Transanal delivery of angiotensin converting enzyme inhibitor prevents colonic fibrosis in a mouse colitis model: development of a unique mode of treatment

Abstract

Background: We have previously shown that angiotensin converting enzyme-inhibitor (ACE-I) improved colonic inflammation and apoptosis in a dextran sodium sulfate (DSS)-induced colitis model. This study attempted to determine whether ACE-I could prevent the development of colonic fibrosis.

Methods: Colitis was induced in C57BL/6 mice with 2.5% DSS water for 7 days, followed by 7 days without DSS (fibrosis development). Study groups: Control (naive or non-treated), DSS+Placebo (polyethylene glycol (PEG), and DSS+ACE-I (using enalaprilat and PEG which are not absorbed through intact mucosa). Placebo and ACE-I were delivered daily via transanal route. Colonic mucosal fibrosis and inflammation were evaluated based on histological findings and cytokine expression.

Results: Transanal administration of ACE-I/PEG dose-dependently decreased the severity of fibrosis and pro-inflammatory cytokine expression. We next investigated if ACE-I acted on the TGF-beta/Smad signaling pathway as a mechanism of this anti-fibrosis action. Results showed a significant down-regulation of TGF-beta1 expression; as well, downstream signaling of the Smad family, known to mediate fibrosis, showed a decline in Smad 3 and 4 expression with ACE-I/PEG.

Conclusion: ACE-I/PEG is effective in preventing colonic fibrosis and pro-inflammatory cytokine expression in a DSS colitis model, most likely by down-regulating the TGF-beta signaling pathway. ACE-I/PEG may be a potential new option for treating inflammatory bowel disease.

Figure 1

(A) Survival rates of DSS-placebo and DSS-ACE-I treated mice. A minimum of N=11 mice in each group were given 2.5% DSS, and survival rates were investigated until the last day of the study. (B) Time course of changes in the disease activity index (DAI). All mice received 2.5% DSS in drinking water. DSS was administrated during the first 7 days, followed by 7 days of standard plain drinking water without DSS. All mice were evaluated daily for weight loss, stool consistency, and occult or gross intestinal bleeding. Results are expressed as mean ± SD. (* P <.05 vs DSS+Placebo). In the ACE-I treatment group, the DAI on day 8 was significantly lower compared to mice in the DSS+Placebo group, and this trend continued until the end of the study.

Figure 2

(A) Representative histologic sections of distal colon are shown after undergoing Hematoxilyn-Eosin staining (x10 magnification). The Control group showed a normal (Naïve) colon without DSS treatment. In the DSS-placebo mice, dense cellular fibrosis was observed in the colonic submucosa with regenerative changes. In ACE-I treated mice, the colon showed normal mucosa architecture and mild fibrosis in the submucosa. (B) Representative histologic sections of distal colon are shown after undergoing Masson’s trichrome staining (x10) of the colon. Note the prominent fibrosis in DSS-placebo mice as represented by a dense blue staining. Whereas only mild fibrosis is noted in ACE-I treated mice.

Figure 3

mRNA expression of pro-collagen I (α1) and (α2) derived from mucosa samples in each group and measured by real time PCR. Result are expressed as 2^{−(−ΔΔCt)} in relation to β-actin gene for PCR. Expression of both pro-collagens were significantly increased in the DSS-placebo group compared to non-treated controls. Administration of ACE-I to DSS-induced colitis significantly reduced the expression of pro-collagens I (α1) and (α2) in a dose-dependent fashion. Note that ACE-I treatment of control mice failed to significantly affect baseline pro-collagen expression. Statistical comparisons are made using ANOVA with a post hoc Bonferroni test.

Figure 4

Expression of mucosal pro-inflammatory cytokines TNF-α and IL-1β as detected by real time PCR. Result are expressed as 2^{−(−ΔΔCt)} in relation to β-actin gene expression. TNF-α and IL-1β mRNA expression were significantly lower in the ACE-I treated groups compared to the DSS-placebo group.

Figure 5

Expression of mucosal –derived TGF-β1 as detected by (A) real time PCR and (B) Western blot techniques. Result are expressed as 2^{−(−ΔΔCt)} in relation to β-actin gene expression for PCR results; and the ratio of TGF-β1/β-actin for immunoblots. Note that TGF-β1 expression was significantly increased with administration of DSS for both mRNA and protein analysis, and both mRNA and protein expression significantly decreased with ACE-I treatment.

Figure 6

mRNA expression of Smad signaling genes form colonic mucosal samples as measured by real-time PCR. mRNA expression of Smad 3 and Smad 4 significantly increased in the DSS-placebo group. Administration of high-dose ACE-I to DSS-treated mice returned the expression of these factors to control levels, and expression was not significantly different from control values. There were no significant differences Smad 2 and Smad 7 expression between study groups.

Figure 7

Expression of phosphorylated Smad 3 (p-Smad 3), Smad 3 and Smad 4 proteins as examined by Western blot methods. Results are reported as the ratio of Smad protein to β-actin protein expression. The expression of p-Smad 3 and Smad 4 were significantly increased in DSS-placebo mice compared with controls. Administration of ACE-I, led to a decline in the expression of these Smad proteins. Interestingly, the increased expression of Smad 3 was confined to the p-Smad (activated protein).