Figure 1.
TUNEL assay shows different patterns in the intestinal mucosa (ICD) and in the mesenteric adipose tissue (MAT) of Crohn’s disease (ACD), compared to the respective control biopsy samples (IC and AC).
(A) Enterocyte and lamina propria cell apoptosis are shown by immunofluorescence staining (overlay image); TUNEL+ cells are showed in orange (co-labeled by PI and FITC). Low numbers of TUNEL+ enterocytes and lamina propria cells were detected in the ICD group compared to IC. (C) Adipocyte apoptosis, shown by immunofluorescence staining (overlay image); TUNEL+ cells are showed in orange (co-labeled by PI and FITC). Low numbers of TUNEL+ adipocytes were detected in the ACD group compared to AC. Note the high density of TUNEL+ adipocytes, in orange, in the control (AC). Images were obtained using a 40x objective. (B) and (D) Quantitative analysis of TUNEL staining in the ICD and ACD groups, compared to the respective controls (IC and AC). The graphs of intestinal tissue show the quantitative analysis for the epithelium and lamina propria TUNEL staining, separately. For ICD, n = 10; for ACD, n = 10; for IC, n = 8; and, for AC, n = 8, *p<0.05 vs control. (E) Representative hematoxylin-eosin (H&E) staining of fixed paraffin-embedded MAT from AC and ACD groups shows lower area and perimeter of the adipocytes in the ACD group, compared to the control (AC). Images were obtained using a 40x objective. (F) Quantitative morphometric histological analysis in the mesenteric adipose tissue (MAT) of Crohn’s disease (ACD), compared to the respective control group (AC). The graphs show the decreased perimeter (µm) and area (µm2) of the adipocytes from the MAT of Crohn’s disease, compared to the control biopsy samples. For ACD, n = 10; for AC, n = 8, *p<0.05 vs control. (G) The graphs dispersion show a significant correlation between the perimeter (µm) and the number of apoptotic cells (TUNEL+), (r = 0.89, p<0.05) and also between the area (µm2) and the number of apoptotic cells (TUNEL+), (r = 0.92, p<0.05). (H) Immunohistochemical staining of Ki67 in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD), compared to the control biopsy samples (AC); no evidence of proliferation were found in all samples (ACD, n = 10; AC, n = 8). Images were obtained using a 40x objective. The positive control was from tissue section of intestinal mucosa.
Figure 2.
Bax and Bcl2 gene expressions, as determined by RT-PCR; low transcript levels of Bax and Bcl2 are observed in the intestinal mucosa of the Crohn’s disease group (ICD), compared to the respective control (IC).
Low transcript levels of Bcl2 are also seen in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD), compared to the respective control (AC), while no differences in Bax transcripts were found in the MAT groups. For ICD, n = 10; for ACD, n = 10; for IC, n = 8; and, for AC, n = 8, *p<0.05 vs control.
Figure 3.
Immunohistochemical staining of Bax in the intestinal tissue (epithelium and lamina propria) of the Crohn’s disease group (ICD) and in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD), compared to the respective control biopsy samples (IC and AC).
(A) Representative staining of fixed paraffin-embedded tissue of terminal ileum from IC and ICD groups showing fewer positive cells (brown) in the lamina propria of ICD, compared to the IC group. (B) Representative staining of fixed paraffin-embedded mesenteric adipose tissue in the AC and ACD groups; no differences were found among the groups. Images were obtained using a 40x objective. The positive control was from tissue section of prostatic cancer. For ACD, n = 10; and for AC, n = 8. (C) Quantitative analysis of immunohistochemical staining for Bax in the intestinal mucosa of the Crohn’s disease group (ICD), compared to the respective control (IC). The graphs show the quantitative analysis for the epithelium and lamina propria immunostainings separately. For ICD, n = 10; for IC, n = 8, *p<0.05 vs control.
Figure 4.
Immunohistochemical staining of Bcl-2 in the intestinal tissue (epithelium and lamina propria) of the Crohn’s disease group (ICD) and in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD), compared to the respective control biopsy samples (IC and AC).
(A) Representative staining of fixed paraffin-embedded tissue of terminal ileum from IC and ICD groups showing similar numbers of positive cells (brown) in the ICD and IC groups. (B) Representative staining of fixed paraffin-embedded mesenteric adipose tissue from the AC and ACD groups, showing a higher intensity in the ACD group, compared to the control (AC). Images were obtained using a 40x objective. (C) and (D) Quantitative analysis of immunohistochemical staining for Bcl-2 of ICD and ACD groups, compared to the respective control groups (IC and AC). The graphs of intestinal tissue show the quantitative analysis for the epithelium and lamina propria immunostainings separately. For ICD, n = 10; for ACD, n = 10; for IC, n = 8; and for AC, n = 8, *p<0.05 vs control.
Figure 5.
Representative Western blot analyses and determination of Bax and Bcl-2 protein expression in the intestinal tissue (mucosa) of the Crohn’s disease group (ICD) and in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD), compared to the respective controls (IC and AC).
Decreased expression of Bax was observed in ICD group, compared to the control group (IC), and higher expression of Bcl-2, an anti-apoptotic protein, was observed in the MAT of Crohn’s disease (ACD) compared to the control group (AC). For illustration purposes, each band represents one patient. For ICD, n = 10; for ACD, n = 10; for IC, n = 8; and, for AC, n = 8, *p<0.05 vs control.
Figure 6.
Immunofluorescence staining of Caspase 3 in the mesenteric adipose tissue (MAT) of the Crohn’s disease group (ACD) compared to the respective control biopsy samples (AC).
(A) Representative staining of fixed paraffin-embedded mesenteric adipose tissue from the AC and ACD groups, showing a higher number of positive cells for FITC (green-fluorescent) in the cytosol, co-labeled with DAPI (nuclear staining: blue-fluorescent) in the ACD group, compared to the control (AC). The arrows show the positive cells. Images were obtained using a 40x objective. (B) Quantitative analysis of immunofluorescence staining for Caspase 3 of ACD group compared to the respective control group (AC). For ACD, n = 10; for AC, n = 8, *p<0.05 vs control.