2,4,6-trinitrobenzene sulfonic acid-induced chronic colitis with fibrosis and modulation of TGF-β1 signaling

Abstract

Aim: To investigate whether targeting proteasome might reverse intestinal fibrosis in rats.

Methods: Chronic colitis was induced in rats by repeated administration of increasing dose of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 15, 30, 45, 60, 60, 60 mg) by rectal injection for 6 wk (from day 0 to day 35), while control rats received the vehicle. TNBS + bortezomib (BTZ) rats received intraperitoneal injections of BTZ twice weekly (from day 37 to day 44) at a dose of 25 mg/kg, whereas the control and TNBS groups received the same amount of the vehicle. Histologic scoring of inflammation and fibrosis was performed. Colonic production of transforming growth factor (TGF)-β was measured by ELISA. Colon fibrosis-related proteins such as phospho-p38, phospho-SMAD2/3, Akt and peroxisome proliferator activated receptor γ (PPARγ) were studied by western blot. Expression of the tight junction proteins, occludin and claudin-1, were assessed by Western blot. Colon proteasome activities (chymotrypsin-like and trypsin-like activities) were assessed.

Results: TNBS-treated rats had a higher colon weight/length ratio compared to control rats (P < 0.01). Furthermore, fibrosis and inflammation scores were higher in TNBS-treated rats compared to control rats (P < 0.01 for both). Colonic production of TGF-β production tended to be higher in TNBS-treated rats (P < 0.06). Fibrosis-related proteins such as phospho-p38, phospho-SMAD2/3, and PPARγ were significantly higher in TNBS-treated rats compared to control rats (all P < 0.05). TNBS rats had a higher expression of Akt compared to control rats (P < 0.01). Tight junction proteins were modified by repeated TNBS challenge: colon occludin expression rose significantly (P < 0.01), whereas claudin-1 expression fell (P < 0.01). Bortezomib inhibition significantly decreased chymotrypsin-like activity (P < 0.05), but had no significant effect on trypsin-like activity (P > 0.05). In contrast, bortezomib had no effect on other studied parameters such as fibrosis score, TGF-β signaling, or tight junction expression (P > 0.05 for all).

Conclusion: Rats with TNBS-induced chronic colitis exhibited colon fibrosis associated with higher TGF-β signaling. Proteasome inhibition by bortezomib had no effect on fibrosis in our experimental conditions.

Keywords: Bortezomib; Colitis; Fibrosis; Proteasome.

Figure 1

Validation of chronic 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Chronic colitis was induced by weekly intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNMS) for 6 wk. A: Colon weight/length (g/m) in control and TNBS groups; B: Colon macroscopy; C: Hematoxylin-eosin stained tissues in control and TNBS groups. In the TNBS sections, chronic inflammation (arrow) and fibrosis (arrow) led to architectural disorders, lymphotic infiltrate and fibrin deposits (magnification: 5× [left], 10× [right]); D: Histologic fibrosis score from 0 (no fibrosis) to 3 (severe fibrosis); E: Histologic inflammation score from 0 (no inflammation) to 3 (severe inflammation). Values are mean ± SE; ^bP < 0.01 vs control.

Figure 2

Colonic production of transforming growth factor-β and associated proteins in rats with chronic 2,4,6-trinitrobenzene sulfonic acid-induced colitis. A: Transforming growth factor (TGF)-β; B: Phospho-Smad2/3; C: Phospho-extracellular regulated kinase (ERK)-1; D: Phospho-ERK2; E: Phospho-p38; F: Peroxisome proliferator activated receptor (PPAR)γ expression in control and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated rats. Values are mean ± SE. ^aP < 0.05, ^bP < 0.01 vs control.

Figure 3

Chronic 2,4,6-trinitrobenzene sulfonic acid challenge induces Akt signaling. A: Akt; B: p70 ribosomal protein S6 kinase (P70S6K) protein expression; C: eIF4E immunoprecipitation; D: Western blot analysis of eIF4E-4E-BP1 association. Values are means ± Standard error. ^bP < 0.01 vs control.

Figure 4

Chronic 2,4,6-trinitrobenzene sulfonic acid alters expression of tight junction proteins. A: Occludin; B: Claudin-1 protein expression in the colons of 2,4,6-trinitrobenzene sulfonic acid (TNBS)- and control-treated rats. Values represent the mean expressed as a percentage of internal β-actin control, mean ± SE. ^bP < 0.01 vs control.

Figure 5

Proteasome inhibition by bortezomib treatment inhibits chymotrypsin-like activity. A: Chymotrypsin-like activity; B: Trypsin-like activity. Values are mean ± SE. ^aP < 0.05 vs control. TNBS: 2,4,6-trinitrobenzene sulfonic acid; BTZ: Bortezomib.