the Caco-2 cell lines and inhibited the effluxes of both the substrates also as concentration-dependent phenomenon (10 - 100 µM) in the p-gp overexpressing CEM/ADR5000 cells through FACS (full form). The treatment of drug-resistant Caco-2, and CEM/ADR5000 cells with doxorubicin (DOX) along with 20 µM of resveratrol in the mixture. It increased the cell sensitivity DOX towards the DOX and enhanced the cytotoxicity. The resveratrol inhibited both CYP3A4 and GST enzymatic activity in a concentration-dependent way and induced apoptosis in the resistance cell lines because of increased levels of caspase-3, -8,-6/9 and incremental phosphatidyl serine (PS) exposure as detected by flow cytometry. The treatment of Caco-2 cells with resveratrol showed significantly lower p-gp, MRP1, BCRP, CYP3A4, GST, and hPXR mRNA levels in a 48 h observation. CONCLUSION: The result confirmed resveratrol mediated inhibition of ABC-transporters' overall efflux functions, and its expression, and apoptosis as well as metabolic enzymes GST and CYP3A4 activity.


Assuntos
Apoptose/efeitos dos fármacos , Células CACO-2/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resveratrol/uso terapêutico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Humanos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Resveratrol/farmacologia
14.
Dig Liver Dis ; 51(1): 47-54, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30055963

RESUMO

BACKGROUND: Gliadins are involved in gluten-related disorders and are responsible for the alteration of the cellular redox balance. It is not clear if the gliadin-related oxidative stress can induce DNA damage in enterocytes. AIM: To investigate any possible genotoxicity caused by gliadin and to assess its relationship with oxidative stress in vitro and ex vivo. METHODS: Caco-2 cells were exposed for 6-12-24 h to increasing concentrations (250 µg/mL-1000 µg/mL) of digested gliadin. We investigated: cytotoxicity, oxidative balance (reactive oxygen species, ROS), DNA damage (comet assay and γ-H2AX detection), transglutaminase type 2 (TG2) activity and annexin V expression. H2AX and 8-OHG immunohistochemistry has been evaluated on duodenal biopsies of celiac subjects and controls. RESULTS: Gliadin induced a significant increase (+50%) of ROS after 12 h of exposition starting with a 500 µg/mL dose of gliadin. Comet assay and γ-H2AX demonstrated DNA damage, evident at the gliadin concentration of 500 µg/mL after 24 h. TG2 activity increased in chromatin and cytoskeleton cellular compartments at different gliadin doses (250/500/1000 µg/mL). The γ-H2AX and 8-OHG immunohistochemistry was altered in the duodenal biopsies of celiac patients. CONCLUSIONS: Gliadin induces cellular oxidative stress, DNA damage and pro-apoptotic stimulation in Caco-2 cells and in the duodenal mucosa of celiac patients.


Assuntos
Doença Celíaca/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Gliadina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Apoptose , Western Blotting , Células CACO-2/efeitos dos fármacos , Ensaio Cometa , Enterócitos/efeitos dos fármacos , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade
15.
Future Microbiol ; 13: 1731-1743, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30526068

RESUMO

AIM: The role of mucus-binding protein (MUB) on the adhesion activity and immunomodulatory effect of Lactobacillus acidophilus. MATERIALS & METHODS: The current research mainly focuses on the adhesion and immune function of MUB from L. acidophilus. The structural characteristics and adhesion properties of MUB were analyzed in the intestinal cell models. RESULTS: MUB can promote the aggregation and formation of a membrane-like morphology in L. acidophilus, which could increase the survival rate of L. acidophilus in gastrointestinal tract (GIT). Furthermore, MUB could trigger immune regulation and intestinal protection through the Toll-like receptor 4 (TLR4) signaling pathway and inhibit the activation of mitogen-activated protein kinase (MAPK) signaling pathway. CONCLUSION: MUB of L. acidophilus is an important component involved in bacterial-mucus interactions and immunomodulatory effect in gastrointestinal tract.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Lactobacillus acidophilus/fisiologia , Muco/imunologia , Muco/microbiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Animais , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Células CACO-2/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Humanos , Fatores Imunológicos/farmacologia , Imunomodulação , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Lactobacillus acidophilus/genética , Camundongos , Interações Microbianas/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Probióticos , Conformação Proteica , Células RAW 264.7/efeitos dos fármacos , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
16.
Microb Pathog ; 123: 183-189, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30017942

RESUMO

Colorectal cancer is the third most common cause of cancer-related death in the world which genetic and environmental agents are responsible for cancer. When cells detach from the tumor and invade surrounding tissues, the tumor is malignant and may form secondary tumors at other locations in a process called metastasis. Probiotics are the largest group of inhabitation bacteria in the colon. Gut microbiota has a central role in prevented the risk colon cancer. Probiotics are beneficial microorganisms, like Lactic acid bacteria and Lactobacilli bacteria which are using in the dairy industry. Probiotics nisin are having the most important category of safe usage. In this study LS180, SW48, HT29 and Caco2 was cultured and treated with different dose of nisin. Cell proliferation was assayed with MTT. The expression of CEA, CEAM6 and MMP2F genes was analyzed with Real-time PCR. Protein expression of CEA was evacuated with ELISA. Our result was shown that the 40-50 IU/mL nisin could suppress proliferation of LS180. Cell proliferation of SW48, HT29, Caco2 cells was decreased in 250-350 IU/mL concentration of nisin. The gene expression of CEA, CEAM6, MMP2F was significantly down-regulated with nisin treatment (p < 0.001, p < 0.01). Also, after cells treated with nisin, CEA protein expression was down regulated (p < 0.01). In conclusion, nisin could suppressed metastatic process via down-regulation of CEA, CEAM6, MMP2F, MMP9F genes. We suggested the new treatment strategies beyond Probiotics, which play a role in the prevention local tumor invasion, metastasis and recurrence.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Nisina/farmacologia , Células CACO-2/efeitos dos fármacos , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Células HT29/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Nisina/administração & dosagem , Probióticos/farmacologia , Proteínas/genética , Proteínas/metabolismo
17.
Microb Pathog ; 120: 79-84, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29715536

RESUMO

Listeria monocytogenes expresses various virulence factors enabling the invasion and multiplying in host cells, and together induces cytokines transcription. In order to explore the relationship between virulence factors of L. monocytogenes wild-type EGD-e and cellular response in human colonic epithelial cell line(Caco-2), we constructed mutant strains with in-frame deletions of critical virulence genes of inlA, inlB, hly, actA and virulence regulatory factor prfA from EGD-e, respectively. Compared with EGD-e, mutant strains showed significantly decreased invasion and apoptosis in Caco-2 cells. However, mutant strains were capable to evoke cytokines transcription of interleukin-8 (IL-8), mononuclear chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and CXCL-2 production in Caco-2 cells. Interestingly, EGD-e Δhly-infected Caco-2 cells showed a significant decrease of IL-6, IL-8 and MCP-1 transcription compared with EGD-e at 1 h post-infection. Simultaneously, EGD-e ΔinlB-infected cells showed a decrease in IL-6 transcription, while EGD-e ΔactA-infected cells reflected a decrease in MCP-1 transcription. Virulence genes play a role in inflammatory transcription, but the interaction between pathogenic bacteria and the host cells predominates in inflammatory transcription. Overall, the data showed cellular response of Caco-2 cells infected with EGD-e mutant strains.


Assuntos
Células CACO-2/efeitos dos fármacos , Células CACO-2/metabolismo , Listeria monocytogenes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Virulência/efeitos adversos , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/efeitos adversos , Toxinas Bacterianas/efeitos adversos , Células CACO-2/imunologia , Quimiocina CCL2/metabolismo , Quimiocina CXCL2/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Listeria monocytogenes/patogenicidade , Proteínas de Membrana/efeitos adversos , Fatores de Terminação de Peptídeos/efeitos adversos , Fator de Necrose Tumoral alfa/metabolismo , Virulência/genética , Fatores de Virulência/genética
18.
Phytomedicine ; 42: 112-125, 2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29655677

RESUMO

BACKGROUND: Metabolic syndrome is the cluster of risk factors that leads to increased episodes of cardiovascular disease (CVD). These risk factors include but are not limited to obesity, non-alcoholic fatty liver (NAFLD), dyslipidemia, and type 2 diabetes. Since the pathogenesis of metabolic syndrome has multiple metabolic origins, there is no single treatment for it. Pharmacological approaches consist of separate drugs which target at individual risk factors which pose various side effects. Functional foods or nutraceuticals which have potentially important anti-obesity properties have thus attracted great attention. Schisandrae Fructus is a Chinese herb traditionally used as a liver tonic. Silymarin, an extract of the milk thistle (Silybum marianum), is a dietary supplement that is widely used in western society for the prevention and treatment of liver problems. Crataegus Fructus (hawthorn) is traditionally used to promote digestion and dissipate food stagnation. Momordica charantia (bitter melon) is traditionally used for treatment of diabetes in Ayurvedic Medicine. HYPOTHESIS/PURPOSE: We aimed to develop a multi-targeted herbal formula to target on the multiple risk factors of metabolic syndrome using individual herbs. This proposed herbal formula include sylimarin and Schisandrae Fructus, for NAFLD; Crataegus Fructus for obesity and hyperlipidemia; and Momordica charantia for hyperglycemia. STUDY DESIGN AND METHODS: For in vitro study, we carried out insulin-induced 3T3-L1 adipocytes differentiation and fluorescent tagged cholesterol-treated Caco-2 cell assay to study for adipogenesis and cholesterol uptake into Caco-2 cells, respectively. Oleic acid-induced HepG2 cell assay was used to study for oleic acid-induced fatty liver, and brush border membrane vesicles (BBMV) assay was used to study for glucose uptake from the gut. For in vivo study, we performed an 8-week and a 12-week treatment studies, with each study comprising of 4 groups of C57Bl/6 male mice given: (i) Normal-chow diet; (ii)-(iv) High-fat diet (contains 21% fat and 0.15% cholesterol). After the initial 8 weeks of normal chow or high-fat diet feeding to induce obesity, animals were given: (i) Normal-chow diet; (ii) High-fat diet; (iii) High-fat diet + 2% herbal formula; or (iv) High-fat diet + 4% herbal formula as treatment for another 8 weeks or 12 weeks. RESULTS: Our in vitro results suggested Crataegus Fructus aqueous extract exerted potent inhibitory effects on 3T3-L1 preadipocytes differentiation and cholesterol uptake into Caco-2 cells. Schisandrae Fructus aqueous extract and milk thistle exerted inhibitory effects on oleic acid-induced fatty liver in HepG2 cells. Momordica charantia extract on the other hand, exerted significant inhibitory effect on glucose uptake into BBMV. Our in vivo results showed that our herbal formula exhibited a trend to reduce diet-induced increase in body weight and fat pad mass (epididymal, perirenal and inguinal fat); and significantly reduced diet-induced increase in liver weight, liver lipid, and plasma lipid dose-dependently. Besides, high-fat diet induced a significant reduction in adiponectin level which was significantly improved by herbal formula supplementation at 4%. There was however no significant effect of the herbal formula on diet-induced increase in plasma glucose or insulin levels at either dose. Herbal formula also significantly reduced diet-induced inflammation in the liver at both doses. CONCLUSIONS: Taken together, these data suggested the potential of our novel multi-targeted herbal formula to be used as a therapeutic agent for diet-induced metabolic syndrome, with special emphasis on NAFLD.


Assuntos
Síndrome Metabólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Extratos Vegetais/farmacologia , Células 3T3-L1 , Animais , Fármacos Antiobesidade/farmacologia , Células CACO-2/efeitos dos fármacos , Colesterol/sangue , Colesterol/metabolismo , Crataegus , Dieta Hiperlipídica/efeitos adversos , Humanos , Hiperlipidemias/tratamento farmacológico , Metabolismo dos Lipídeos , Masculino , Síndrome Metabólica/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Cardo-Mariano/química , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Extratos Vegetais/química
19.
Zhonghua Shao Shang Za Zhi ; 34(4): 214-218, 2018 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-29690739

RESUMO

Objective: To investigate the effects of short chain fatty acid (SCFA) on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide (LPS) and the related mechanism. Methods: The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. Cells were divided into control group, LPS group, and SCFA+ LPS group according to the random number table. Cells in control group were only routinely cultured with DMEM medium. Cells in LPS group were cultured with DMEM medium and LPS with final mass concentration of 10 µg/mL. Cells in SCFA+ LPS group were cultured with DMEM medium, LPS and SCFA (consisting of 0.5 mmol/L acetate, 0.01 mmol/L propionate, and 0.01 mmol/L butyrate) with final mass concentration of 10 µg/mL. At post culture hour (PCH) 0, 1, 2, 6, 12, and 24, transepithelial electrical resistance (TER) of cells was determined with an ohmmeter, with sample number of 72. Another portion of cells were divided and treated as above, and then Western blotting was employed to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, and claudin-1 at PCH 24, with sample number of 6. Another portion of cells were divided and treated as above and then immunofluorescence was used to observe cellular morphology and distribution of ZO-1. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) Compared with that in control group, TER of cells in LPS group was significantly reduced from PCH 1 to 24 (P<0.01), while TER of cells in SCFA+ LPS group showed no obvious change (P>0.05). TER of cells in SCFA+ LPS group was significantly higher than that in LPS group from PCH 1 to 24 (P<0.01). (2) Compared with the protein expressions of ZO-1, occludin, and claudin-1 of cells in control group (1.25±0.10, 1.17±0.04, and 1.24±0.20), those of cells in LPS group (0.74±0.23, 0.76±0.11, and 0.77±0.11) at PCH 24 were significantly decreased (P<0.05), while those of cells in SCFA+ LPS group (1.23±0.46, 1.05±0.09, and 1.01±0.13) showed no significant differences (P>0.05). Protein expressions of occludin and claudin-1 of cells in SCFA+ LPS group were significantly higher than those in LPS group at PCH 24 (P<0.05). Protein expression of ZO-1 of cells in SCFA+ LPS group was higher than that in LPS group at PCH 24 with no significant difference (P>0.05). (3) At PCH 24, cells in control group were compact in arrangement with pebble-like appearance, and ZO-1 was distributed smoothly and continuously along the cell membrane. In LPS group, cells were sparse in arrangement with change in appearance, and ZO-1 was distributed uncontinuously along the cell membrane with curls and breaks. In SCFA+ LPS group, the appearance of cells and distribution of ZO-1 were remarkably ameliorated compared with those in LPS group. Conclusions: SCFA can alleviate the barrier disruption of human intestinal epithelial cell induced by LPS through interdicting the abnormal distribution of ZO-1 and decrease of TER and tight junction proteins' expressions.


Assuntos
Células CACO-2/fisiologia , Células Epiteliais/citologia , Ácidos Graxos Voláteis , Mucosa Intestinal/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Junções Íntimas/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo , Western Blotting , Células CACO-2/efeitos dos fármacos , Claudina-1 , Células Epiteliais/efeitos dos fármacos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestinos , Ocludina , Distribuição Aleatória , Junções Íntimas/metabolismo
20.
Food Funct ; 9(4): 2251-2260, 2018 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-29557438

RESUMO

Casein phosphopeptides (CPPs) have been demonstrated to be calcium chelators. Unfortunately, few studies have been reported on the effects of CPPs on the mechanism of the uptake and absorption of Ca2+ and bone metabolism. In this study, a monomeric peptide fraction isolated by RP-HPLC (F6-1) that possessed high calcium transport capacity in Caco-2 cell monolayers was separated and characterized. The effects of F6-1 on the absorption mechanisms of Ca2+ in a Caco-2 monolayer model and bone metabolism in rats were investigated. F6-1 was isolated by preparative and analytical RP-HPLC. Results for calcium transport suggested that the rates of Ca2+ transportation by F6-1 were approximately 2.57, 2.87 and 2.38 times higher than those in the control group at 30, 60 and 120 min, respectively. Results of ultraviolet (UV) spectroscopy indicated that the intensity of UV absorption changed because of the binding of Ca2+ to F6-1. Analysis of transepithelial electrical resistance (TEER) and the expression of TRPV6 in Caco-2 cells showed that F6-1 was likely to influence the transcellular pathway of intestinal absorption of Ca2+ rather than the paracellular pathway. Furthermore, the F6-1 group (1% Ca, 0.03% F6-1) exhibited increases in serum Ca2+ levels, femur length and femur Ca and decreases in serum alkaline phosphatase (ALP) levels and urinary pyridinoline content in a Sprague-Dawley rat model, which implied that F6-1 was beneficial for bone calcification. Overall, our results suggested that F6-1 enhanced the transport of Ca2+ in Caco-2 cells by affecting the transcellular pathway by upregulating the expression of TRPV6. F6-1 also improved bone formation and prevented bone resorption to benefit bone health in rats, which provided a basis for using F6-1 in calcium supplements or functional foods.


Assuntos
Cálcio/metabolismo , Caseínas/farmacologia , Absorção Intestinal/efeitos dos fármacos , Fosfopeptídeos/farmacologia , Animais , Células CACO-2/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Organismos Livres de Patógenos Específicos
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