Effects Of IGF-1 And Resveratrol On Intestinal Fibrosis In Rats With Crohn's Disease And Its Mechanism

Crohn’s disease (CD) is a disorder of chronic, transmural inflammation that can affect any part of the gastrointestinal tract from the mouth to the anus. Intestinal fibrosis is a common and serious complication of CD and increases the risk of intestinal stenosis or obstruction, ultimately leads to surgical intervention at substantial personal and economic cost.The pathological process of intestinal fibrosis is characterized by mesenchymal cell proliferation and extracellular matrix (ECM) accumulation in interstitial space. A chronic or recurrent inflammation causing chronic or recurrent tissue damageand tissue degradation is considered a necessary condition for the initiation of intestinal fibrosis. In addition, previous studies demostrate that epithelial-mesenchymal transition (EMT) contribute to intestinal fibrosis through genetic lineage tracing technique in TNBS-induced model of crohn colitis. However, the underline mechanism for ECM synthesis and EMT signaling is unclear. Future intervention should involve stronger and more selective prevention of the continuous tissue damage by modulation of myofibroblast migration and ECM synthesis.In recent years, the role of IGFs (insulin-like growth factors) in gastrointesinal tract has gained more and more attention. IGF-1 is a paracrine/autocrine regulator of gastrointestinal (GI) physiology. Several lines of investigation have shown the profibrogenic actions of IGF-1 in the intestinal tract. IGF-1 not only stimulates proliferation and inhibits apoptosis of fibroblasts, myofibroblasts, and smooth muscle cells but also increases collagen expression and production in intestinal smooth muscle cells. However, there is no data show the molecular mechanism(s) of IGF-1 effect on fibroblast migration and collagen I in intestinal fibroblasts.Until now, no effective therapy exists for averting such fibrogenic events, because the fibrotic process becomes autopropagative and fails to respond to antiinflammatory interventions. New therapeutic approaches are urgently needed. Resveratrol (3,4,5-trihydroxy-trans-stilbene) is a polyphenol naturally occurring in grapes and red wine that exhibits beneficial health effects such as extending the life span, regulating tumor growth and oxidation. Resveratrol activates silent information regulator-1 (SIRT1), a NAD+-dependent deacetylase, which has many biological functions by deacetylating a number of key transcription factors, including p53, nuclear factor-icB (NF-κB) and so on. In addition, resveratrol also has dramatic antifibrotic effect in rodent models of renal fibrosis, cardiac fibrosis, and hepatic fibrosis, but the molecular mechanism(s) are currently unknown. Previous studies showed that resveratrol has antifibrotic effect in the peptidoglycan-polysaccharide (PG-PS) rat model of Crohn’s disease and can diminish IGF-1-stimulated collagen production in intestinal smooth muscle cells. Therefore, we further speculate that resveratrol may play a protective role in intestinal fibrosis by activating SIRT1.Part I Insulin-like growth factor-I induces epithelial to mesenchymal transition in rat intestinal epithelial cellsObjectives:To explore the effect and the possible molecular mechanisms of IGF-1 on epithelial to mesenchymal transition in rat intestinal epithelial cells.Methods:To evaluate the dose and time effect of IGF-1 on e-cadherin and vimentin expression, rat intestinal epithelial cells (IEC-6) were treated with IGF-1 at different time points of 0,6,12,24,48 h and at the concentration of 100,200 ng/ml, respectively. The expression of E-cadherin and vimentin were then determined by immunofluorescent staining in IEC-6, and the cell morphology was also observed. The effect of IGF-1 on the activities of signaling moleculers (PI3K/Akt, ERK1/2, p38, JNK) was examined by western blot after IGF-1 treatment at different time points. IGF-IRβ siRNA was used to examine whether IGF-1 inhibited e-cadherin expression through IGF-1 receptor. MEK1/2 inhibitor (U0126) and PI3K inhibitror (LY294002) were used to determine whether IGF-1-induced e-cadherin down-regulation was mediated by MAPK/ERK1/2 or PI3K/Akt-dependent mechanism via western blot and immunofluorescent staining.Results:IGF-1 induced the down-regulation of e-cadherin and up-regulation of vimentin in a time-dependent manner. IEC-6 cells in control medium displayed cuboidal morphology and had robust e-cadherin expression by both western blot and immunofluorescent labeling. In contrast, the same cells becames pindle-shaped, lost e-cadherin expression, and increased vimentin expression upon incubation with IGF-1. IGF-1 dose-dependently inhibited the expression of e-cadherin. All these results suggested that IGF-1 induced EMT in intestinal epithelial cells in a time and dose-dependent manner. IGF-1 significantly induced the insulin-like growth factor-1 receptor (IGF-1R) and ERK1/2 phosphorylation levels, which were increased at 5 min and at 15min respectively, and both of them were became noticeable at 30 min. Akt phosphorylation level was increased at 30min. Knockdown of IGF-1 R(3 markedly diminished the IGF-1-induced e-cadherin down-regulation. Treatment with a specific MEK 1/2 inhibitor (U0126 at 50μM) significantly increased e-cadherin expression of IGF-1 down-regulated cells by both western blot and immunofluorescent labeling, whereas treatment with DMSO vehicle control and PI3K inhibitor (LY294002 at 20μM) had no effect. All these results suggested that IGF-1 down-regulated e-cadherin expression through MAPK/ERK signaling pathway.Conclusion:IGF-1 down-regulated e-cadherin expression and induced EMT in intestinal epithelial cells. And the IGF-1R/MAPK/ERK signaling might play a major role in the expression of e-cadherin.Part II IGF-1 promots cell migration and collagen I synthesis in intestinal fibroblastsObjectives:To explore the effect of IGF-1 on cell migration and collagen I synthesis in intestinal fibroblasts.Methods:(1) To evaluate the time effect of IGF-1 on cell migration, human intestinal fibroblasts (CCD-18Co) and mouse primary intestinal fibroblasts (MIFs) were treated with IGF-1 at different time points of 0,6,12,24,48 h, then the ability of fibroblasts migrate was mesured using a scratch wound assay. The expression of N-cadherin was determined by western blot and real-time PCR. The effect of IGF-1 on the activities of signaling moleculers (IGF-1R, PI3K/Akt, p38, JNK, ERK1/2) was examined by western blot after IGF-1 treatment at different time points of 0,5,15,30,60 min. To determine the possible signaling pathway invovled in the expression of N-cadherin, we examined the potential role of PI3K/Akt and MAPK/ERK signaling. CCD-I8Co cells were treated with the PI3K/Akt inhibitor LY294002 and MEK1/2 inhibitor U0126 for 30min before IGF-1 sitmulation for 24 h. (2) To evaluate the dose and time effect of IGF-1 on collagen I expression, CCD-I8Co cells and MIFs were treated with IGF-1 at different time points of 0,24,48,72 h and at the concentration of 50,100, 150 ng/ml, respectively. The expression of collagen I was measured by western blot and real-time PCR. A MEK1/2 inhibitor U0126 was used to determine whether MAPK/ERK signaling pathway was involved in collagen I synthesis induced by IGF-1.Results:(1) IGF-1 treatment induced intestinal fibroblasts migration in a time dependent manner. Both protein and mRNA levels of N-cadherin were up-regulated after IGF-1 treatment. A 30-min treatment with IGF-1 alone showed the highest levels of IGF-1R, ERK1/2 and Akt phosphorylation. The up-regulation effect of IGF-1 on N-cadherin was inhibited by a PI3K/Akt inhibitor LY294002, not the MEK inhibitor U0126 by both western blot and cell scratch wound assay. (2) IGF-1 increased the expression of collagen I in a dose and time dependent manner in intestinal fibroblasts. IGF-1 significantly induced the phosphorylation of IGF-1R and ERK1/2. Collagen I expression was decreased when cells were treated with U0126 for 1 h followed by IGF-1 treatment.Conclusion:(1)IGF-1 might promot fibroblasts migration and N-cadheirn expression via PI3K/Akt signaling pathway. (2) IGF-1/IGF-1R/ERK axis may be contributed to the induction of IGF-1 on collagen I.Part Ⅲ The protect role of resveratrol in IGF-1-treated intestinal fibroblasts and TNBS-induced intestinal fibrosisObjectives:To investigate whether resveratrol (3,4,5-trihydroxy-trans-stilbene) inhibits collagen I synthesis induced by insulin-like 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 21d of TNBS administration, the degree of inflammation and fibrosis in colon were measured by HE staining and Masson’s trichrome staining. First, western blot was used to examine collagen I, IGF-1 and SIRT1 (silent information regulator 1) protein expressions in colitis tissues. SIRT1 expression and distribution were examined by immunohistochemistry. Then, CCD-I8Co cells were transfected with wild-type SIRT1 (SIRT1 WT) and deacetylase-inactive mutant SIRT1 (SIRT1 H363Y), western blot was used to examine collagen I protein expression. Then, western blot and quantitative real-time PCR were used to characterize collagen I protein and mRNA expression in mouse intestinal fibroblasts and CCD-I8Co cells treated with resveratrol. To evaluate whether resveratrol-induced collagen I down-regulation is an SIRT1-dependent manner, we used SIRT1 siRNA (siSIRT1) to specifically knockdown SIRT1. To further investigate the molecular mechanism of resveratrol, insulin growth factor-1 receptor (IGF-1R) and ERK1/2 phosphorylation levels were also examined after resveratrol treatment in the absence or presence of IGF-1 in fibroblasts.Results:Collagen I and IGF-1 expression were increased, and SIRT1 expression was decreased (0.67 ±0.04 vs 1.05 ±0.07, P＜0.001) in TNBS-induced colitis in rats compared with the control group. In vitro, IGF-1 could induce collagen I expression, mainly through the ERK 1/2 signal pathway. Result from immunohistochemistry also shown that SIRT1 was mainly located in the cytoplasm in the muscularis mucosa and muscularis propria. Compare to control group, SIRT1 expression was decreased in TNBS group. Overexpression of wild-type SIRT1, not deacetylase-inactive mutant SIRT1, decreased expression of collagen I induced by IGF-1. Resveratrol reduced basal and IGF-1-induced collagen I gene and protein expression in intestinal fibroblasts. Moreover, silencing SIRT1 restored collagen I expression in fibroblasts challenged with resveratrol. However, disruption of SIRT1 did not influence the anti-fibrotic effect of resveratrol in IGF-1-induced collagen I expression. Further analysis revealed that resveratrol significantly decreased phosphorylation of IGF-1R and its downstream signaling molecules in SIRT1-independent manner.Conclusion:(1) Our data in vivo suggested that SIRT1 might be involved in TNBS-induced intestinal fibrosis. (2) In vitro, resveratrol suppressed collagen I expression possibly via activating SIRT1. And resveratrol effectively inhibited collagen I synthesis induced by IGF-1, partly by inhibiting IGF-1R activation.