Dietary salt exacerbates intestinal fibrosis in chronic TNBS colitis via fibroblasts activation

Abstract

Intestinal fibrosis is a frequent complication in inflammatory bowel diseases (IBD). It is a challenge to identify environmental factors such as diet that may be driving this risk. Intestinal fibrosis result from accumulation of extracellular matrix (ECM) proteins secreted by myofibroblasts. Factors promoting intestinal fibrosis are unknown, but diet appears to be a critical component in its development. Consumption of salt above nutritional recommendations can exacerbate chronic inflammation. So far, high salt diet (HSD) have not been thoroughly investigated in the context of intestinal fibrosis associated to IBD. In the present study, we analyze the role of dietary salt in TNBS chronic colitis induced in rat, an intestinal fibrosis model, or in human colon fibroblast cells. Here, we have shown that high-salt diet exacerbates undernutrition and promoted ECM-associated proteins in fibroblasts. Taken together, our results suggested that dietary salt can activate intestinal fibroblasts, thereby contributing to exacerbation of intestinal fibrosis. Dietary salt may be considered as a putative environmental factor that drives intestinal fibrosis risk.

Figure 1

Dietary salt exacerbates undernutrition in rats with chronic colitis. Male Sprague–Dawley rats underwent weekly trinitrobenzene sulfonic acid (TNBS; n = 10) enemas for 3 weeks to induce chronic colitis-induced intestinal fibrosis, whereas control rats received a saline solution (CT; n = 10). Rats were subjected to either a standard diet or high-salt diet (4%, w/w) for 4 weeks (TNBS + salt, n = 10). (A) Experimental design. (B) Body weight. (C) Food intake. Body composition by echoMRI measurement: (D) fat mass and (E) lean mass. Control (CT, n = 10); TNBS (n = 6); TNBS + salt (n = 9). (B) Two-way ANOVA followed Bonferroni post-test: §means p < 0.05: CT vs TNBS + salt, #p < 0.05: CT vs TNBS, @@p < 0.01: TNBS vs TNBS + salt. (C,D,E) Kruskal–Wallis test followed post-test de Dunn: *p < 0.05, ***p < 0.001 respectively.

Figure 2

Colon weight/length ratio in chronic colitis. Male Sprague–Dawley rats underwent weekly trinitrobenzene sulfonic acid (TNBS; n = 10) enemas for 3 weeks to induce chronic colitis-induced intestinal fibrosis, whereas control rats received a saline solution (CT; n = 10). Rats were subjected to either a standard diet or high-salt diet (4%, w/w) for 4 weeks (TNBS + salt, n = 10). (A) Colon weight/length ratio. (B) Representative images of rat colon. Control (CT, n = 10); TNBS (n = 6); TNBS + salt (n = 9). One way ANOVA followed Tukey post-test: * means p < 0.05, ***p < 0.001 respectively.

Figure 3

Dietary salt promotes collagen 1 and MMP-9 in chronic colitis-induced intestinal fibrosis. Male Sprague–Dawley rats underwent weekly trinitrobenzene sulfonic acid (TNBS; n = 10) enemas for 3 weeks to induce chronic colitis-induced intestinal fibrosis, whereas control rats received a saline solution (CT; n = 10). Rats were subjected to either a standard diet or high-salt diet (4%, w/w) for 4 weeks (TNBS + salt, n = 10). Relative colon mRNA levels encoding for (A) α-sma (alpha smooth muscle actin), (B) Ctgf (connective tissue growth factor) and (C) Col1a1 (collagen 1). Relative protein expression of colon α-SMA (D) and MMP-9 (E). GAPDH is used as an internal control. Control (CT, n = 10); TNBS (n = 6); TNBS + salt (n = 9). One-way ANOVA followed by Tukey post-test: * means p < 0.05, **p < 0.001 respectively.

Figure 4

Dietary salt induces MMP-9 and MMP-2 activity in chronic colitis-induced intestinal fibrosis. Male Sprague–Dawley rats underwent weekly trinitrobenzene sulfonic acid (TNBS; n = 10) enemas for 3 weeks to induce chronic colitis-induced intestinal fibrosis, whereas control rats received a saline solution (CT; n = 10). Rats were subjected to either a standard diet or high-salt diet (4%, w/w) for 4 weeks (TNBS + salt, n = 10). (A) Representative gelatin zymography of colon MMP-9 and MMP-2 activity. Quantification of colon MMP-9 (B) and MMP-2 (C) activity. (D) Representative gelatin zymography for plasma MMP-9 and MMP-2 activity. Quantification of plasma MMP-9 (E) and MMP-2 (F) activity. Control (CT, n = 10); TNBS (n = 6); TNBS + salt (n = 9). Kruskal–Wallis test followed post-test de Dunn: *means p < 0.05, **p < 0.01, ***p < 0.001 respectively.

Figure 5

NaCl effect on CCD-18Co fibroblast cells in response to TGF-β. CCD-18Co cells were incubated with increasing concentration of dietary salt (0, 20 and 40 mM) in response to TGF-β (10 ng/mL) for 24 h. Representative western blot image (A) and quantification of alpha smooth muscle actin (α-SMA) in cell lysates (B). Relative mRNA level of α-sma (alpha smooth muscle actin) (C), Ctgf (connective tissue growth factor) (D), Col1a1 (collagen 1) (E) and Col3a1 (collagen 3) (F) in cell lysates. Representative gelatin zymography image or quantification of MMP-2 activity from cell supernatant (G) or from cell lysate (H). Two-way ANOVA test followed Bonferroni post-test: *means p < 0.05, **p < 0.01 respectively, ***p < 0.001.(n = 4 from independent experiments).