Grupo: Farmacología de Productos Naturales (CTS164)
https://hdl.handle.net/10481/54496
2024-03-29T11:41:12ZMice carrying an epithelial deletion of the glucocorticoid receptor NR3C1 develop a higher tumor load in experimental colitis associated cancer
https://hdl.handle.net/10481/77920
Mice carrying an epithelial deletion of the glucocorticoid receptor NR3C1 develop a higher tumor load in experimental colitis associated cancer
Arredondo Amador, María; González Pérez, Raquel; Aranda, Carlos J.; Martínez Augustín, María Olga; Sánchez De Medina López-Huertas, Fermín
The glucocorticoid receptor NR3C1 is expressed in multiple cell types in the gut and
elsewhere. Intestinal epithelial cells both produce and respond to glucocorticoids in
different physiological and pathological contexts. In experimental colitis
glucocorticoids have been shown to exert a dual role, dampening inflammation while
producing a deterioration in animal status, including death. Mice with tamoxifen
inducible, intestinal epithelial specific deletion of NR3C1 (NR3C1IEC mice) are
protected against experimental colitis, suggesting glucocorticoid epithelial actions are
deleterious. Since glucocorticoids modulate epithelial proliferation it follows that they
may affect the development of colon cancer. In this study we set out to test this
hypothesis using the dextran sulfate sodium - azoxymethane model of colitis-associated
cancer. KO mice were found to exhibit a 2-fold higher tumor load but similar incidence
and tumor size. Tumors had a higher trend to extend to the submucosal layer (36% vs.
0%) in NR3C1IEC mice, and overexpressed Lgr5, Egfr and Myc, consistent with
increased proliferation and neoplastic transformation. Snai1 and Snai2 were upregulated
specifically in tumors of NR3C1ΔIEC mice, suggesting enhanced epithelial to
mesenchymal transition in the absence of the intestinal epithelial GC receptor. We
conclude that endogenous GC epithelial signaling is involved in colitis associated
cancer.
Leptin-resistant Zucker rats with trinitrobenzene sulfonic acid colitis present a reduced inflammatory response but enhanced epithelial damage
https://hdl.handle.net/10481/76498
Leptin-resistant Zucker rats with trinitrobenzene sulfonic acid colitis present a reduced inflammatory response but enhanced epithelial damage
Rivero Gutiérrez, Belén; Arredondo Amador, María; Gámez Belmonte, María de los Reyes; Sánchez De Medina López-Huertas, Fermín; Martínez Augustín, María Olga
The role of leptin in the development of intestinal inflammation remains controversial, since proinflammatory and anti-inflammatory
effects have been described. This study describes the effect of the absence of leptin signaling in intestinal inflammation.
Experimental colitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to lean and obese Zucker
rats (n = 10). Effects on inflammation and mucosal barrier were studied. Bacterial translocation and LPS concentration were evaluated
together with colonic permeability to 4-kDa FITC-dextran. Obese Zucker rats showed a lower intestinal myeloperoxidase
and alkaline phosphatase activity, reduced alkaline phosphatase sensitivity to levamisole, and diminished colonic expression of
Nos2, Tnf, and Il6, indicating attenuated intestinal inflammation, associated with attenuated STAT3, AKT, and ERK signaling in
the colonic tissue. S100a8 and Cxcl1 mRNA levels were maintained, suggesting that in the absence of leptin signaling neutrophil
activation rather than infiltration is hampered. Despite the lower inflammatory response, leptin resistance enhanced intestinal
permeability, reflecting an increased epithelial damage. This was shown by augmented LPS presence in the portal vein of colitic
obese Zucker rats, associated with induction of tissue nonspecific alkaline phosphatase, LPS-binding protein, and CD14 hepatic
expression (involved in LPS handling). This was linked to decreased ZO-1 immunoreactivity in tight junctions and lower occludin
expression. Our results indicate that obese Zucker rats present an attenuated inflammatory response to TNBS, but increased intestinal epithelial damage allowing the passage of bacterial antigens.
The study was funded the following grants of the Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional FEDER (SAF2011-22922, SAF2011-22812, BFU2014- 57736-P, and AGL2014-58883-R) and Junta de Andalucía (CTS164, CTS235, and CTS6736). B. Rivero-Guti errez, R. Gámez-Belmonte, and M. Arredondo-Amador were supported by fellowships from the Ministerio de Educación.
Epithelial deletion of the glucocorticoid receptor protects the mouse intestine against experimental inflammation
https://hdl.handle.net/10481/76482
Epithelial deletion of the glucocorticoid receptor protects the mouse intestine against experimental inflammation
Sánchez De Medina López-Huertas, Fermín; Martínez Augustín, María Olga; Arredondo Amador, María; Aranda, Carlos J.; Ocón, Borja; González Pérez, Raquel
Intestinal epithelium glucocorticoid receptor knockout mice (NR3C1 IEC) were treated
with dextran sulfate sodium (DSS, 2.5%) to induce colitis. Inflammatory status was
assessed by morphological and biochemical methods and corticoid production was
measured in colonic explants.
Key Results.
After 7 days of DSS NR3C1 mice exhibited lower weight loss and tissue damage,
reduced colonic expression of S100A9, attenuated phosphorylation of STAT3 and a better
overall state compared with WT. Ki67 immunoreactivity was also shifted, indicating an
effect on epithelial proliferation. A subgroup of mice were treated with budesonide and
showed completely prevented budesonide induced weight loss. Epithelial deletion of the
glucocorticoid receptor also protected mice in a protracted colitis protocol. Conversely
knockout mice presented a worse status compared to the control group at 1 day post DSS,
as shown by blood in feces and increased inflammatory parameters. In a separate
experiment colonic corticosterone production was shown to be significantly increased in
knockout mice at 7 days of colitis but not at earlier stages.
Conclusions and Implications.
The intestinal epithelial glucocorticoid receptor has deleterious effects in experimental
colitis induced by dextran sodium sulfate, probably related to inhibition of epithelial
proliferative responses leading to impaired wound healing and reduced endogenous
corticosterone production.
Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting
https://hdl.handle.net/10481/63191
Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting
Rivero Gutiérrez, Belén; Anzola Santander, Andrea; Martínez Augustín, María Olga; Sánchez De Medina López-Huertas, Fermín
It is currently a routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, β-tubulin, among others, in Western blots, as it is the rule in RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control.
The authors want to thank the input of Arnaud Remy and Enrique Orozco of Bio-Rad Laboratories. This study was supported by the Ministry of Science and Innovation (SAF2011-22922 and SAF2011-22812), and by funds from Junta de Andalucia (CTS6736 and CTS164). B. Rivero and A. Anzola were supported by fellowships of the Ministry of Education and Science of Spain and Junta de Andalucia, respectively. CIBERehd is funded by the Instituto de Salud Carlos III.
Exploring the Role of CYP3A4 Mediated Drug Metabolism in the Pharmacological Modulation of Nitric Oxide Production
https://hdl.handle.net/10481/58343
Exploring the Role of CYP3A4 Mediated Drug Metabolism in the Pharmacological Modulation of Nitric Oxide Production
Pérez del Palacio, José; Díaz, Caridad; Vergara, Noemi; Algieri, Francesca; Rodríguez-Nogales, Alba; De Pedro, Nuria; Rodríguez Cabezas, María Elena; Genilloud Rodríguez, Olga; Gálvez Peralta, Julio Juan; Vicente, Francisca
Nitric-oxide synthase, the enzyme responsible for mammalian nitric oxide generation,
and cytochrome P450, the major enzymes involved in drug metabolism, share
striking similarities. Therefore, it makes sense that cytochrome P450 drug mediated
biotransformations might play an important role in the pharmacological modulation
of nitric oxide synthase. In this work, we have undertaken an integrated in vitro
assessment of the hepatic metabolism and nitric oxide modulation of previously
described dual inhibitors (imidazoles and macrolides) of these enzymes in order assess
the implication of CYP450 activities over production of nitric oxide. In vitro systems
based in human liver microsomes and activated mouse macrophages were developed
for these purposes. Additionally in vitro production the hepatic metabolites of dual
inhibitor, roxithromycin, was investigated achieving the identification and isolation of
main hepatic biotransformation products. Our results suggested that for some macrolide
compounds, the cytochrome P450 3A4 derived drug metabolites have an important
effect on nitric oxide production and might critically contribute to the pharmacological
immunomodulatory activity observed.