Figure 2.

Effect of CPZ rectal administration on intestinal permeability and tight junction proteins. (A) in-vitro intestinal permeability—representative images for release of FITC-loaded nanoparticles from colon at different time points (n = 3), (B) time-based plot for radiance efficiency and (C) AUC of the radiance efficiency. Representative images for immunofluorescence in colon tissues for expression of—(D) Zona occludens-1 (ZO-1), (E) Occludin (OCC), and (F) Claudin-1 (CLDN-1) (n = 3). Fluorescence intensity was measured using ImageJ, and data are presented after normalization with DAPI; (G) gene expression analysis for tight junction proteins. Mice were divided into two groups—Control (administered vehicle rectally) and CPZ (administered 531 µM CPZ rectally). Treatment was given for two weeks. After sacrifice, colon tissues (n = 3) were fixed in carnoy fixative, paraffin embedded moulds were prepared, and 5 µm sections were obtained. The sections were subjected to IHC with antigen retrieval using citrate buffer pH 6.0 (90 °C, 60 min) followed by blocking in 5% goat serum in PBS-Tween20, overnight incubation in primary antibodies, secondary antibody treatment for 2 h and DAPI for 30–60 s. High resolution images at 40× magnification were obtained using a confocal microscope, and florescence intensity was measured using ImageJ. For gene expression studies, RNA was extracted from colon tissues, and gene expression was performed for tight junction proteins using a Nanostring nCounter multiplex gene expression assay. All data are represented as mean ± SEM. Intergroup variations were assessed using a Student’s unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001 versus Control.