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1.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 52(2): 202-206, 2021 Mar.
Artículo en Chino | MEDLINE | ID: mdl-33829692

RESUMEN

Objective: To examine the infectivity of human adenovirus type 55 (HAdV-55) in human intestinal cells. Methods: Caco-2 cells were cultured in vitro, and infected with HAdV-3, 7, 14 and 55. The expression of viral proteins in infected cells was detected with immunofluorescence method. The intracellular and supernatant viral DNA levels were determined with fluorescent quantitative PCR at different points of time. The level of infectious virus particles in the supernatant of Caco-2 cells was determined with adenovirus sensitive HEp-2 infection assay. Results: Immunofluorescence assay showed positive result for the expression of HAdV-55 virus protein in Caco-2 cells 48 h post infection. HAdV-3, 7, 14, and 55 showed sustained replication and proliferation in Caco-2 cells. The level of viral DNA in infected cells and the supernatant increased with the infection time, and the viral DNA level of HAdV-55 was significantly higher than those of HAdV-3, 7 and 14. The infectious virus particles of HAdV-55 in Caco-2 supernatant were more than those of HAdV-3, 7 and 14, showing statistically significant difference ( P<0.05). Caco-2 cells were infected with low doses of virus (1×TCID 50), and the cytopathic effect (CPE) of HAdV-55 infection wells was more significant than that of HAdV-3, 7 and 14 infection wells. Conclusion: This study found that human intestinal cells were susceptible to HAdV-55, and the infection level was higher than that of other common respiratory infections caused by adenovirus types 3, 7 and 14.


Asunto(s)
Adenovirus Humanos , Adenoviridae/genética , Adenovirus Humanos/genética , Células CACO-2 , ADN Viral , Humanos , Replicación Viral
2.
Zhongguo Zhong Yao Za Zhi ; 46(5): 1120-1127, 2021 Mar.
Artículo en Chino | MEDLINE | ID: mdl-33787105

RESUMEN

To evaluate the effects of Hydroxypropyl methylcellulose acetate succinate(HPMCAS MF) on absorption of silybin(SLB) from supersaturable self-nanoemulsifying drug delivery system which was pre-prepared at the early stage experiment. The cell toxicity of self-emulsifying preparation was evaluated by the MTT method, and the in vitro membrane permeability and absorption promoting effect of the self-emulsifying preparation were evaluated by establishing a Caco-2 cell monolayer model. The in vivo and in vitro supersaturation correlation was evaluated via the blood concentration of SLB. The results of MTT showed that the concentration of the preparation below 2 mg·mL~(-1)(C_(SLB) 100 µg·mL~(-1)) was not toxic to Caco-2 cells, and the addition of polymer had no significant effect on Caco-2 cells viability. As compared with the solution group, the transport results showed that the P_(app)(AP→BL) of the self-emulsifying preparation had a very significant increase; the transport rate of silybin can be reduced by polymer in 0-30 min; however, there was no difference in supersaturated transport between supersaturated SLB self-nanoemulsion drug delivery system(SLB-SSNEDDS) and SLB self-nanoemulsion drug delivery system(SLB-SNEDDS) within 2 hours. As compared with SLB suspension, pharmacokinetic parameters showed that the blood concentration of both SLB-SNEDDS and SLB-SSNEDDS groups were significantly increased, and C_(max) was 5.25 times and 9.69 times respectively of that in SLB suspension group, with a relative bioavailability of 578.45% and 1 139.44% respectively. C_(max) and relative bioavailability of SLB-SSNEDDS were 1.85 times and 197% of those of SLB-SNEDDS, respectively. Therefore, on the one hand, SSNEDDS can increase the solubility of SLB in gastrointestinal tract by maintaining stability of SLB supersaturation state; on the other hand, the osmotic transport process of SLB was regulated through the composition of its preparations, and both of them could jointly promote the transport and absorption of SLB to improve the oral bioavailability of SLB.


Asunto(s)
Sistemas de Liberación de Medicamentos , Nanopartículas , Administración Oral , Disponibilidad Biológica , Células CACO-2 , Emulsiones , Humanos , Metilcelulosa/análogos & derivados , Tamaño de la Partícula , Silibina , Solubilidad
3.
Food Chem ; 351: 129315, 2021 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-33647686

RESUMEN

Mangiferin-loaded nanobilosomes (MGF-NBSs) were developed using microfluidic-based techniques to improve aqueous solubility, digestive stability, and cellular antioxidant activity (CAA) of mangiferin. Preliminary experiments showed that optimal formation conditions were 5:1 aqueous (water) to solvent (ethanol) phase ratio and 85 mL/min total flow rate. Further optimization using response surface methodology provided the optimal formulation (200 mg encapsulant consisting of 90.91% phosphatidylcholine and 9.09% sodium glycocholate, and 25.89 mg mangiferin), achieving 9.25% mangiferin loading and 80.65% encapsulation efficiency. Mono-dispersed MGF-NBSs with an average size of around 48.14 nm and zeta potential of -30.1 mV were obtained. FTIR and DSC results confirmed the successful encapsulation of mangiferin into the nanobilosomes and revealed interactions among the components. MGF-NBSs showed a 7-fold increase in the aqueous solubility compared with non-encapsulated mangiferin. CAA of MGF-NBSs in Caco-2 cells was 2 times higher than that of mangiferin and the in vitro digestive stability was improved.


Asunto(s)
Antioxidantes/química , Antioxidantes/farmacología , Nanoestructuras/química , Agua/química , Xantonas/química , Xantonas/farmacología , Antioxidantes/metabolismo , Células CACO-2 , Digestión , Composición de Medicamentos , Humanos , Dispositivos Laboratorio en un Chip , Tamaño de la Partícula , Solubilidad , Xantonas/metabolismo
4.
Nat Commun ; 12(1): 1799, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741965

RESUMEN

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.


Asunto(s)
Neisseria gonorrhoeae/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Ribosomas/efectos de los fármacos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Células CACO-2 , Femenino , Gonorrea/microbiología , Gonorrea/prevención & control , Humanos , Ratones , Neisseria gonorrhoeae/genética , Biosíntesis de Proteínas/genética , Inhibidores de la Síntesis de la Proteína/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo
5.
Sheng Li Xue Bao ; 73(1): 42-50, 2021 Feb 25.
Artículo en Chino | MEDLINE | ID: mdl-33665659

RESUMEN

This study was designed to evaluate the role of short-chain fatty acid butyrate acid on intestinal morphology and function, and atherosclerotic plaque formation in apolipoprotein E-knockout (ApoE-/-) mice. ApoE-/- mice on high-fat, high-cholesterol diet were treated with butyrate acid (200 mmol/L) or NaCl (control) in the drinking water for 12 weeks, followed by histological evaluations of atherosclerotic lesion in aorta. Real-time PCR analysis and ELISA were used to measure the expression levels of proinflammatory cytokines. Butyrate acid significantly attenuated high-fat, high-cholesterol diet-induced atherosclerotic plaque formation in ApoE-/- mice. Butyrate acid prevented high-fat, high-cholesterol diet-induced inflammation in both the aorta and the circulation, as evidenced by reduced expression of proinflammatory cytokines. These changes were accompanied by a marked attenuation in metabolic endotoxemia lipopolysaccharide (LPS). Butyrate acid induced intestinal expression of the tight junction proteins (Occludin and zona occuldens protein-1), thereby preventing the gut permeability. Butyrate acid dose-dependently upregulated the expression of the tight junction proteins in Caco-2 cells in GPR41-dependent manner. In conclusion, butyrate acid attenuates atherosclerotic lesions by ameliorating metabolic endotoxemia-induced inflammation through restoration of the gut barrier.


Asunto(s)
Aterosclerosis , Placa Aterosclerótica , Animales , Apolipoproteínas E/genética , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/prevención & control , Butiratos/farmacología , Células CACO-2 , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos Volátiles , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
6.
Zhongguo Zhong Yao Za Zhi ; 46(4): 845-854, 2021 Feb.
Artículo en Chino | MEDLINE | ID: mdl-33645089

RESUMEN

Network pharmacology and liver fibrosis(LF) model in vitro were used to analyze the underly mechanism of anti-liver fibrosis effect that induced by Piperis Longi Fructus and its major active compounds. TCMSP and TCMIP were used to search for the chemical constituents of Piperis Longi Fructus, as well as the oral bioavailability(OB), drug-likeness(DL), intercellular permeability of intestinal epithelial cells(Caco-2) and Drug-likeness grading were set as limiting conditions. The related target genes of Piperis Longi Fructus were queried by TCMSP database, while related targets of LF were screened by GeneCards databases. Interaction network was constructed using Cytoscape 3.7.1. These above data were imported into STRING database for PPI network analysis. Enrichment of gene ontology(GO) and pathway analysis(KEGG) within Bioconductor database were utilized to note functions of related targets of Piperis Longi Fructus. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The effects of piperlongumine(PL), the major active component of Piperis Longi Fructus, on proliferation of rat liver stellate cells(HSC-T6) and expression of α smooth muscle actin(α-SMA) and collagen Ⅰ were investigated. The major factors TNF-α of tumor necrosis factor(TNF) pathway and NF-κB p65, IL-6 protein expressions of LF process were examined. A total of 12 active compounds such as PL were obtained by analyzing the bioavailability and drug-like properties, which inferred to 48 targets. The functional enrichment analysis of GO obtained 1 240 GO items, mainly involving in process of biology and molecular function. A total of 99 signaling pathways were enriched in the KEGG pathway enrichment analysis, including TNF signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathways. CCK-8 assay showed that PL inhibited proliferation of HSC-T6 induced by transforming growth factor-ß1(TGF-ß1). Western blot analysis found that treated with PL suppressed the protein expressions of α-SMA, collagen Ⅰ, TNF-α and p65 in HSC-T6. Enzyme linked immunosorbent assay(ELISA) showed that PL inhibited the expressions of TNF-α and IL-6 in the cluture supertant of HSC-T6 cells. In conclusion, PL could play an anti-liver fibrosis role by regulating TNF/NF-κB signaling pathway. This study provided the mechanism basis of anti-LF effects induced by Piperis Longi Fructus and its major active compounds, which might help for the further study of the mechanism and key targets of Piperis Longi Fructus.


Asunto(s)
Células Estrelladas Hepáticas , Cirrosis Hepática , Animales , Células CACO-2 , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/genética , FN-kappa B/metabolismo , Ratas , Transducción de Señal
7.
ACS Infect Dis ; 7(3): 586-597, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33645977

RESUMEN

As the COVID-19 pandemic continues to unfold, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII, and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. In this study, we further characterized the mechanism of action of these four compounds using the SARS-CoV-2 pseudovirus neutralization assay. It was found that GC-376 and calpain inhibitors II and XII have a dual mechanism of action by inhibiting both viral Mpro and host cathepsin L in Vero cells. To rule out the cell-type dependent effect, the antiviral activity of these four compounds against SARS-CoV-2 was also confirmed in type 2 transmembrane serine protease-expressing Caco-2 cells using the viral yield reduction assay. In addition, we found that these four compounds have broad-spectrum antiviral activity in inhibiting not only SARS-CoV-2 but also SARS-CoV, and MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift-binding assay and enzymatic fluorescence resonance energy transfer assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 might be promising starting points for further development against existing human coronaviruses as well as future emerging CoVs.


Asunto(s)
Antivirales/farmacología , Carbonatos/farmacología , Glicoproteínas/farmacología , Leucina/farmacología , Oligopéptidos/farmacología , Prolina/análogos & derivados , Ácidos Sulfónicos/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Células CACO-2 , Catepsina L/antagonistas & inhibidores , Línea Celular , Chlorocebus aethiops , Coronavirus Humano 229E/efectos de los fármacos , Coronavirus Humano NL63/efectos de los fármacos , Coronavirus Humano OC43/efectos de los fármacos , Combinación de Medicamentos , Células HEK293 , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Prolina/farmacología , Serina Endopeptidasas/metabolismo , Células Vero
8.
Carbohydr Polym ; 260: 117832, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33712170

RESUMEN

Vagina atrophy is a common symptom in women after menopause owing to decreasing estrogen levels. The most conventional treatment for this condition is estrogen cream. The shortcoming is its weak adhesion to the vagina mucus, thus requiring frequent daily application. In this study, BDDE was selected to crosslink and graft chitosan with thioglycolic acid, to form thiolated chitosan (CT) and improve the mucoadhesive properties of chitosan. Genistein was selected as the bioactive molecule that could exhibit estrogen-like properties for long-term treatment of vaginal atrophy. The efficacies of the materials were characterized and evaluated both in vitro and in vivo. Results showed that the mucoadhesive property of CT was approximately two-fold stronger against the constant flow than unmodified chitosan. CT with genistein (CT-G) was administered intravaginally every three days in vivo. It showed that the developed CT-G recover 54 % of the epithelium thickness of an atrophic vagina and ease vaginal atrophy.


Asunto(s)
Vaginitis Atrófica/tratamiento farmacológico , Quitosano/química , Genisteína/uso terapéutico , Hidrogeles/química , Tioglicolatos/química , Animales , Vaginitis Atrófica/patología , Células CACO-2 , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Femenino , Genisteína/química , Genisteína/metabolismo , Genisteína/farmacología , Humanos , Hidrogeles/síntesis química , Hidrogeles/farmacología , Ratones , Ratas , Ratas Sprague-Dawley , Vagina/patología
9.
Nat Commun ; 12(1): 1726, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741941

RESUMEN

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.


Asunto(s)
/tratamiento farmacológico , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , alfa 1-Antitripsina/farmacología , Anticuerpos Antivirales/sangre , Antivirales/farmacología , Células CACO-2 , Humanos , Inmunoglobulina G/sangre , Simulación del Acoplamiento Molecular , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos
10.
Environ Pollut ; 274: 116524, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33548667

RESUMEN

Increasing attention has been brought to microplastics pollution recently, while emerging evidences indicate that nano-plastics degraded from microplastics are more of research significance owing to stronger toxicity. However, there is little study focused on the prevention of nano-plastics induced toxicity until now. Canidin-3-glucoside (C3G), a natural anthocyanin proved to possess multiple functions like antioxidant and intestinal tissue protection. Thus, we proposed whether C3G could act as a molecular weapon against nano-plastics induced toxicity. In Caco2 cell and Caenorhabditis elegans (C. elegans) models, we found that polystyrene (PS) nano-plastics exposure resulted in physiological toxicity and oxidative damage, which could be restored by C3G. More significantly in Caco2 cells, we observed that autophagy was activated via Sirt1-Foxo1 signaling pathway to attenuate PS induced toxicity after C3G intervention and further verified by adding autophagy inhibitor 3-Methyladenine (3-MA). Meanwhile, PS co-localization with lysosomes was observed, indicating the encapsulation and degradation of PS. In C. elegans, by detecting LGG-1/LC3 expression in GFP-targeted LGG-1 report gene (LGG-1:GFP) labeled transgenic DA2123 strain, the co-localization of LGG-1:GFP with PS was found as well, means that autophagy is involved in C3G's beneficial effects. Furthermore, we were surprised to find that C3G could promote the discharge of PS from N2 nematodes, which reduces PS toxicity more directly.


Asunto(s)
Caenorhabditis elegans , Plásticos , Animales , Autofagia , Células CACO-2 , Glucósidos/toxicidad , Humanos , Microplásticos
11.
Biochem Biophys Res Commun ; 545: 203-207, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33571909

RESUMEN

The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In this study, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative agent of COVID-19, in cell culture. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is worth to be further investigated as an antiviral drug candidate for COVID-19.


Asunto(s)
Antivirales/farmacología , Ácidos Levulínicos/farmacología , Animales , Antivirales/administración & dosificación , /virología , Células CACO-2 , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Compuestos Ferrosos/farmacología , Humanos , Ácidos Levulínicos/administración & dosificación , Células Vero
12.
J Vis Exp ; (167)2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33554962

RESUMEN

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.


Asunto(s)
Neoplasias del Colon/patología , Células Madre Neoplásicas/patología , Esferoides Celulares/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Enterocitos/efectos de los fármacos , Enterocitos/patología , Técnica del Anticuerpo Fluorescente , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Coloración y Etiquetado
13.
Gut Microbes ; 13(1): 1-9, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33550892

RESUMEN

Microbiota-derived molecules called short-chain fatty acids (SCFAs) play a key role in the maintenance of the intestinal barrier and regulation of immune response during infectious conditions. Recent reports indicate that SARS-CoV-2 infection changes microbiota and SCFAs production. However, the relevance of this effect is unknown. In this study, we used human intestinal biopsies and intestinal epithelial cells to investigate the impact of SCFAs in the infection by SARS-CoV-2. SCFAs did not change the entry or replication of SARS-CoV-2 in intestinal cells. These metabolites had no effect on intestinal cells' permeability and presented only minor effects on the production of anti-viral and inflammatory mediators. Together our findings indicate that the changes in microbiota composition of patients with COVID-19 and, particularly, of SCFAs do not interfere with the SARS-CoV-2 infection in the intestine.


Asunto(s)
/virología , Ácidos Grasos Volátiles/metabolismo , Microbioma Gastrointestinal , Mucosa Intestinal/virología , Adulto , Anciano , Células CACO-2 , Colon/virología , Células Epiteliales/virología , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , /fisiología , Carga Viral , Internalización del Virus , Adulto Joven
14.
Sci Data ; 8(1): 70, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33637768

RESUMEN

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic, in which acute respiratory infections are associated with high socio-economic burden. We applied high-content screening to a well-defined collection of 5632 compounds including 3488 that have undergone previous clinical investigations across 600 indications. The compounds were screened by microscopy for their ability to inhibit SARS-CoV-2 cytopathicity in the human epithelial colorectal adenocarcinoma cell line, Caco-2. The primary screen identified 258 hits that inhibited cytopathicity by more than 75%, most of which were not previously known to be active against SARS-CoV-2 in vitro. These compounds were tested in an eight-point dose response screen using the same image-based cytopathicity readout. For the 67 most active molecules, cytotoxicity data were generated to confirm activity against SARS-CoV-2. We verified the ability of known inhibitors camostat, nafamostat, lopinavir, mefloquine, papaverine and cetylpyridinium to reduce the cytopathic effects of SARS-CoV-2, providing confidence in the validity of the assay. The high-content screening data are suitable for reanalysis across numerous drug classes and indications and may yield additional insights into SARS-CoV-2 mechanisms and potential therapeutic strategies.


Asunto(s)
Antivirales/farmacología , Reposicionamiento de Medicamentos , /efectos de los fármacos , Células CACO-2 , Cetilpiridinio , Evaluación Preclínica de Medicamentos , Ésteres , Guanidinas , Humanos , Lopinavir , Mefloquina , Papaverina
15.
Methods Mol Biol ; 2273: 279-296, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604861

RESUMEN

In vitro epithelial models are valuable tools for both academic and industrial laboratories to investigate tissue physiology and disease. Epithelial tissues comprise the surface epithelium, basement membrane, and underlying supporting stromal cells. There are various types of epithelial tissue and they have a diverse and intricate architecture in vivo, which cannot be successfully recapitulated using two-dimensional (2D) cell culture. Tissue engineering strategies can be applied to bioengineer the organized, multilayered, and multicellular structure of epithelial tissues in vitro. Alvetex® is a porous, polystyrene scaffold that enables fibroblasts to synthesize a complex network of endogenous, humanized extracellular matrix proteins. This creates a physiologically relevant three-dimensional (3D) subepithelial microenvironment, enriched with mechanical and chemical cues, which supports the organization and differentiation of epithelial cells. Such technology has been used to bioengineer different epithelial architectures in vitro, including the simple, columnar structure of the intestine and the stratified, squamous, and keratinized structure of skin. Epithelial tissue models provide a useful platform for fundamental and translational research, with multifaceted applications including disease modeling, drug discovery, and product development.


Asunto(s)
Células Epiteliales/citología , Poliestirenos/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Células CACO-2 , Línea Celular , Fibroblastos/citología , Humanos , Queratinocitos/citología , Porosidad , Piel/citología
16.
Mol Carcinog ; 60(3): 188-200, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33544929

RESUMEN

Interaction between a tumor and its microenvironment is important for tumor initiation and progression. Cancer stem cells (CSCs) within the tumor interact with a microenvironmental niche that controls their maintenance and differentiation. We investigated the CSC-promoting effect of factors released from myofibroblasts into the microenvironment of early colorectal cancer tumors and its molecular mechanism. By messenger RNA microarray analysis, expression of HES1, a Notch signaling target, significantly increased in Caco-2 cells cocultured with 18Co cells (pericryptal myofibroblasts), compared to its expression in Caco-2 cells cultured alone. Caco-2 cells cultured in 18Co-conditioned media (CM) showed a significant increase in CD133+CD44+ cells and HES1 expression compared to that in Caco-2 cells cultured in regular media. Significant amounts of interleukin-6 (IL-6) and IL-8 were detected in 18Co-CM compared to levels in regular media. The 18Co-CM-induced increase in CD133+CD44+ cells was attenuated by IL-6- and IL-8-neutralizing antibodies. Furthermore, these neutralizing antibodies and inhibitors of STAT3 and gamma-secretase reduced the expression of HES1 induced in Caco-2 cells cultured in 18Co-CM. Immunohistochemical analysis of human tissues revealed that IL-6, IL-8, and HES1 expression increased from normal to adenoma, and from adenoma to cancer tissues. In addition, IL-6 and HES1 expression was positively correlated in early colorectal cancer tissues. In conclusion, the increase of CSCs by myofibroblasts could be mediated by IL-6/IL-8-induced HES1 activation in the tumor microenvironment. Based on these data, the IL-6/IL-8-mediated Notch/HES1 and STAT3 pathway, through which CSCs interact with their microenvironment, might be a potential target for the prevention and treatment of colorectal tumors.


Asunto(s)
Neoplasias Colorrectales/patología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Células Madre Neoplásicas/patología , Factor de Transcripción HES-1/metabolismo , Células CACO-2 , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Medios de Cultivo Condicionados/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Organoides/patología , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción HES-1/genética , Microambiente Tumoral/efectos de los fármacos
17.
Drug Dev Ind Pharm ; 47(2): 259-267, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33501858

RESUMEN

OBJECTIVE: Bromotetrandrine (W198) was reported as a P-glycoprotein (P-gp) inhibitor. We aimed to prepare oral W198 micelles following by paclitaxel (PTX) micelles (W198/PTX micelles) to improve the clinical application of PTX. SIGNIFICANCE: The poor water solubility, intestinal permeability, and multidrug resistance (MDR) of PTX can be improved in the multistage oral delivery system. METHODS: The novel W198/PTX oral micelles were developed by water-bath ultrasound method and were evaluated in vivo and in vitro in 4T1 orthotopic tumor-bearing mice model. RESULTS: PTX micelles and W198 micelles were prepared to be round and uniform. W198 micelles pre-administrated group showed higher cellular uptake efficiency of PTX on Caco-2 cells and more prominent cytotoxicity compared with W198-untreated group on 4T1 cells. The oral bioavailability of W198/PTX micelles group was nearly 5.7-folds higher than the PTX micelles only group. In addition, W198/PTX micelles showed enhanced anticancer efficacy. CONCLUSIONS: We established a multistage oral delivery system to improve oral bioavailability and anticancer efficacy of PTX.


Asunto(s)
Antineoplásicos Fitogénicos , Células CACO-2/química , Paclitaxel , Administración Oral , Animales , Disponibilidad Biológica , Línea Celular Tumoral , Portadores de Fármacos , Humanos , Ratones , Micelas
18.
J Med Chem ; 64(3): 1593-1610, 2021 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-33470812

RESUMEN

PEGylation of therapeutic agents is known to improve the pharmacokinetic behavior of macromolecular drugs and nanoparticles. In this work, we performed the conjugation of polyethylene glycols (220-5000 Da) to a series of non-steroidal small agonists of the bile acids receptor TGR5. A suitable anchoring position on the agonist was identified to retain full agonistic potency with the conjugates. We describe herein an extensive structure-properties relationships study allowing us to finely describe the non-linear effects of the PEG length on the physicochemical as well as the in vitro and in vivo pharmacokinetic properties of these compounds. When appending a PEG of suitable length to the TGR5 pharmacophore, we were able to identify either systemic or gut lumen-restricted TGR5 agonists.


Asunto(s)
Hipoglucemiantes/síntesis química , Hipoglucemiantes/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Barrera Hematoencefálica/metabolismo , Células CACO-2 , Células HEK293 , Humanos , Hipoglucemiantes/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Polietilenglicoles/química , Receptores Acoplados a Proteínas G/química , Relación Estructura-Actividad
19.
New Microbiol ; 44(1): 42-50, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33453704

RESUMEN

This work aims to assess the in vitro adhesion of two type strains of Lactobacillus plantarum (ATCC 14917 and ATCC BAA-793) (now Lactiplantibacillus plantarum). The experiments were conducted both in vitro on colon cells lines (Caco-2 and HT-29) and in vivo by adopting Galleria mellonella, a well-known alternative preclinical model. Data comparison obtained from in vitro and in vivo assays showed that adhesion performance is comparable in both models. Moreover, the type strain BAA-793, originally isolated from human saliva, showed enhanced adhesion performance, either in vitro to the low mucus-producing cell line (HT-29) or in vivo into the G mellonella gut. These results suggest a possible adaptation of this strain to its ecological niche compared to ATCC 14917. This preliminary pilot study, once again, showed the reliability of G. mellonella oral administration model as a first-line screening tool for in vitro to in vivo translation. Also, for the first time, the permanence of Lactobacillus strains into G. mellonella gut has been reported, reinforcing the claim that this preclinical model can be used, together with standardised in vitro and in vivo procedures already accepted across the scientific community, for the evaluation and investigation of new potential probiotic strains.


Asunto(s)
Lactobacillus plantarum , Lepidópteros , Probióticos , Administración Oral , Animales , Adhesión Bacteriana , Células CACO-2 , Humanos , Proyectos Piloto , Reproducibilidad de los Resultados
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