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1.
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
2.
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 de Medicamentos , Toxinas Marinhas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Furanos/farmacologia , Humanos , Inflamação , Intestinos/citologia , 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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
Pharm Res ; 35(4): 74, 2018 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-29484506

RESUMO

PURPOSE: To identify conditions allowing the use of cell-based models for studies of drug absorption during in vitro lipolysis of lipid-based formulations (LBFs). METHODS: Caco-2 was selected as the cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer. RESULTS: Most undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, I-LC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco-2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion. CONCLUSIONS: Most LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products.


Assuntos
Células CACO-2/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Liberação Controlada de Fármacos , Lipólise , Preparações Farmacêuticas/metabolismo , Administração Oral , Células CACO-2/metabolismo , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/toxicidade , Excipientes/química , Absorção Gastrointestinal , Humanos , Lipase/metabolismo , Lipase/toxicidade , Lipídeos/química , Mucinas/metabolismo , Pancrelipase/metabolismo , Pancrelipase/toxicidade , Proteínas Recombinantes/metabolismo
13.
Expert Opin Drug Deliv ; 15(3): 213-222, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29257904

RESUMO

INTRODUCTION: The oral route is widely accepted as the most physiological path for exogenous administration of insulin, as it closely mimic the endogenous insulin pathway. Thus, in this work it is proposed an innovative lipid-polymeric nanocarrier to delivery insulin orally. Areas covered: Nanoparticles were produced through a modified solvent emulsification-evaporation method, using ethyl palmitate and hydroxypropylmethylcellulose acetate succinate as matrix. Lipid-polymeric nanoparticles were around 300 nm in size, negatively charged (-20 mV) and associated insulin with efficiency higher than 80%. Differential scanning calorimetry suggested thermal stability of nanoparticles. In vitro release assays under simulated gastrointestinal conditions resulted in 9% and 14% of insulin released at pH 1.2 during 2 h and at pH 6.8 for 6 h, respectively, demonstrating the ability of those nanoparticles to protect insulin against premature degradation. Importantly, nanoparticles were observed to be safe at potential therapeutic concentrations as did not originate cytotoxicity to intestinal epithelial cells. Lastly, the permeability of nanoencapsulated insulin through Caco-2 monolayers and a triple Caco-2/HT29-MTX/Raji B cell model correlated well with slow release kinetics, and fosters the effectiveness of nanoparticles to promote intestinal absorption of peptidic drugs. Expert opinion: Lipid-polymeric nanoparticles were developed to encapsulate and carry insulin through intestine. Overall, nanoparticles provide insulin stability and intestinal permeability.


Assuntos
Sistemas de Liberação de Medicamentos , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Metilcelulose/análogos & derivados , Nanopartículas/química , Ácidos Palmíticos/química , Administração Oral , Animais , Células CACO-2/efeitos dos fármacos , Varredura Diferencial de Calorimetria , Cromatografia Líquida de Alta Pressão , Humanos , Concentração de Íons de Hidrogênio , Absorção Intestinal , Metilcelulose/química , Microscopia Eletrônica de Varredura , Permeabilidade , Difração de Raios X
14.
Int J Nanomedicine ; 12: 8671-8680, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29263662

RESUMO

Diabetes mellitus is an incurable metabolic disorder that seriously threatens human health. At present, there is no effective medication available to defeat it. This work intended to develop selenium-coated nanostructured lipid carriers (SeNLCs) for enhancing the oral bioavailability and the curative effect of berberine, an antidiabetic phytomedicine. Berberine-loaded SeNLCs (BB-SeNLCs) were prepared by hot-melt dispersion/homogenization procedure followed by in situ reduction. BB-SeNLCs were characterized by particle size, morphology, entrapment efficiency (EE) and in vitro release. Pharmacokinetics of berberine solution, berberine-loaded NLCs (BB-NLCs) and BB-SeNLCs were studied in Sprague Dawley rats administered by oral gavage. The prepared BB-SeNLCs were around 160 nm in particle size with an EE of 90%. In addition, BB-SeNLCs exhibited a better sustained release of berberine compared to the plain NLCs. After oral administration, BB-SeNLCs greatly enhanced the oral bioavailability of berberine, which was approximately 6.63 times as much as that of berberine solution. The hypoglycemic effect of BB-SeNLCs was also significantly superior to that of BB-NLCs and berberine solution. It turned out that sustained drug release and good intestinal absorption, plus the synergy of selenium, were basically responsible for enhanced oral bioavailability and hypoglycemic effect. Our findings show that SeNLCs are promising nanocarriers for oral delivery of berberine to strengthen the antidiabetic action.


Assuntos
Berberina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Hipoglicemiantes/farmacologia , Nanoestruturas/administração & dosagem , Selênio/administração & dosagem , Administração Oral , Animais , Berberina/farmacocinética , Berberina/farmacologia , Disponibilidade Biológica , Células CACO-2/efeitos dos fármacos , Preparações de Ação Retardada/farmacologia , Portadores de Fármacos/química , Sinergismo Farmacológico , Humanos , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacocinética , Absorção Intestinal/efeitos dos fármacos , Lipídeos/química , Nanoestruturas/química , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Selênio/química , Selênio/farmacologia
15.
Clin Sci (Lond) ; 131(21): 2611-2626, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28954820

RESUMO

OBJECTIVE: We sought to quantify the anti-inflammatory effects of two cannabinoid drugs, cannabidiol (CBD) and palmitoylethanolamide (PEA), in cultured cell lines and compared this effect with experimentally inflamed explant human colonic tissue. These effects were explored in acutely and chronically inflamed colon, using inflammatory bowel disease and appendicitis explants. DESIGN: Caco-2 cells and human colonic explants collected from elective bowel cancer, inflammatory bowel disease (IBD) or acute appendicitis resections, and were treated with the following drug treatments: vehicle, an inflammatory protocol of interferon γ (IFNγ) and tumour necrosis factor α (TNFα; 10 ng/ml), inflammation and PEA (10 µM), inflammation and CBD (10 µM), and PEA or CBD alone, CBD or vehicle were added simultaneously with IFNγ. Nine intracellular signalling phosphoproteins were determined by multiplex. Inflammatory cytokine secretion was determined using ELISA. Receptor mechanisms were investigated using antagonists for CB1, CB2, PPARα, PPARγ, TRPV1 and GPR55. RESULTS: IFNγ and TNFα treatment increased phosphoprotein and cytokine levels in Caco-2 cultures and colonic explants. Phosphoprotein levels were significantly reduced by PEA or CBD in Caco-2 cultures and colonic explants. CBD and PEA prevented increases in cytokine production in explant colon, but not in Caco-2 cells. CBD effects were blocked by the CB2 antagonist AM630 and TRPV1 antagonist SB366791. PEA effects were blocked by the PPARα antagonist GW6471. PEA and CBD were anti-inflammatory in IBD and appendicitis explants. CONCLUSION: PEA and CBD are anti-inflammatory in the human colon. This effect is not seen in cultured epithelial cells. Appropriately sized clinical trials should assess their efficacy.


Assuntos
Amidas/química , Anti-Inflamatórios/farmacologia , Canabidiol/farmacologia , Colo/efeitos dos fármacos , Etanolaminas/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Ácidos Palmíticos/farmacologia , Doença Aguda , Células CACO-2/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
16.
Int J Mol Sci ; 18(5)2017 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-28505081

RESUMO

Rice bran lectins, named as RBA1 and RBA2, were isolated from Oryza sativa in two chromatography steps: affinity chromatography and cation-exchange chromatography. RBA1 was found to be composed of a covalently linked heterodimer of 20- and 12-kDa subunits, and RBA2 was a noncovalently linked dimer of 12-kDa subunits. Both RBA1 and RBA2 bound to desialylated complex glycoproteins such as fetuin, α1-acid glycoprotein, and transferrin, and agalactosylated complex glycoproteins such as agalacto fetuin, agalacto-α1-acid glycoprotein, and agalacto-transferrin, in addition to chitooligosacchrides. RBAs were heat stable up to 80 °C and stable at pH 4-10. RBA1 increased the transport of the fluorescent marker, rhodamine 123, which is known to be transported via the P-glycoprotein-mediated efflux pathway across human intestinal Caco-2 cell monolayers. Furthermore, RBA1 itself was transported to the basolateral side of the monolayers via an endocytotic pathway.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Células CACO-2/efeitos dos fármacos , Carboidratos/química , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Fetuínas/metabolismo , Glicoproteínas/metabolismo , Humanos , Oryza/química , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Transferrina/metabolismo
17.
Int J Nanomedicine ; 12: 3471-3483, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28496323

RESUMO

A simple and reproducible water-in-oil (W/O) nanoemulsion technique for making ultrasmall (<15 nm), monodispersed and water-dispersible nanoparticles (NPs) from chitosan (CS) is reported. The nano-sized (50 nm) water pools of the W/O nanoemulsion serve as "nano-containers and nano-reactors". The entrapped polymer chains of CS inside these "nano-reactors" are covalently cross-linked with the chains of polyethylene glycol (PEG), leading to rigidification and formation of NPs. These NPs possess excessive swelling properties in aqueous medium and preserve integrity in all pH ranges due to chemical cross-linking with PEG. A potent and newly developed cell-penetrating peptide (CPP) is further chemically conjugated to the surface of the NPs, leading to development of a novel peptide-conjugated derivative of CS with profound tight-junction opening properties. The CPP-conjugated NPs can easily be loaded with almost all kinds of proteins, peptides and nucleotides for oral delivery applications. Feasibility of this nanoparticulate system for oral delivery of a model peptide (insulin) is investigated in Caco-2 cell line. The cell culture results for translocation of insulin across the cell monolayer are very promising (15%-19% increase), and animal studies are actively under progress and will be published separately.


Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Quitosana/química , Portadores de Fármacos/química , Nanopartículas/química , Administração Oral , Células CACO-2/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Quitosana/administração & dosagem , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Emulsões/administração & dosagem , Emulsões/química , Excipientes/química , Humanos , Insulina/administração & dosagem , Insulina/química , Nanopartículas/administração & dosagem , Polietilenoglicóis/química , Água/química
18.
J Med Chem ; 60(12): 5193-5208, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28541707

RESUMO

PI3Kδ plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4'-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.


Assuntos
Artrite Experimental/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Relação Estrutura-Atividade , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Células CACO-2/efeitos dos fármacos , Células CACO-2/imunologia , Cães , Canal de Potássio ERG1/metabolismo , Inibidores Enzimáticos/química , Feminino , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Lectinas Tipo C/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Coelhos
19.
FEBS J ; 284(11): 1726-1737, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28391610

RESUMO

Cancer cells require both energy and material to survive and duplicate in a competitive environment. Nutrients, such as amino acids (AAs), are not only a caloric source, but can also modulate cell metabolism and modify hormone homeostasis. Our hypothesis is that the environmental messages provided by AAs rule the dynamics of cancer cell life or death, and the alteration of the balance between essential amino acids (EAAs) and non-essential amino acids (NEAAs) (lower and higher than 50%, respectively) present in nutrients may represent a key instrument to alter environment-dependent messages, thus mastering cancer cells destiny. In this study, two AA mixtures, one exclusively consisting of EAAs and the other consisting of 85% EAAs and 15% NEAAs, were tested to explore their effects on the viability of both normal and cancer cell lines and to clarify the molecular mechanisms involved. Both mixtures exerted a cell-dependent anti-proliferative, cytotoxic effect involving the inhibition of proteasome activity and the consequent activation of autophagy and apoptosis. These results, besides further validating the notion of the peculiar interdependence and extensive crosstalk between the ubiquitin-proteasome system (UPS) and autophagy, indicate that variation in the ratio of EAAs and NEAAs can deeply influence cancer cell survival. Consequently, customization of dietary ratios among EAAs and NEAAs by specific AA mixtures may represent a promising anticancer strategy able to selectively induce death of cancer cells through the induction of apoptosis via both UPS inhibition and autophagy activation.


Assuntos
Aminoácidos Essenciais/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Células HCT116/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Mama/citologia , Células CACO-2/efeitos dos fármacos , Células CACO-2/enzimologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Feminino , Células HCT116/enzimologia , Células HeLa/enzimologia , Células Hep G2/efeitos dos fármacos , Células Hep G2/enzimologia , Humanos
20.
J Crohns Colitis ; 11(5): 593-602, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28453766

RESUMO

Background and Aims: CT-P13 is the first biosimilar monoclonal antibody to infliximab, and was recently approved in the European Union, Japan, Korea, and USA for all six indications of infliximab. However, studies directly assessing the biologic activity of CT-P13 versus inflximab in the context of inflammatory bowel disease [IBD] are still scanty. In the present study, we aimed to compare the biological activities of CT-P13 and infliximab with specific focus on intestinal cells so as to gain insight into the potential biosimilarity of these two agents for treatment of IBD. Methods: CT-P13 and infliximab were investigated and compared by in vitro experiments for their neutralisation ability of soluble tumour necrosis factor alpha [sTNFα] and membrane-bound tumour necrosis factor alpha [mTNFα], suppression of cytokine release by reverse signalling, induction of regulatory macrophages and wound healing, and antibody-dependent cell cytotoxicity [ADCC]. Results: CT-P13 showed similar biological activities to infliximab as gauged by neutralisation of soluble TNFα, as well as blockade of apoptosis and suppression of pro-inflammatory cytokines in intestinal Caco-2 cells. Infliximab and CT-P13 equally induced apoptosis and outside-to-inside signals through transmembrane TNFα [tmTNFα]. Moreover, regulatory macrophage induction and ensuing wound healing were similarly exerted by CT-P13 and infliximab. However, neither CT-P13 nor infliximab exerted any significant ADCC of ex vivo-stimulated peripheral blood monocytes or lamina propria mononuclear cells from IBD patients. Conclusions: These findings indicate that CT-P13 and infliximab exert highly similar biological activities in intestinal cells, and further support a mechanistic comparability of these two drugs in the treatment of IBD.


Assuntos
Anticorpos Monoclonais/farmacologia , Medicamentos Biossimilares/farmacologia , Fármacos Gastrointestinais/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab/farmacologia , Intestinos/efeitos dos fármacos , Anticorpos Monoclonais/uso terapêutico , Medicamentos Biossimilares/uso terapêutico , Células CACO-2/efeitos dos fármacos , Citocinas/metabolismo , Fármacos Gastrointestinais/uso terapêutico , Humanos , Técnicas In Vitro , Infliximab/uso terapêutico , Intestinos/citologia , Intestinos/imunologia , Macrófagos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Cicatrização/efeitos dos fármacos
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