IBD

IBD is a term used to describe  disorders that involve chronic inflammation of the digestive tract. Ulcerative colitis is mainly localised into the colon (large intestine), while Crohn’s disease can affect any part of the digestive system. In both cases, symptoms may range from mild to severe, with periods of active illness followed by temporary remission. There is currently no cure for IBD, and medications tend to reduce the symptoms preventing severe complications, which include colon cancer.

Caco2 mono-culture IBD model

Caco-2 Model for IBD: Tight Junction Proteins

Caco-2 cells were differentiated for 21 days and stained for tight junction proteins such as ZO-1 (purple) and Occludin (orange) along with DAPI (blue– nucleus). The images were captured at 20X magnification using high content imager and the intensities of each fluorescence analysed.

Caco-2 Model for IBD: TEER Reported

Caco-2 cells were seeded onto trans-well inserts and allowed to differentiate for 21 days and TEER was measured regularly. On day 21 (Basal), the monolayer was treated Dexamethasone and stimulated with LPS+ IL1beta cocktail for 48 hours. TEER measurements were continued at 24 and 48 hours post treatment (pt) and reported as Ω.cm2 (n=3±SEM). 

Caco-2 Model for IBD: Tight Junction Proteins

Caco-2 cells differentiated for 21 days were treated with Dexamethasone followed by stimulation with LPS+ IL1beta cocktail for 48 hours. The monolayers were stained for tight junction proteins such as ZO-1 (green) with DAPI (blue– nucleus). The images were captured using ImageXpress Pico Imager and the fluorescent intensities analysed to obtain Percentage (%) of Positive Cells.

Caco-2 Model for IBD: Permeability

Caco-2 cells were differentiated for 21 days and treated with Dexamethasone followed by stimulation with LPS+ IL1beta cocktail for 48 hours. FITC-Dextran was added to the apical chamber along with the stimulants. Breaks in the monolayer were detected by the permeability of FITC-Dextran into the basal chamber. A significant reduction in FITC dextran was observed in Dexamethasone when compared to stimulated condition (****p<0.0001; n=3±SEM). The RFU of all the conditions were normalised to the untreated control (100). A red dotted line indicates the level of the untreated.

Caco-2 Model for IBD: Inflammatory Mediators

Caco-2 cells were differentiated for 21 days and subsequently treated with Dexamethasone, followed by stimulation with LPS+ IL1beta cocktail for 8 hours. mRNA expression in the cell lysates was quantified using the Luminex Quantigene™ Assay. A second set of differentiated Caco-2 cells were maintained for 48 hours post treatment. The supernatants were analysed after 48h for inflammatory mediators using a Luminex Multiplex Assay (**p<0.01; ***p<0.001; ****p<0.0001; n=3 mean±SEM).  

Caco2/THP-1 co-culture IBD model

Caco-2 cells differentiated for 21 days and PMA differentiated THP-1 cells were grown in co-culture and stimulated with LPS and IL-1β for 48hrs in the presence or absence of Sulfasalazine. The monolayers were stained after 48hrs for tight junction protein such as ZO-1 (pink) with DAPI (blue– nucleus). The images show a reduction in the ZO-1 protein in the stimulated cells compared to the well defined protein structure seen in the unstimulated cells. Also, prior treatment with sulfasalazine before stimulation prevented damage to the tight junction protein when compared to the stimulated cells.

The images were captured using ImageXpress Pico Imager at a x20 magnification.

Caco-2 cells differentiated for 21 days and PMA differentiated THP-1 cells were grown in co-culture and stimulated with LPS and IL-1β for 48hrs to induce an Inflammatory Bowel Disease phenotype in the presence or absence of Sulfasalazine and 5-Aminosalicylic Acid. The TEER reported for all the conditions (compounds and time of treatment) were normalised to the TEER reported on day 21 of differentiation (Basal) for each of the transwells. This data was analysed for both compounds using Two-way ANOVA followed by Dunnett’s multiple comparisons test [95% CI of diff.] (*p<0.05, **p<0.01,****p<0.0001). For each time point, all the conditions are compared to the Stimulated control. The co-culture model treated with Sulfasalazine, was significantly different at 48hours and showed an improvement in TEER values when compared to the stimulated co-culture model. Note: Unstimulated = Co-culture (Caco-2 +THP-1) alone, Stimulated= Co-culture (Caco-2 +THP-1) + LPS + IL-1β.

Pro-inflammatory marker suppression by Sulfasalazine and 5-Aminosalicylic Acid

Significantly increased levels of pro-inflammatory markers were observed after stimulation of the differentiated Caco-2 monolayers with IL-1β and LPS when compared to unstimulated control. The levels of these markers were suppressed when the Caco-2 monolayers were pre-treated with both compounds. Data was analyzed using Ordinary One-way ANOVA followed by Dunnett’s multiple comparisons test [95% CI of diff.] (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns= not significant). All the conditions were compared to the stimulated control for each of the markers and showed significant differences. However, the levels for 5-Aminosalicylic Acid  for TNF- alpha and Sulfasalazine and 5-Aminosalicylic Acid for MCP-1 were not significant (ns).

Note: Unstimulated = Co-culture (Caco-2 +THP-1), Stimulated= Co-culture (Caco-2 +THP-1) + LPS + IL-1β.

Lactate Dehydrogenase (LDH) release by Caco-2 cells

Data was analyzed using Ordinary One-way ANOVA followed by Dunnett’s multiple comparisons test [95% CI of diff.] (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns= not significant). The LDH positive control, provided with the assay, generated significant OD 450 values (p<0.0001) compared to the unstimulated control. Both Sulfasalazine and 5-Aminosalicylic Acid showed no significant LDH release when compared to the unstimulated control.

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