Dig Dis Sci DOI 10.1007/s10620-014-3398-6
ORIGINAL ARTICLE
Vitamin D Prevents the Intestinal Fibrosis Via Induction of Vitamin D Receptor and Inhibition of Transforming Growth Factor-Beta1/Smad3 Pathway Qingsong Tao • Baochai Wang • Yu Zheng Xiaohua Jiang • Zheng Pan • Jianan Ren
•
Received: 9 February 2014 / Accepted: 10 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Background and Aims Vitamin D deficiency in patients with inflammatory bowel disease (IBD) is associated with greater disease activity and lower quality of life. Intestinal fibrosis is a main complication of IBD. However, the effect of vitamin D on intestinal fibrosis remains unclear. We investigated the prophylactic effect and the underlying mechanism of vitamin D on the intestinal fibrosis in vitamin D-deficient mice with chronic colitis. Methods Vitamin D-deficient mice were randomized into two groups receiving the vitamin D-deficient or vitamin D-sufficient diet from weaning (week 4). Intestinal fibrosis was induced by six-weekly 2,4,6-trinitrobenzene sulfonic acid administrations from week 8. At week 14, the productions of extracellular matrix (ECM) and total collagen were measured
in the colons, and TGF-b1/Smad3 signal transduction was examined in isolated colonic subepithelial myofibroblasts (SEMF). The expression of vitamin D receptor (VDR), a-SMA and Collagen I in normal SEMF and VDR-null SEMF exposed to TGF-b1 and/or 1,25(OH)2D3 was measured. Results Vitamin D significantly reduced the histological scoring, ECM and collagen productions in the colons and decreased the levels of TGF-b1, Smad-3, p-Smad3 and Collagen I in SEMF. 1,25(OH)2D3-induced VDR expression and decreased TGF-b1-stimulated a-SMA and Collagen I expressions in SEMF. Knocking down VDR expression in SEMF abolished the effect of 1,25(OH)2D3. Conclusions Vitamin D has prophylactic effect on intestinal fibrosis in the vitamin D-deficient mice with chronic colitis, which may be associated with the inhibited activation of TGF-b1/Smad3 pathway in the SEMF via VDR induction.
Q. Tao (&) B. Wang X. Jiang Z. Pan Department of General Surgery, Affiliated Zhongda Hospital, Southeast University Medical School, 87 Ding Jia Qiao Road, Nanjing 210089, Jiangsu, China e-mail: [email protected]; [email protected]
Keywords Vitamin D Intestinal fibrosis Myofibroblast TGF-b1 Smad3 VDR
B. Wang e-mail: [email protected] X. Jiang e-mail: [email protected] Z. Pan e-mail: [email protected] Y. Zheng Department of Surgery, Haian People’s Hospital, Nantong University Medical School, Nantong 226600, Jiangsu, China e-mail: [email protected] J. Ren Research Institute of General Surgery, Jinling Hospital, Nanjing University Medical School, Nanjing 210093, Jiangsu, China e-mail: [email protected]
Abbreviations IBD Inflammatory bowel disease CD Crohn’s disease SEMF Subepithelial myofibroblasts VDR Vitamin D receptor ECM Extracellular matrix a-SMA a-Smooth muscle actin p-Smad3 Smad3 phosphorylation Vit D Vitamin D
Introduction Vitamin D deficiency, defined as the serum 25-hydroxyvitamin D level of less than 20 ng/ml (50 nmol/L) [1], is
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common in inflammatory bowel disease (IBD) [2] and is independently associated with lower health-related quality of life (HRQOL) and greater disease activity [3]. Supplemental vitamin D, as an adjunctive treatment, may help in controlling IBD [4]. Intestinal fibrosis is a common and potentially serious complication of IBD, especially in Crohn’s disease (CD), that results from the abnormal wound healing of intestinal tissue following chronic inflammation [5]. Past studies have shown that vitamin D may help in liver and renal fibrosis [6, 7] and revealed that high levels of vitamin D receptor (VDR) ligands contribute to these effects [6, 8]. However, the effects of vitamin D on intestinal fibrosis in IBD still remain unclear [9]. Subepithelial myofibroblasts (SEMF) play an important role in intestinal fibrosis by regulating inflammatory responses and extracellular matrix metabolism [10]. These create a plausible link between mucosal inflammation and destruction of the subepithelial matrix. Thus, the inhibition of these processes represents a lucrative target for IBD therapies. Based on the potential immune-regulatory [11] and gut barrier-protective [12] functions of vitamin D, we investigated the prophylactic efficacy and underlying mechanism of vitamin D on the intestinal fibrosis in the vitamin D-deficient mice with chronic colitis.
Methods Vitamin D-Deficient and Vitamin D-Sufficient Diet The vitamin D-deficient diet (TD.89123) from Harlan Teklad (Madison, WI) uses vitamin-free test casein (VFT casein) as the protein source. VFT casein is extracted with denatured alcohol to reduce the fat content from about 1–0.1 %. In the process, the content of a number of vitamins is further reduced. The vitamin D-sufficient diet is the standard open formula vitamin D diet (7017 NIH-31, Harlan Teklad) containing 4.2 IU/g vitamin D3 (1 IU vitamin D3 = 25 ng cholecalciferol), which would be considered as high daily dose when calculated to human equivalency dose (HED) [13]. The special nutrient formulas are shown in Table 1. Induction of Intestinal Fibrosis and Chronic Colitis in Vitamin D-Deficient Mice Vitamin D-deficient offspring were bred from CD-1 mice (Charles River Laboratories Inc., Wilmington, MA) that were maintained on the vitamin D-deficient diet in a room with artificial UV-free light [14]. Pups were randomized into two groups receiving vitamin D-deficient or vitamin
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Table 1 Selected nutrient formulas of diets Vitamin D-deficient diet (TD.89123)
Vitamin D-sufficient diet (NIH-31-7017)
Calories from carbohydrate (%)
60.3
62
Calories from protein (%)
16.7
24
Calories from fat (%) Energy density (Kcal/g)
22.9 3.9
14 3.6
VFT casein (wg %)
18
–
Vitamin D3 (IU/g)
–
4.2
VFT vitamin-free test
D-sufficient diets from weaning (week 4). From week 8, the mice were administrated weekly with TNBS (SigmaAldrich, St. Louis, MO) for 6 weeks for induction of chronic colitis and intestinal fibrosis according to the previously described method [15]. The CD-1 strain was chosen because of its susceptibility to extracellular matrix (ECM) changes [16]. TNBS was enema administered via a 3F catheter attached to a 1-ml syringe into the rectum until the tip was 5 cm proximal to the anal verge. To ensure distribution of TNBS, mice were held in a vertical position for 60 s after the intrarectal injection. The mice received increasing doses of TNBS throughout the 6-week study: 1, 1, 1.5, 1.5, 2, 2 mg/0.1 ml per week in 45 % EtOH. The control mice received a 0.1 ml enema of phosphate buffer solution (PBS). All mice were euthanized 42 days post the initiation of TNBS induction. Animal use protocols were approved by Animal Care and Use Committee for Southeast University, and all animal studies were performed according to the institutional ethical guidelines stipulated by the Review Board of Southeast University. Histological Evaluation and Image Analysis of ECM Content in the Colon The paraffin embedded blocks representing the similar positions of colon were sectioned and stained with H&E and Masson trichrome, respectively. Histological scoring and quantitative ECM analysis were performed by two pathologists in blinded manner according to the previously described protocols [17, 18]. Histological scoring was graded determined with the H&E staining. For quantitative analysis of ECM with Trichrome staining, 6–12 randomly chosen fields for each section were photographed and transformed into digital readings by using Olympus Image Analysis Software (Olympus, Tokyo, Japan), which is allowed for quantification of the various color wavelengths with pixels as the unit of measurement. The percentage of ECM was then calculated by dividing the pixel area of the ECM by the pixel area corresponding to the total tissue in the field of view.
Dig Dis Sci
Sircol Collagen Assay Total collagen content in the colon was detected with Sirius red collagen detection kit (Chondrex, Redmond, WA). Tissues were homogenized in T-PER buffer (Thermal Science, Amarillo, TX), incubated on ice for 15 min and centrifuged for 5 min at 10,6009g at 4 °C. Each protein sample was diluted in 0.5 M acetic acid to a final concentration (100 lg/ml). Optical density was read at 530 nm. Results were calculated based on collagen per 100 lg/ml protein. Analyses of Vitamin D Metabolism Considering the limit amount of murine blood, sera were diluted for assay. The levels of 25(OH)D3 and calcium in serum were evaluated by liquid chromatography–tandem mass spectroscopy (LC–MS/MS) and atomic spectroscopy (Agilent Technologies, Santa Clara, CA), respectively, according to the manufacturer’s protocols. LC–MS/MS was performed on Waters 2695 Alliance HPLC System (Waters, Milford, MA) and an analytical column (2.1 9 100 mm, 1.7 lm; Acquity UPLC CSH; Waters, Milford, MA). Isolation of Subepithelial Myofibroblasts We have previously described the isolation of the subepithelial myofibroblasts [19, 20]. Briefly, fresh mucosal samples were obtained from the colons of the mice. After washing with calcium- and magnesium-free Hanks’ balanced salt solution (HBSS; GIBCO-BRL, Gaithersburg, MD), the mucosa samples were completely denuded of epithelial cells by three 30-min incubations at 37 °C in 1 mM EDTA (Sigma). The de-epithelialized mucosal samples were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS), 50 U/ml penicillin and 50 lg/ml streptomycin (GIBCO-BRL), and incubated at 37 °C in 5 % CO2-atmosphere. The cells in suspension were removed after every 24- to 72-h culture period, and the denuded mucosal tissue was maintained in culture for up to 2 week. Myofibroblasts possess the contractile characteristics of, with immunostaining for, a-smooth muscle actin (SMA) and vimentin, but completely negative for desmin [21].
phospho-Smad3 (Cell Signaling), VDR (Abcam, Cambridge, MA), Collagen I (Sigma) and b-Actin (Sigma) using standard methods. Band intensities were quantified normalized relative to the quantity of their respective bactin bands. Reverse Transcription PCR Total RNA isolated from SEMF using Trizol reagent kit was reverse transcribed with Superscript II (Invitrogen, Carlsbad, CA). Extracted RNA was quantified by spectrophotometry (Nanodrop 2000C, Thermo, Pittsburgh, PA). PCR products were obtained after 30–40 cycles of amplification with an annealing temperature of 50–57 °C. PCR primer sequences were TGF-b1 (680 bp), 50 -GCG ATT CGA TGA TCA G-30 and 50 -AGC GGA TCT TGA GCT30 ; Smad3 (425 bp), 50 -GGA CGC TAC TAG GTC A-30 and 50 -CGT TAG CAG TGG TCG A-30 ; b-actin (155 bp) 50 -TAT GAC TTA GTT GCG TTA CAC C-30 and 50 -CCT TCA CCG TTC CAG TTT-30 . Quantitative Real-Time PCR (RT-qPCR) RT-qPCR was used to measure RNA transcripts of a-SMA (Acta2) and Collagen Ia1 (Col1a1) using the 2-DDCt method based on GAPDH amplification as previously reported [19]. Real-time quantitative Taqman PCR analysis was performed using an ABI PRISM 7700 (Perkin–Elmer, CA, USA). Specific primers and dual-labeled fluorescent probes were purchased from Applied Biosystems (Foster City, CA): Acta2 50 -CTG ACA GAG GCA CCA CTG AA-3 and 50 -ACG CAT GAT GGC ATG AGG CA-30 ; Col1a1 50 -AAG GAG TTT CAT CTG GCC CT-30 and 50 -AGC AGG TCC TTG GAA ACC TT-30 ; GAPDH 50 -TGA GGC CGG TGC TGA GTA TGT CG-30 and 50 -CCA CAG TCT TCT GGG TGG CAG TG-30 . RNA Interference SEMF at 30–40 % confluency were transfected with 20 nM of VDR-specific siRNAs (Invitrogen, 50 -CCC ACC TGG CTG ATC TTG TCA GTT A-30 and 50 -GGA CAT GAT GGA ACC GGC CAG CTT T-30 ) using Lipofectamine RNAi max (Invitrogen) according to the manufacture’s protocol.
Western Blotting
Statistical Analysis
Protein lysates were prepared in 1 9 lysis buffer (Cell Signaling Technology, Billerica, MA). Equal amounts of protein from cells were determined by the Bio-Rad Protein assay (Bio-Rad, Waltham, MA) and then immunoblotted with the appropriate antibodies: Smad3 (Santa Cruz),
Statistical significance was determined by Student’s t test or ANOVA followed by Fisher’s protected least significant difference post hoc test, as appropriate, using IBM SPSS Statistics v20 (IBM, New York, NY). Data are expressed as mean ± SE. Statistical significance was set at P \ 0.05.
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Results Vitamin D Prevented Intestinal Fibrosis Via Inhibition of TGF-b1/Smad3 Signal Transduction As shown in Fig. 1, repeated TNBS administration caused more severe intestinal inflammation and fibrosis in the mice without vitamin D supplementation (Fig. 1c). The colons showed pockets of inflammation, thickened colonic wall, persistent edematous swelling, as well as obstruction (Figs. 1c, 2 top). Histological scoring, as shown in Fig. 3a, significantly decreased by vitamin D treatment. The colons exhibited more collagen deposition in the mucosa, submucosa and serosal areas determined with Masson trichrome staining (Fig. 2, middle and bottom). In contrast, the mice with vitamin D treatment developed less severe colonic fibrosis by the quantitation of ECM (Fig. 3b). The total collagen productions measured with the Sircol assay consistently confirmed the prophylactic effect of supplemental vitamin D on the intestinal fibrosis (P \ 0.01, Fig. 3c). Meanwhile, vitamin D treatment also significantly increased the serum 25(OH)D3 level (Fig. 3d). Serum levels of calcium were also increased with vitamin D supplementation without significant difference (Fig. 3e), which reflected the calcium homeostasis was affected, but without serious adverse consequences in this study. Previous chronic TNBS colitis showed increased levels of TGF-b1 and Smad3 phosphorylation (p-Smad3) [22]. Then, we isolated the colonic subepithelial myofibroblasts (SEMF, Fig. 4) from the mice and examine the TGF-b1/
Fig. 1 Macroscopic findings of the colons from the mice in four different groups. a Control group without vitamin D treatment (Vehicle - Vit D). b Control group with vitamin D treatment (Vehicle ? Vit D). c Model group without vitamin D treatment (TNBS - Vit D). d Model group with vitamin D treatment (TNBS ? Vit D)
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Smad3 expressions in the primary SEMF before the first passage. As shown in Fig. 5, the expressions of TGF-b1, Smad3 and p-Smad3 normalized by b-actin or total Smad3, as well as the mRNA levels of TGF-b1 and Smad3 in the SEMF from the mice with or without vitamin D supplementation were strongly activated with repeated TNBS administration. Western blotting showed that type I collagen levels were increased in the model versus control colons (Fig. 5), indicative of activation of TGF-b1-Smad3 signal transduction. Vitamin D treatment decreased the expression of TGF-b1, Smad3, p-Smad3 and type I collagen (Fig. 5). These results suggest that Vitamin D ameliorates TGF-b1/Smad3 signal transduction, which may lead to suppression of intestinal fibrosis. 1,25(OH)2D3 Inhibits TGF-b1-Induced Expression of Pro-fibrotic Genes Via Up-Regulation of Vitamin D Receptor To further test the possibility and explore the mechanism underlying inhibition of intestinal fibrosis and TGF-b1/ Smad3 signal transduction, we evaluated the expressions of profibrotic genes in the SEMF from the normal colons of the mice exposed to TGF-b1 (5 ng/ml) and/or 1,25(OH)2D3 (0.01, 0.1, 10 nM) for 8 h. As expected, TGF-b1-stimulated Acta2 and Col1a1 mRNA expression, which was dose-dependently decreased by 1,25(OH)2D3 treatment (Fig. 6a, b). Western blot analysis revealed that TGF-b-induced elevation of p-Smad3, Smad3, collagen I, a-SMA levels was dose-dependently decreased by 1,25(OH)2D3 (Fig. 6c). It has been reported that Vitamin D receptor (VDR) signaling inhibits colitis by protecting the mucosal epithelial barrier [23]. To test the potential role of VDR in the antifibrosis effect of Vitamin D, We next examined the effects of 1,25(OH)2D3 on expression of VDR in SEMF. Western blotting confirmed that 1,25(OH)2D3 dosedependently induced the expression of VDR (Fig. 6c). Then, the VDR expression was knocked down in SEMF using VDR-specific siRNA (SiVDR). VDR-null or control SEMF were cultured in the presence or absence of 5 ng/ml TGF-b1 and 10 nM 1,25(OH)2D3 for 8 h. Real-time qPCR revealed that 1,25(OH)2D3 failed to decrease TGF-b1induced expressions of Acta2 and Col1a1 mRNA in VDRnull SEMF. Western blotting confirmed TGF-b1-induced expressions of collagen I and a-SMA were unaffected by 1,25(OH)2D3 in VDR-null SEMF.
Discussion In the present study, we demonstrated the prophylactic effect of vitamin D on the colonic fibrosis in the vitamin
Dig Dis Sci Fig. 2 Histology of colonic morphology. Top H&E staining (920). Middle Masson trichrome staining (920). Bottom Masson trichrome staining (94). Red box focused the areas showed in middle. ECM deposition (blue area) was decreased with vitamin D treatment
Fig. 3 Vitamin D decrease the histological scoring (a) and productions of ECM (b) and total collagen (c) in the colons, associated with the increased levels of 25(OH)D3 (d) and calcium (e) in the serum.
Results are expressed as mean ± SD, from 8 mice each group. *P \ 0.05; **P \ 0.01; ***P \ 0.001
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Fig. 4 Characterization of isolated subepithelial myofibroblasts (a, 9100), immunostaining with monoclonal antibodies against vimentin (b, positive, 9400), a-SMA (c, positive, 9400), and Desmin (d, negative, 9400)
Fig. 5 Vitamin D inhibits of TGF-b1/Smad3 signal transduction in primary isolated subepithelial myofibroblasts. a TGF-b1 mRNA. b Smad3 mRNA. c Expressions of TGFb1, Smad3, p-Smad3 and Collagen I determined by Western blotting
D-deficient mice with chronic colitis, and the effects were associated with the inhibited TGF-b1/Smad3 signal transduction via VDR induction in the colonic SEMF. Legishetty et al. [24] found that vitamin D deficiency in mice dysregulated colonic antimicrobial activity and impaired homeostasis of enteric bacteria. Kong et al. [25] found that vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility to mucosal damage and increased risk of IBD. These may link vitamin D deficiency with chronic colitis and answer the question that who should be the chicken or egg [26]. The effects of the vitamin D on regulating immune responses, including macrophages, dendritic cells, T cells and B cells
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have been studied previously [27–29]. But few study investigated the effect of vitamin D on the intestinal fibrosis, which is a common complication of IBD. Liu et al. [23] demonstrated that VDR signaling inhibited colitis by protecting the mucosal epithelial barrier. Gocek et al. [30] reported that 1,25(OH)2D3 induced a rapid increase in synthesis of VDR protein and its transport to the nucleus. The up-regulation of VDR levels may increase the responsiveness to 1,25(OH)2D3 [31]. The present study demonstrated for the first time that vitamin D may have prophylactic effect on intestinal fibrosis through the inhibition of TGF-b1/Smad3 pathway and up-regulation of VDR in the SEMF.
Dig Dis Sci
Fig. 6 1,25(OH)2D3 inhibits TGF-b1-induced expression of profibrotic genes via up-regulation of VDR. Normal SEMF were cultured in the presence or absence of 5 ng/ml TGF-b1 and 0.01, 0.1, or 10 nM 1,25(OH)2D3 for 8 h. The levels of Acta2 (encoding a-SMA, a) and Col1a1 (encoding collagen Ia1, b) mRNA were determined by qPCR. c VDR, a-SMA, Collagen I, Smad3, p-Smad3 and b-actin protein levels were determined by Western blotting. VDR-null or control SEMF were cultured with 5 ng/ml TGF-b1 and/or 10 nM
1,25(OH)2D3 for 8 h. Knocking down VDR expression by VDRspecific siRNA in SEMF abolished the effect of 1,25(OH)2D3 on TGF-b1-induced Acta2 (d) and Col1a1 (e) mRNA expressions. Western blotting confirmed no expression of VDR protein in response to VDR-specific siRNA (f, top). f Collagen I and a-SMA protein levels were determined by Western blotting. Results are expressed as mean ± SD, from 8 mice each group. *P \ 0.05; **P \ 0.01; ***P \ 0.001
TGF-b1 acts as a potent growth inhibitor for most types of cells, inducing the apoptosis of epithelial cells, stimulating the production of extracellular matrix and inducing fibrosis in various tissues in vivo [32]. Activation of TGF-b type I receptor (TGF-b1RI) on TGF-b1 binding will in turn phosphorylate Smad2 and Smad3. Targeted disruption of Smad3 is associated with diminished cell responsiveness to TGF-b1 [33]. In this study, decreased levels of Smad3 and p-Smad3 in the colonic SEMF were associated with the inhibition of intestinal fibrosis in the chronic colitis, as indicated by the decreased expression of TGF-b1-target genes, a-SMA and type I collagen. There are some limitations in this study. One of the inhibitory proteins of Smad family, Smad7, occupies the ligand-activated TGF-b1RI and interferes with the phosphorylation of Smad2/3. Up-regulation of Smad7 is associated with inhibition of TGF-b1-induced Smad signaling [34]. Thus, Smad7 and other proteins of Smad family would be examined in the further study. Furthermore, the
underlying interaction between VDR and Smad3 also needs to be determined. Although vitamin D may be effective for intestinal fibrosis, the associated dysfunction of serum calcium homeostasis would prevent its clinical application. In this study, the increased serum levels of calcium by vitamin D treatment were non-significantly different from the controls; the drug safety of vitamin D should be examined in the future. In summary, our data demonstrate that vitamin D has prophylactic effect on the intestinal fibrosis via induction of VDR and inhibition of TGF-b1/Smad3 pathway. It may provide a novel adjunctive treatment against IBD. However, further research is required to understand the underlying mechanisms and potential limitations of treatment. Acknowledgments This study was supported by grants from National Foundation for Natural Scientific Research of China (No 81000153) and Foundation for Natural Scientific Research of Jiangsu Province (BK2010415).
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None.
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ORIGINAL ARTICLE
Vitamin D Prevents the Intestinal Fibrosis Via Induction of Vitamin D Receptor and Inhibition of Transforming Growth Factor-Beta1/Smad3 Pathway Qingsong Tao • Baochai Wang • Yu Zheng Xiaohua Jiang • Zheng Pan • Jianan Ren
•
Received: 9 February 2014 / Accepted: 10 October 2014 Ó Springer Science+Business Media New York 2014
Abstract Background and Aims Vitamin D deficiency in patients with inflammatory bowel disease (IBD) is associated with greater disease activity and lower quality of life. Intestinal fibrosis is a main complication of IBD. However, the effect of vitamin D on intestinal fibrosis remains unclear. We investigated the prophylactic effect and the underlying mechanism of vitamin D on the intestinal fibrosis in vitamin D-deficient mice with chronic colitis. Methods Vitamin D-deficient mice were randomized into two groups receiving the vitamin D-deficient or vitamin D-sufficient diet from weaning (week 4). Intestinal fibrosis was induced by six-weekly 2,4,6-trinitrobenzene sulfonic acid administrations from week 8. At week 14, the productions of extracellular matrix (ECM) and total collagen were measured
in the colons, and TGF-b1/Smad3 signal transduction was examined in isolated colonic subepithelial myofibroblasts (SEMF). The expression of vitamin D receptor (VDR), a-SMA and Collagen I in normal SEMF and VDR-null SEMF exposed to TGF-b1 and/or 1,25(OH)2D3 was measured. Results Vitamin D significantly reduced the histological scoring, ECM and collagen productions in the colons and decreased the levels of TGF-b1, Smad-3, p-Smad3 and Collagen I in SEMF. 1,25(OH)2D3-induced VDR expression and decreased TGF-b1-stimulated a-SMA and Collagen I expressions in SEMF. Knocking down VDR expression in SEMF abolished the effect of 1,25(OH)2D3. Conclusions Vitamin D has prophylactic effect on intestinal fibrosis in the vitamin D-deficient mice with chronic colitis, which may be associated with the inhibited activation of TGF-b1/Smad3 pathway in the SEMF via VDR induction.
Q. Tao (&) B. Wang X. Jiang Z. Pan Department of General Surgery, Affiliated Zhongda Hospital, Southeast University Medical School, 87 Ding Jia Qiao Road, Nanjing 210089, Jiangsu, China e-mail: [email protected]; [email protected]
Keywords Vitamin D Intestinal fibrosis Myofibroblast TGF-b1 Smad3 VDR
B. Wang e-mail: [email protected] X. Jiang e-mail: [email protected] Z. Pan e-mail: [email protected] Y. Zheng Department of Surgery, Haian People’s Hospital, Nantong University Medical School, Nantong 226600, Jiangsu, China e-mail: [email protected] J. Ren Research Institute of General Surgery, Jinling Hospital, Nanjing University Medical School, Nanjing 210093, Jiangsu, China e-mail: [email protected]
Abbreviations IBD Inflammatory bowel disease CD Crohn’s disease SEMF Subepithelial myofibroblasts VDR Vitamin D receptor ECM Extracellular matrix a-SMA a-Smooth muscle actin p-Smad3 Smad3 phosphorylation Vit D Vitamin D
Introduction Vitamin D deficiency, defined as the serum 25-hydroxyvitamin D level of less than 20 ng/ml (50 nmol/L) [1], is
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common in inflammatory bowel disease (IBD) [2] and is independently associated with lower health-related quality of life (HRQOL) and greater disease activity [3]. Supplemental vitamin D, as an adjunctive treatment, may help in controlling IBD [4]. Intestinal fibrosis is a common and potentially serious complication of IBD, especially in Crohn’s disease (CD), that results from the abnormal wound healing of intestinal tissue following chronic inflammation [5]. Past studies have shown that vitamin D may help in liver and renal fibrosis [6, 7] and revealed that high levels of vitamin D receptor (VDR) ligands contribute to these effects [6, 8]. However, the effects of vitamin D on intestinal fibrosis in IBD still remain unclear [9]. Subepithelial myofibroblasts (SEMF) play an important role in intestinal fibrosis by regulating inflammatory responses and extracellular matrix metabolism [10]. These create a plausible link between mucosal inflammation and destruction of the subepithelial matrix. Thus, the inhibition of these processes represents a lucrative target for IBD therapies. Based on the potential immune-regulatory [11] and gut barrier-protective [12] functions of vitamin D, we investigated the prophylactic efficacy and underlying mechanism of vitamin D on the intestinal fibrosis in the vitamin D-deficient mice with chronic colitis.
Methods Vitamin D-Deficient and Vitamin D-Sufficient Diet The vitamin D-deficient diet (TD.89123) from Harlan Teklad (Madison, WI) uses vitamin-free test casein (VFT casein) as the protein source. VFT casein is extracted with denatured alcohol to reduce the fat content from about 1–0.1 %. In the process, the content of a number of vitamins is further reduced. The vitamin D-sufficient diet is the standard open formula vitamin D diet (7017 NIH-31, Harlan Teklad) containing 4.2 IU/g vitamin D3 (1 IU vitamin D3 = 25 ng cholecalciferol), which would be considered as high daily dose when calculated to human equivalency dose (HED) [13]. The special nutrient formulas are shown in Table 1. Induction of Intestinal Fibrosis and Chronic Colitis in Vitamin D-Deficient Mice Vitamin D-deficient offspring were bred from CD-1 mice (Charles River Laboratories Inc., Wilmington, MA) that were maintained on the vitamin D-deficient diet in a room with artificial UV-free light [14]. Pups were randomized into two groups receiving vitamin D-deficient or vitamin
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Table 1 Selected nutrient formulas of diets Vitamin D-deficient diet (TD.89123)
Vitamin D-sufficient diet (NIH-31-7017)
Calories from carbohydrate (%)
60.3
62
Calories from protein (%)
16.7
24
Calories from fat (%) Energy density (Kcal/g)
22.9 3.9
14 3.6
VFT casein (wg %)
18
–
Vitamin D3 (IU/g)
–
4.2
VFT vitamin-free test
D-sufficient diets from weaning (week 4). From week 8, the mice were administrated weekly with TNBS (SigmaAldrich, St. Louis, MO) for 6 weeks for induction of chronic colitis and intestinal fibrosis according to the previously described method [15]. The CD-1 strain was chosen because of its susceptibility to extracellular matrix (ECM) changes [16]. TNBS was enema administered via a 3F catheter attached to a 1-ml syringe into the rectum until the tip was 5 cm proximal to the anal verge. To ensure distribution of TNBS, mice were held in a vertical position for 60 s after the intrarectal injection. The mice received increasing doses of TNBS throughout the 6-week study: 1, 1, 1.5, 1.5, 2, 2 mg/0.1 ml per week in 45 % EtOH. The control mice received a 0.1 ml enema of phosphate buffer solution (PBS). All mice were euthanized 42 days post the initiation of TNBS induction. Animal use protocols were approved by Animal Care and Use Committee for Southeast University, and all animal studies were performed according to the institutional ethical guidelines stipulated by the Review Board of Southeast University. Histological Evaluation and Image Analysis of ECM Content in the Colon The paraffin embedded blocks representing the similar positions of colon were sectioned and stained with H&E and Masson trichrome, respectively. Histological scoring and quantitative ECM analysis were performed by two pathologists in blinded manner according to the previously described protocols [17, 18]. Histological scoring was graded determined with the H&E staining. For quantitative analysis of ECM with Trichrome staining, 6–12 randomly chosen fields for each section were photographed and transformed into digital readings by using Olympus Image Analysis Software (Olympus, Tokyo, Japan), which is allowed for quantification of the various color wavelengths with pixels as the unit of measurement. The percentage of ECM was then calculated by dividing the pixel area of the ECM by the pixel area corresponding to the total tissue in the field of view.
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Sircol Collagen Assay Total collagen content in the colon was detected with Sirius red collagen detection kit (Chondrex, Redmond, WA). Tissues were homogenized in T-PER buffer (Thermal Science, Amarillo, TX), incubated on ice for 15 min and centrifuged for 5 min at 10,6009g at 4 °C. Each protein sample was diluted in 0.5 M acetic acid to a final concentration (100 lg/ml). Optical density was read at 530 nm. Results were calculated based on collagen per 100 lg/ml protein. Analyses of Vitamin D Metabolism Considering the limit amount of murine blood, sera were diluted for assay. The levels of 25(OH)D3 and calcium in serum were evaluated by liquid chromatography–tandem mass spectroscopy (LC–MS/MS) and atomic spectroscopy (Agilent Technologies, Santa Clara, CA), respectively, according to the manufacturer’s protocols. LC–MS/MS was performed on Waters 2695 Alliance HPLC System (Waters, Milford, MA) and an analytical column (2.1 9 100 mm, 1.7 lm; Acquity UPLC CSH; Waters, Milford, MA). Isolation of Subepithelial Myofibroblasts We have previously described the isolation of the subepithelial myofibroblasts [19, 20]. Briefly, fresh mucosal samples were obtained from the colons of the mice. After washing with calcium- and magnesium-free Hanks’ balanced salt solution (HBSS; GIBCO-BRL, Gaithersburg, MD), the mucosa samples were completely denuded of epithelial cells by three 30-min incubations at 37 °C in 1 mM EDTA (Sigma). The de-epithelialized mucosal samples were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS), 50 U/ml penicillin and 50 lg/ml streptomycin (GIBCO-BRL), and incubated at 37 °C in 5 % CO2-atmosphere. The cells in suspension were removed after every 24- to 72-h culture period, and the denuded mucosal tissue was maintained in culture for up to 2 week. Myofibroblasts possess the contractile characteristics of, with immunostaining for, a-smooth muscle actin (SMA) and vimentin, but completely negative for desmin [21].
phospho-Smad3 (Cell Signaling), VDR (Abcam, Cambridge, MA), Collagen I (Sigma) and b-Actin (Sigma) using standard methods. Band intensities were quantified normalized relative to the quantity of their respective bactin bands. Reverse Transcription PCR Total RNA isolated from SEMF using Trizol reagent kit was reverse transcribed with Superscript II (Invitrogen, Carlsbad, CA). Extracted RNA was quantified by spectrophotometry (Nanodrop 2000C, Thermo, Pittsburgh, PA). PCR products were obtained after 30–40 cycles of amplification with an annealing temperature of 50–57 °C. PCR primer sequences were TGF-b1 (680 bp), 50 -GCG ATT CGA TGA TCA G-30 and 50 -AGC GGA TCT TGA GCT30 ; Smad3 (425 bp), 50 -GGA CGC TAC TAG GTC A-30 and 50 -CGT TAG CAG TGG TCG A-30 ; b-actin (155 bp) 50 -TAT GAC TTA GTT GCG TTA CAC C-30 and 50 -CCT TCA CCG TTC CAG TTT-30 . Quantitative Real-Time PCR (RT-qPCR) RT-qPCR was used to measure RNA transcripts of a-SMA (Acta2) and Collagen Ia1 (Col1a1) using the 2-DDCt method based on GAPDH amplification as previously reported [19]. Real-time quantitative Taqman PCR analysis was performed using an ABI PRISM 7700 (Perkin–Elmer, CA, USA). Specific primers and dual-labeled fluorescent probes were purchased from Applied Biosystems (Foster City, CA): Acta2 50 -CTG ACA GAG GCA CCA CTG AA-3 and 50 -ACG CAT GAT GGC ATG AGG CA-30 ; Col1a1 50 -AAG GAG TTT CAT CTG GCC CT-30 and 50 -AGC AGG TCC TTG GAA ACC TT-30 ; GAPDH 50 -TGA GGC CGG TGC TGA GTA TGT CG-30 and 50 -CCA CAG TCT TCT GGG TGG CAG TG-30 . RNA Interference SEMF at 30–40 % confluency were transfected with 20 nM of VDR-specific siRNAs (Invitrogen, 50 -CCC ACC TGG CTG ATC TTG TCA GTT A-30 and 50 -GGA CAT GAT GGA ACC GGC CAG CTT T-30 ) using Lipofectamine RNAi max (Invitrogen) according to the manufacture’s protocol.
Western Blotting
Statistical Analysis
Protein lysates were prepared in 1 9 lysis buffer (Cell Signaling Technology, Billerica, MA). Equal amounts of protein from cells were determined by the Bio-Rad Protein assay (Bio-Rad, Waltham, MA) and then immunoblotted with the appropriate antibodies: Smad3 (Santa Cruz),
Statistical significance was determined by Student’s t test or ANOVA followed by Fisher’s protected least significant difference post hoc test, as appropriate, using IBM SPSS Statistics v20 (IBM, New York, NY). Data are expressed as mean ± SE. Statistical significance was set at P \ 0.05.
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Results Vitamin D Prevented Intestinal Fibrosis Via Inhibition of TGF-b1/Smad3 Signal Transduction As shown in Fig. 1, repeated TNBS administration caused more severe intestinal inflammation and fibrosis in the mice without vitamin D supplementation (Fig. 1c). The colons showed pockets of inflammation, thickened colonic wall, persistent edematous swelling, as well as obstruction (Figs. 1c, 2 top). Histological scoring, as shown in Fig. 3a, significantly decreased by vitamin D treatment. The colons exhibited more collagen deposition in the mucosa, submucosa and serosal areas determined with Masson trichrome staining (Fig. 2, middle and bottom). In contrast, the mice with vitamin D treatment developed less severe colonic fibrosis by the quantitation of ECM (Fig. 3b). The total collagen productions measured with the Sircol assay consistently confirmed the prophylactic effect of supplemental vitamin D on the intestinal fibrosis (P \ 0.01, Fig. 3c). Meanwhile, vitamin D treatment also significantly increased the serum 25(OH)D3 level (Fig. 3d). Serum levels of calcium were also increased with vitamin D supplementation without significant difference (Fig. 3e), which reflected the calcium homeostasis was affected, but without serious adverse consequences in this study. Previous chronic TNBS colitis showed increased levels of TGF-b1 and Smad3 phosphorylation (p-Smad3) [22]. Then, we isolated the colonic subepithelial myofibroblasts (SEMF, Fig. 4) from the mice and examine the TGF-b1/
Fig. 1 Macroscopic findings of the colons from the mice in four different groups. a Control group without vitamin D treatment (Vehicle - Vit D). b Control group with vitamin D treatment (Vehicle ? Vit D). c Model group without vitamin D treatment (TNBS - Vit D). d Model group with vitamin D treatment (TNBS ? Vit D)
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Smad3 expressions in the primary SEMF before the first passage. As shown in Fig. 5, the expressions of TGF-b1, Smad3 and p-Smad3 normalized by b-actin or total Smad3, as well as the mRNA levels of TGF-b1 and Smad3 in the SEMF from the mice with or without vitamin D supplementation were strongly activated with repeated TNBS administration. Western blotting showed that type I collagen levels were increased in the model versus control colons (Fig. 5), indicative of activation of TGF-b1-Smad3 signal transduction. Vitamin D treatment decreased the expression of TGF-b1, Smad3, p-Smad3 and type I collagen (Fig. 5). These results suggest that Vitamin D ameliorates TGF-b1/Smad3 signal transduction, which may lead to suppression of intestinal fibrosis. 1,25(OH)2D3 Inhibits TGF-b1-Induced Expression of Pro-fibrotic Genes Via Up-Regulation of Vitamin D Receptor To further test the possibility and explore the mechanism underlying inhibition of intestinal fibrosis and TGF-b1/ Smad3 signal transduction, we evaluated the expressions of profibrotic genes in the SEMF from the normal colons of the mice exposed to TGF-b1 (5 ng/ml) and/or 1,25(OH)2D3 (0.01, 0.1, 10 nM) for 8 h. As expected, TGF-b1-stimulated Acta2 and Col1a1 mRNA expression, which was dose-dependently decreased by 1,25(OH)2D3 treatment (Fig. 6a, b). Western blot analysis revealed that TGF-b-induced elevation of p-Smad3, Smad3, collagen I, a-SMA levels was dose-dependently decreased by 1,25(OH)2D3 (Fig. 6c). It has been reported that Vitamin D receptor (VDR) signaling inhibits colitis by protecting the mucosal epithelial barrier [23]. To test the potential role of VDR in the antifibrosis effect of Vitamin D, We next examined the effects of 1,25(OH)2D3 on expression of VDR in SEMF. Western blotting confirmed that 1,25(OH)2D3 dosedependently induced the expression of VDR (Fig. 6c). Then, the VDR expression was knocked down in SEMF using VDR-specific siRNA (SiVDR). VDR-null or control SEMF were cultured in the presence or absence of 5 ng/ml TGF-b1 and 10 nM 1,25(OH)2D3 for 8 h. Real-time qPCR revealed that 1,25(OH)2D3 failed to decrease TGF-b1induced expressions of Acta2 and Col1a1 mRNA in VDRnull SEMF. Western blotting confirmed TGF-b1-induced expressions of collagen I and a-SMA were unaffected by 1,25(OH)2D3 in VDR-null SEMF.
Discussion In the present study, we demonstrated the prophylactic effect of vitamin D on the colonic fibrosis in the vitamin
Dig Dis Sci Fig. 2 Histology of colonic morphology. Top H&E staining (920). Middle Masson trichrome staining (920). Bottom Masson trichrome staining (94). Red box focused the areas showed in middle. ECM deposition (blue area) was decreased with vitamin D treatment
Fig. 3 Vitamin D decrease the histological scoring (a) and productions of ECM (b) and total collagen (c) in the colons, associated with the increased levels of 25(OH)D3 (d) and calcium (e) in the serum.
Results are expressed as mean ± SD, from 8 mice each group. *P \ 0.05; **P \ 0.01; ***P \ 0.001
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Fig. 4 Characterization of isolated subepithelial myofibroblasts (a, 9100), immunostaining with monoclonal antibodies against vimentin (b, positive, 9400), a-SMA (c, positive, 9400), and Desmin (d, negative, 9400)
Fig. 5 Vitamin D inhibits of TGF-b1/Smad3 signal transduction in primary isolated subepithelial myofibroblasts. a TGF-b1 mRNA. b Smad3 mRNA. c Expressions of TGFb1, Smad3, p-Smad3 and Collagen I determined by Western blotting
D-deficient mice with chronic colitis, and the effects were associated with the inhibited TGF-b1/Smad3 signal transduction via VDR induction in the colonic SEMF. Legishetty et al. [24] found that vitamin D deficiency in mice dysregulated colonic antimicrobial activity and impaired homeostasis of enteric bacteria. Kong et al. [25] found that vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility to mucosal damage and increased risk of IBD. These may link vitamin D deficiency with chronic colitis and answer the question that who should be the chicken or egg [26]. The effects of the vitamin D on regulating immune responses, including macrophages, dendritic cells, T cells and B cells
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have been studied previously [27–29]. But few study investigated the effect of vitamin D on the intestinal fibrosis, which is a common complication of IBD. Liu et al. [23] demonstrated that VDR signaling inhibited colitis by protecting the mucosal epithelial barrier. Gocek et al. [30] reported that 1,25(OH)2D3 induced a rapid increase in synthesis of VDR protein and its transport to the nucleus. The up-regulation of VDR levels may increase the responsiveness to 1,25(OH)2D3 [31]. The present study demonstrated for the first time that vitamin D may have prophylactic effect on intestinal fibrosis through the inhibition of TGF-b1/Smad3 pathway and up-regulation of VDR in the SEMF.
Dig Dis Sci
Fig. 6 1,25(OH)2D3 inhibits TGF-b1-induced expression of profibrotic genes via up-regulation of VDR. Normal SEMF were cultured in the presence or absence of 5 ng/ml TGF-b1 and 0.01, 0.1, or 10 nM 1,25(OH)2D3 for 8 h. The levels of Acta2 (encoding a-SMA, a) and Col1a1 (encoding collagen Ia1, b) mRNA were determined by qPCR. c VDR, a-SMA, Collagen I, Smad3, p-Smad3 and b-actin protein levels were determined by Western blotting. VDR-null or control SEMF were cultured with 5 ng/ml TGF-b1 and/or 10 nM
1,25(OH)2D3 for 8 h. Knocking down VDR expression by VDRspecific siRNA in SEMF abolished the effect of 1,25(OH)2D3 on TGF-b1-induced Acta2 (d) and Col1a1 (e) mRNA expressions. Western blotting confirmed no expression of VDR protein in response to VDR-specific siRNA (f, top). f Collagen I and a-SMA protein levels were determined by Western blotting. Results are expressed as mean ± SD, from 8 mice each group. *P \ 0.05; **P \ 0.01; ***P \ 0.001
TGF-b1 acts as a potent growth inhibitor for most types of cells, inducing the apoptosis of epithelial cells, stimulating the production of extracellular matrix and inducing fibrosis in various tissues in vivo [32]. Activation of TGF-b type I receptor (TGF-b1RI) on TGF-b1 binding will in turn phosphorylate Smad2 and Smad3. Targeted disruption of Smad3 is associated with diminished cell responsiveness to TGF-b1 [33]. In this study, decreased levels of Smad3 and p-Smad3 in the colonic SEMF were associated with the inhibition of intestinal fibrosis in the chronic colitis, as indicated by the decreased expression of TGF-b1-target genes, a-SMA and type I collagen. There are some limitations in this study. One of the inhibitory proteins of Smad family, Smad7, occupies the ligand-activated TGF-b1RI and interferes with the phosphorylation of Smad2/3. Up-regulation of Smad7 is associated with inhibition of TGF-b1-induced Smad signaling [34]. Thus, Smad7 and other proteins of Smad family would be examined in the further study. Furthermore, the
underlying interaction between VDR and Smad3 also needs to be determined. Although vitamin D may be effective for intestinal fibrosis, the associated dysfunction of serum calcium homeostasis would prevent its clinical application. In this study, the increased serum levels of calcium by vitamin D treatment were non-significantly different from the controls; the drug safety of vitamin D should be examined in the future. In summary, our data demonstrate that vitamin D has prophylactic effect on the intestinal fibrosis via induction of VDR and inhibition of TGF-b1/Smad3 pathway. It may provide a novel adjunctive treatment against IBD. However, further research is required to understand the underlying mechanisms and potential limitations of treatment. Acknowledgments This study was supported by grants from National Foundation for Natural Scientific Research of China (No 81000153) and Foundation for Natural Scientific Research of Jiangsu Province (BK2010415).
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None.
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