Étude des protéines de jonctions serrées au cours de l'inflammation intestinale (impact des acides aminés)

Intestinal barrier is regulated in part by tight junctions (Ds) which are multiprotein structures. Recent studies have suggested that an increased intestinal permeability is involved in the pathophysiology of several intestinal bowel diseases. We aimed to study underlying mechanisms involved in the regulation of three tight junction (Lis) proteins, claudin-1, occludin and ZO-1 in colonic biopsies of IBS patients and in experimental models of Methotrexate (MTX)-induced intestinal mucositis. In addition, we also evaluated the effects of amino acids on MTX-treated models. In colonic mucosa of patients with IBS, we have shown alterations of claudin-1, occludin II proteins in IBS patients with differences according to the IBS subsets and symptoms, specifically, in IBS patients with predominant diarrhea. This suggested that these changes may be involved in the pathophysiology of IBS. In a rodent model of mucositis, we observed that the increase of intestinal permeability induced by MTX, related in part to alteration of T.'s, ZO-1, occludin and claudin-1 proteins expression and cellular distribution. The in vitro data suggest that not only NF-KB, but also MEK1 &2 and INK pathways are involved in the gut barrier disruption induced by MTX. Glutamine supplementation limited the increase of chemotherapy-induced intestinal permeability and restored the expression of proteins of tight junctions probably via the erk and NF-x_13 pathways. Combined glutamine and arginine seemed associated with a stronger mortality. Further studies are needed to confirm the preventive andlor therapeutic interest of the glutamine.ROUEN-BU Médecine-Pharmacie (765402102) / SudocSudocFranceF

Correlation analysis of TLR2 and TLR4 and duration of symptoms according to the subtype of IBS.

<p>r_s and p values for Spearman correlation between TLR2 and TLR4 mRNA expression and duration of symptoms.</p

Expression levels of TLR2 and TLR4 in the colonic mucosa of controls and IBS patients.

<p><i>(</i><b><i>A</i></b><i>)</i> Correlation between TLR2 and TLR4 mRNA expression in the whole group of IBS patients (r_s = 0.78, p<0.0001). <i>(</i><b><i>B</i></b><i>)</i> Changes in mRNA expression levels of TLR2 and <i>(</i><b><i>C</i></b><i>)</i> TLR4 in controls and in patients with predominant diarrhea (IBS-D), or constipation (IBS-C) or mixed (IBS-M) assessed by real-time PCR. Values are expressed as mean ± SEM. * Represents <i>p<0.05</i> using Mann-Whitney test.</p

Correlation analysis of TLR2 and TLR4 mRNA levels and symptoms.

<p>Correlation between TLR2 <i>(</i><b><i>A</i></b><i>)</i> and TLR4 <i>(</i><b><i>B</i></b><i>)</i> expressions in the colonic mucosa and the duration of symptoms of IBS patients (n = 46, r_s = 0.34, p = 0.02 for TLR2 and r_s = 0.37, p = 0.01 for TLR4). Data were correlated by using non-parametric Spearman test.</p

TLR2, TLR4 and CD14 expression in IECs (Epcam+ cells).

<p>Epithelials cells obtained from the colonic mucosa were assessed by flow cytometry as described in Patients and Methods. Mucosal colonic cells were double-stained with isotype IgG control or mAbs to TLR2, TLR4 or CD14 together with APC-conjugated anti Epcam mAbs. <i>(</i><b><i>A</i></b><i>)</i> Representative histogram showing the fluorescence intensity of surface TLR4 expression in gated Epcam+ cells from all IBS subtypes and controls patients. Changes in TLR4 surface expression <i>(</i><b><i>B</i></b><i>)</i>, intracellular TLR4 expression <i>(</i><b><i>C</i></b><i>)</i>, CD14 expression <i>(</i><b><i>D</i></b><i>)</i> and TLR2 surface expression <i>(</i><b><i>E</i></b><i>)</i> in IECs in control and IBS subtypes. Values are expressed as mean intensities fluorescence ± SEM. (*) Represents <i>p<0.05</i> and (**) represents p<0.005 using Mann-Whitney test. Kruskal-Wallis p values are *p = 0.01 (B); p = 0.5 (C); p = 0.95 (D) and *p = 0.03 (E) respectively.</p

Levels of pro-inflammatory cytokines and chemokines in colonic mucosa.

<p>Cytokines were measured by multiplex cytokine bead immunoassays as described previously in Patients and Methods. Protein extracts of colonic mucosa were evaluated in duplicate for the expression of IL-8 <i>(</i><b><i>A</i></b><i>)</i>, IL1β <i>(</i><b><i>B</i></b><i>)</i>, TNF-α <i>(</i><b><i>C</i></b><i>)</i>, IL-6 <i>(</i><b><i>D</i></b><i>)</i> and IFN γ <i>(</i><b><i>E</i></b><i>)</i> in IBS patients and controls. Results were expressed as pg/mg protein. Differences in protein secretion were determined to be statistically significant by Mann-Whitney test with a value of p<0.05 (*), p<0.005 (**) and p<0.0005 (***).</p

TLR2 and TLR4 expression in the colonia mucosa of control patients according to the gender and age.

<p>Values are medians (range).</p

Immunofluorescence microscopy analysis of TLR2 and TLR4 in the colonic mucosa of IBS subgroups.

<p>Representative photomicrographs show the distribution of TLR2 and TLR4 proteins in the colonic mucosa of controls and IBS patients according to the disease subtype (IBS-Constipated, IBS-Diarrhea or IBS-Mixed alternating constipation with diarrhea) Red, TLR staining; blue, DAPI nuclear staining. Original magnification, ×20.</p