Resveratrol inhibits collagen I synthesis by suppressing IGF-1R activation in intestinal fibroblasts

Abstract

Aim: To investigate whether resveratrol (3,4,5-trihydroxy-trans-stilbene) inhibits collagen I synthesis induced by insulin growth factor-1 (IGF-1) in intestinal fibroblasts, and to explore the possible molecular mechanisms.

Methods: Male Sprague-Dawley rats were randomly divided into two groups: a control group and a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis group. After 21 d of TNBS administration, the degree of inflammation and fibrosis in colon was measured by HE staining and Masson's trichrome staining. Western blotting was used to examine collagen I, IGF-1 and silent information regulator 1 (SIRT1) protein expression in colitis tissues. Western blotting and quantitative real-time polymerase chain reaction were used to characterize collagen I protein and col1a2 mRNA expression in mouse intestinal fibroblasts and CCD-(18)Co cells treated with IGF-1. A MEK inhibitor (U0126) was used to determine whether IGF-1-induced collagen I expression was mediated by extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent mechanism. Effects of resveratrol on collagen I protein level, insulin growth factor-1 receptor (IGF-1R) and ERK1/2 phosphorylation levels were also examined after IGF-1 treatment in fibroblasts. To evaluate whether SIRT1 was necessary for the anti-fibrosis effect of resveratrol, cells were transfected with SIRT1-specific small interfering RNAs, wild-type SIRT1, and deacetylase-inactive mutant SIRT1.

Results: Collagen I and IGF-1 expression was increased, and SIRT1 expression was decreased (0.67 ± 0.04 vs 1.05 ± 0.07, P < 0.001) in TNBS-induced colitis compared with the control group. In vitro, IGF-1 could induce collagen I expression, mainly through the ERK 1/2 signal pathway. Resveratrol reduced basal and IGF-1-induced collagen I gene and protein expression in intestinal fibroblasts. Overexpression of wild-type SIRT1, not deacetylase-inactive mutant SIRT1, decreased expression of collagen I induced by IGF-1. Moreover, silencing SIRT1 restored collagen I expression in fibroblasts challenged with resveratrol. However, disruption of SIRT1 did not influence the anti-fibrotic effects of resveratrol and IGF-1-induced collagen I expression. Further analysis revealed that resveratrol significantly decreased phosphorylation of IGF-1R and its downstream signaling molecules by inhibiting IGF-1 binding to its receptor.

Conclusion: Our data suggest that resveratrol effectively inhibits collagen I synthesis in IGF-1-stimulated fibroblasts, partly by inhibiting IGF-1R activation, and SIRT1 is also responsible for the process.

Keywords: Fibroblasts; Insulin-like growth factor-1; Intestinal fibrosis; Resveratrol; Silent information regulator 1.

Figure 1

Silent information regulator 1 expression is decreased in 2,4,6-trinitrobenzenesulfonic acid-induced colitis. A, B: Change of clinical symptoms shown by disease activity index (DAI) and histological characterization of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis (original magnification × 200); C: The proteins collagen I, insulin growth factor-1 (IGF-1) and silent information regulator 1 (SIRT1) levels were measured by Western blotting. Data are expressed as mean ± SD (n = 7 in each group). ^bP < 0.01 vs those of normal rats. MTS: Masson’s trichrome staining; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 2

Insulin growth factor-1 induces collagen I expression via mitogen-activated protein-kinase pathway. A, B: Collagen I protein and col1a2, col1a1 mRNA expression in mouse intestinal fibroblasts (MIFs) and CCD-¹⁸Co cells stimulated with increasing concentrations of insulin growth factor-1 (IGF-1) for 24 h; C: Phosphorylation of IGF-1 receptor (IGF-1R) (p-IGF-1R) and extracellular signal-regulated kinase 1/2 (ERK1/2) (p-ERK1/2) in MIFs and CCD-¹⁸Co exposed to 100 ng/mL IGF-1 for 0, 15, 30, or 60 min. The bar graph represents the quantitation of the Western blotting normalized to the control; D: Cells were pretreated with 50 μmol/L mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/ERK inhibitor U0126 or vehicle for 1 h and then coincubated with 100 ng/mL IGF-1 for 24 h. Collagen I protein level was measured. The experiment was repeated three times and obtained similar results. Values represent mean ± SD. ^bP < 0.01 vs no treatment group. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

Resveratrol inhibits insulin growth factor-1-induced collagen I expression partly through silent information regulator 1. A, B: Collagen I protein and col1a2 mRNA expression were measured in fibroblasts stimulated with insulin growth factor-1 (IGF-1) (100 ng/mL) or IGF-1 plus resveratrol (RSV 50, 100 μmol/L) for 24 h; C: Wild type silent information regulator 1 (SIRT1) (WT) or enzyme deficient SIRT1 (HY) constructs were transfected into CCD-¹⁸Co cells followed by treatment with IGF-1. Collagen I protein was measured by Western blotting; D: Cells were transfected with the empty vector (pcDNA3.0) or SIRT1 WT, then collagen I, SIRT1, phosphorylation of extracellular signal-regulated kinase (ERK) (p-ERK) or total ERK1/2 (t-ERK1/2) were examined by Western blotting; E: 293T cells were transiently transfected with SIRT1-specific small interfering RNAs (siRNAs) (1, 2, 3) or scrambled siRNA (SCR) for 48 h, then SIRT1 protein and mRNA expression was measured by Western blotting and quantitative real-time polymerase chain reaction; F: CCD-¹⁸Co cells were transfected with SIRT1 siRNAs (1, 3) or SCR followed by treatment with resveratrol. The collagen I/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio shows the quantification of the expression levels of collagen I (Relative Amount). The experiment was repeated three times and obtained similar results. Values represent mean ± SD. ^aP < 0.05, ^bP < 0.01 vs control.

Figure 4

Resveratrol attenuates insulin growth factor-1 receptor and extracellular signal-regulated kinase 1/2 phosphorylation induced by insulin growth factor-1 via a silent information regulator 1-independent pathway. A: Mouse intestinal fibroblasts (MIFs) and CCD-¹⁸Co cells were treated with 100 ng/mL insulin growth factor-1 (IGF-1) in the absence or presence of resveratrol (100 μmol/L) for 30 min; B: CCD-¹⁸Co cells were transfected with silent information regulator 1 (SIRT1) expression constructs [wild type SIRT1 (WT) or enzyme deficient SIRT1 (HY)] followed by exposure to 100 ng/mL IGF-1 for 30 min. The phosphorylated levels of IGF-1R and extracellular signal-regulated kinase (ERK)1/2 were measured; C: Serum-starved quiescent CCD-¹⁸Co cells were pretreated with 100 μmol/L of resveratrol for 24 h, then removed and stimulated with IGF-1 alone (column 4) or IGF-1 plus resveratrol (column 5) for 30 min. The bar graph represents the quantitation of the Western blotting normalized to the control. The experiment was repeated three times and obtained similar results. Values represent mean ± SD. ^bP < 0.01 vs control.

Figure 5

Proposed mechanism by which resveratrol inhibits insulin growth factor-1-induced collagen I synthesis in intestinal fibroblasts. SIRT1: Silent information regulator 1; IGF-1: Insulin growth factor-1; ERK: Extracellular signal- regulated kinase; MEK: Mitogen-activated protein/extracellular signal-regulated kinase kinase; IGF-1R: IGF-1 receptor.