Molecular Mechanisms of Transforming Growth Factor-β/Smad7 Signaling Pathway in Ulcerative Colitis

Background and Aims Abnormal transforming growth factor-β (TGF-β)/Smad7 signaling pathway may be an important mechanism of IBD.Therefore, this study was to investigate whether anti-colitis drugs modulate intestinal epithelial permeability in experimental colitis and to determine its TGF-β/Smad7 signaling pathway. Methods A murine colitis model was induced, and then anti-TNF-α and 5-ASA were administered intraperitoneally and orally respectively. Myeloperoxidase(MPO) activity, histological index(HI) of colon and the disease activity index(DAI) scores of mice were detected. Transmission electron microscopy (TEM), immunohistochemical and functional tests which included two methods: one was Evans blue(EB) and the other was FITC-dextran(FD-4), were used to evaluate intestinal mucosal permeability. The expression of epithelial E-cad, Occludin, ZO-1, TGF –β and Smad7 were analyzed. Epithelial MLCK expression and activity were determined.


Introduction
The intestinal mucosal barrier dysfunction, characterized by increased intestinal mucosal permeability, has an important effect on the initiation of in ammation in infection and immunity-induced ulcerative colitis (UC) [1][2][3] . Intestinal epithelial cells (IECs) form the barrier, which mainly consists of two parts, one is the epithelial tight junction (TJ) and the other is the apical enterocyte membrane. While TJs are located at the top of IECs, and their constituent proteins include occludin, claudin, ZO, JAM-1, etc. Adhesion junction (AJ) is a cell structure adjacent to TJ, and its constituent proteins include E-cadherin, catenin, etc. In transgenic animal model, the absence of E-cadherin can cause the dysfunction of AJs, which is similar to the pathophysiological process of in ammatory bowel disease (IBD) [4] . Moreover, the enhancement of pro-in ammatory cytokines and the degradation of TJ protein lead to increased permeability of intestinal mucosa in IBD [5] . And the contraction of cytoskeleton involved in actin in IECs could destroy TJs between cells, open the cell space and increase the permeability of intestinal mucosa, while this process requires myosin light chain kinase (MLCK) to phosphorylate myosin light chain(MLC) [6][7][8] . The abnormality of transforming growth factor-β (TGF-β)/Smad7 signaling pathway may be an important pathogenesis for IBD. Especially the high expression of Smad7 and the imbalance between Smad7 and Smad2, Smad3 can lead to the loss of anti-in ammatory effect of TGF-β, resulting in the sustainability of chronic in ammation in the intestinal tract of UC [9][10][11][12] . It has been shown that TGF-β/Smad7 signaling pathway affects the expression of MLCK in vascular smooth muscle cells [13,14] . Furthermore, TGF-β has been shown to affect the expression of MMP-9 in squamous cell carcinoma cell line of human head and neck via Smad/MLCK pathway [15] . And during in ammation of UC patients, MMP-9 could regulate MLCK expression [16,17] . Meanwhile, Su LP et al have found that the increase of intestinal permeability caused by TNF-α is due to activation of MLCK via ERK1/2 signalingenhancement of MMPs and apoptosis [18,19] . Inhibitors targeting MMP has verti ed desirable anti-in ammatory effect in mice colitis models, but less powerful on humans [20] . Recent studies suggest that mucosal healing is a new goal of UC [21] . Anti-TNF-α has a de nite property on promoting mucosal healing, but the speci c mechanism is unclear [22] . The effect of 5-ASA on intestinal mucosal healing is still controversial, and there are great differences among individuals. While Salazosulfapyridine and Balsalazide can improve intestinal mucosal permeability [23] . here is no report on whether the e cacious treatment of anti-TNF-α in refractory UC and the effect of

Induction of DSS-Colitis model
A murine colitis model induced by 5%(w/v) DSS for 7 days of free drinking [24] .

Experimental protocols
Mice were equally-randomly classi ed into the normal group, the DSS-treated group, the 5-ASA-treated group and anti-TNF-α-treated group. The latter two groups were set as treated groups. All groups were treated accordingly for 7 days.
Assessment of Disease Activity Index (DAI) Two observers recorded the following daily: body weight, fecal blood and consistency. The average daily DAI score per mouse was calculated according to the standard method [24] .

Assessment of In ammation
After laparotomy, the rst step was to examine the gross mucosal morphology of mouse colon and then two continuous pieces of distal colon were collected. For histological analysis, this study used 10% neutral buffered formalin to immobilize one of the colon to maintain the original morphological structure of the cell, followed by para n embedding for sections (4 um), and nally HE staining. The severity of in ammation was assessed by histological index (HI) [24] . The other was homogenized for assessing MPO activity [25] .

Assessment of TEM
A 0.5-cm distal ileal segment within 1cm of the ileocecal junction was xed in 2.5% glutaraldehyde at 4℃ for 6h, and in osmic acid, and then embedded in Epon as the specimen for TEM.
Assessment of E-cadherin, ZO-1 and occludin Protein Expression 1cm ileum was xed in formalin at the above concentrations, and preserved in liquid nitrogen after electron microscopic examination. The expression of E-cadherin, occludin and ZO-1 in ileum epithelial cells was detected by immuno-histochemistry.

Intestinal Permeability Assay
According to the previous method, a 6-cm segment of small intestine was used as sac by ligating both ends, and then 1.5 % (w/v) EB in PBS of 0.2 ml was injected into the sac.What's more, the sac was incubated in 20mL Krebs buffer and removed after 30 min. Then the intestinal lumen was rinsed with physiological saline until the rinse solution was clari ed, dried at 37℃ for 24h, weighed on the dry weight of the intestinal tissue, and incubated with formamide. The estimated wavelength of dye eluting amount is 655nm. And the permeability of intestine was assessed by EB and the amount of EB was calculated according to the standard curve. FITC was detected in vivo.Ligated the ileum of 6cm after anaesthesia and injected into the cavity with 0.2ml FITC solution. The portal vein blood was extracted after 30min to determine the concentration of FITC in plasma.

Assessment of MLCK Enzymatic Activity
The intestinal mucosa homogenate was prepared at 4 ℃ by taking out liquid nitrogen frozen intestinal mucosa and adding a proper amount of extract buffer. MLCK enzymatic activity of small intestine was detected according to the ELISA kit.

Detection of MLCK via immunohistochemistry
Intestinal mucosa specimens were collected, xed with 10% formaldehyde solution and embedded in para n. The expression of MLCK protein in ileum epithelial cells was detected by SP method of immunohistochemistry. The experimental steps were carried out according to the kit instructions. The control group was treated with phosphate buffered saline (PBS) instead of the primary antibody. Three visual elds were randomly selected from each slice under light microscope (40 ×), and the distribution of positive particles in the cells was observed under high power microscope (200 ×). According to the blind score of two pathologists, brown granules were found to be positive in the cells. The evaluation standard of cell staining were as follows: ≥75% was +++, 50%-< 75% was ++, 10%-< 50% was +, and < 10% was negative. All the experiments were repeated 3 times.

Detection of MLCK, TGF-β and Smad7 via Western blot
The intestinal mucosae specimens were cut into pieces in ice bath and added protein extraction buffer to prepare homogenate, frozen at-80 ℃ and thawed for three times to fully release MLCK,TGF-β, Smad7.
Then the homogenate was transferred into Ep tube, and 14000 r/min centrifugation for 15 min to extract the supernatant. Protein concentration was determined. Specimens were prepared by the following process: the extract quanti ed and adjusted for protein concentration was mixed with the sample buffer for 2 × protein electrophoresis and boiled for 5 min. Polyacrylamide gel electrophoresis (SDS-PAGE) included 10% separated gel and 5% concentrated gel. And the sample size of each lane was equal.
Protein transfer: electrophoretic bands was transferred to PVDF membranes by electric transfer. Blocking and antibody binding: blocking non-speci c antigen of PVDF membrane, in turn adding primary antibody, incubating, washing, adding secondary antibody, incubating, exposure after washing.
Assessment of the mRNA contents of TGF-β and Smad7 The contents of TGF-β and Smad7 mRNA in IECs were detected by RT-PCR. RNA was extracted from samples by Trizol method to establish RT-PCR reaction system and conditions. The samples were well mixed with Trizol, placed in static condition, and then centrifuged at 12000 r/min for 15 min at 4 ℃ to take supernatant. The supernatant was mixed with equal isopropanol well, centrifuged as above to take the precipitate.And then the precipitate was washed with 75% alcohol, dried, dissolved and frozen at-80 ℃. The concentration, purity, quantity and quality of RNA were determined. Reverse transcription cDNA was prepared by PCR cycle. And PCR reaction system consisted of cDNA 4.00ul, 10 × PCR buffer 1.96ul, MgCl2 (25Mm) 2.40ul, upstream primer (20pM) 1.00ul, downstream primer (20pM) 1.00ul, dNTP (10mM) 0.36ul, Taq DNA polymerase (5IU/ul) 0.10ul and the deionized water 10.08ul.

Ethical Considerations
The local ethics committee approved these experiments and conducted in accordance with laboratory animal management and use guidelines.

Statistical Analysis
Using SPSS 20.0 for data statistics, one-way analysis of variance (ANOVA) was used to test overall statistical differences. All results were presented in the form of mean ± standard deviation (SD). P<0.050 was considered statistically signi cant.

General situation of mice
In DSS group, the mice showed reduced activity, mental deterioration and weight loss. At the end of the experiment, there were different degrees of blood in feces, and the appearance of feces was soft or thinshaped. The DAI scores increased gradually with time ( Fig. 1). Compared with DSS group, the activity and mental state of mice in treated groups were better while the weight loss was lower and a few mice had slight bloody fecal or OB(+). DAI scores of the treated groups were between DSS group and normal group (Fig. 2).

Gross observation and pathological examination of colonic tissue
Colon mucosa was characterized by extensive hyperemia and edema in DSS group. Meanwhile, multiple erosion, bleeding spots and super cial ulcer were observed. But no obvious abnormality was found in normal group. Comfortingly, there were only scattered hyperemia and erosion without obvious bleeding and ulcer in treated groups.
HE pathological examination showed that the colonic mucosa had multiple super cial ulcers, a large number of crypt glands were destroyed and a slew of in ammatory cells in ltrated in DSS group (Fig. 3). While in the normal group, the colonic IECs were intact, the crypt glands were neatly arranged and no in ammatory cell in ltrated (Fig. 4). In treated groups, a few colon mucosa of mice were scattered with super cial ulcer, and the structure of crypt gland decreased and destroyed compared with normal group, but it was signi cantly less than that in DSS group. At the same time, the degree of in ltration of in ammatory cells in mucosa and submucosa was mild (Fig. 5). Moreover, compared with DSS group, the HI scores of the treated groups were signi cantly lower (P < 0.050, Fig. 6).
The activity of MPO in colonic homogenate of DSS group was higher, while MPO activity of the treated group decreased than that in DSS group, which suggests that the colon in ammation in DSS group was serious (P < 0.050, Fig. 7) and anti-TNF-α and 5-ASA could reduce in ammatory injury of colon (P<0.050, Fig. 8).

Ultrastructure of intestinal mucosal barrier
TEM was used to observe the ultrastructure of the IECs in the ileum of mice. From the result it can be observed that in the normal group the IECs were intact, the surface microvilli were long and dense, the arrangement was regular, and the cells were closely connected. While the DSS group showed edema or even shedding of IECs, atrophy and sparseness of surface microvilli, enlargement of intercellular space and opening of some TJs. Then the treated groups were mainly characterized by edema of some IECs, reduction of microvilli and opening of TJs, which is much better than DSS group (Fig. 9).

Intestinal mucosal barrier function
Compared with these in the normal group, the intestinal EB content and blood FITC level in DSS group increased (P < 0.050), suggesting that the intestinal mucosal barrier in DSS group was damaged and the high molecular weight EB entered the intestinal mucosa through expanded TJs, while FITC was absorbed into the portal vein system. Anti-TNF-α and 5-ASA could decrease the level of intestinal EB and blood FITC in different degrees ( P < 0.050, Fig. 10, 11).

The protein expression of TJ and AJ
The expression of Occludin, ZO-1 and E-cadherin in small intestinal mucosal epithelial cells of mice in treated groups was decreased to some extent, but was higher than that of DSS group (Fig. 12 -14), suggesting that anti-TNF-α and 5-ASA could improve the destroyed structures of TJ, AJ and other epithelial cell barriers.

Expression, distribution and activity of MLCK protein
The expression and activity of MLCK protein in small intestinal epithelial cells were higher in DSS group than those in normal group, but lower in treated group than in DSS group (P < 0.050, Fig. 15). The results of IHC were consistent with those of the Western blot (P < 0.050), suggesting that anti-TNF-α and 5-ASA intervention can downregulate the MLCK expression and activity in IECs, and thus facilitate the improvement of intestinal mucosal permeability in colitis mice. However, no signi cant difference was detected between anti-TNF-α and 5-ASA (P > 0.050, Fig. 15).

mRNA and protein levels of TGF-β and Smad7
Compared with the normal group, the mRNA and protein levels of TGF-β and Smad7 were decreased and increased respectively in DSS group. The intervention of anti-TNF-α and 5-ASA improved the situation above (Fig. 16, 17). These results suggest that anti-TNF-α and 5-ASA can enhance the expression of TGF-β and attenuate the expression of Smad7 in IECs of colitis mice, and may regulate the expression of TJassociated protein through TGF-β/Smad7 signaling pathway, which ultimately regulate the intestinal mucosal permeability in colitis mice.

Discussion
UC is a recurrent non-speci c in ammatory disease and intestinal mucosal barrier damage may be an important reason for the recurrence of UC [26,27] . In SAMP1/Yit and IL-10 (-/ -) mouse models of colitis, intestinal mucosal permeability increased signi cantly before intestinal in ammatory changes [28,29] . Repair of intestinal mucosal barrier, which can block an important part of the pathogenesis of IBD, is bene cial to control or reduce intestinal mucosal in ammation and immune response, control the condition, improve curative effect, promote mucosal healing, and maintain remission, and even play the role of etiological treatment in some cases [30][31][32][33] . Anti-TNF-α could observably decrease neutrophils in ltration in in ammatory mucosa of IBD patients and downregulate the activity of T cells and in ammatory mediators, while inhibiting neutrophils from producing pro-in ammatory mediators including ROS,TNF-α,IL-8 and so on [34,35] . Above all, through binding to the antibody, TNF-α receptor activation is blocked, leading to reduction of intestinal permeability basically due to the decrease in paracellular permeability across the TJs and apoptosis of IECs. Furthermore, upon blocking lipid rafts, in iximab could repair the colonic barrier of adhesive invasive Escherichia coli in Crohn's disease [36,37] . In this study, we observed whether anti-TNF-α and 5-ASA could improve intestinal permeability and regulate TGF-β/Smad7 signaling pathway in mice with DSS colitis, which provides a basis for exploring the molecular mechanism of TGF-β/Smad7 signaling pathway in regulating intestinal permeability of UC.
And it turned out that anti-TNF-α and 5-ASA could reduce MPO activity, alleviate IECs injury, upregulate TJs and improve intestinal mucosal structure and function, thus reducing clinical symptoms and DAI and HI scores. However, its speci c mechanism is not clear.
By blocking the adjacent intestinal epithelial spaces, TJs prevent bacteria, antigens and other substances from entering the intestinal mucosal lamina propria to activate immune cells [6,38,39] . Clayburgh DR et al found that TJs were signi cantly damaged, and the permeability of intestinal mucosa was increased in IBD patients [6] . TJs and AJs are destroyed due to internalization or loss of forming proteins such as occludin (TJs) or E-cadherin (AJs) [40] . And TJ proteins' recruitment to the apical lateral membrane is critical for closing the paracellular space. However, TJ assembly relies heavily on AJ formation [41] . 5-ASA could increase intercellular adhesion via membranous restoration of AJ proteins such as β-catenin and Ecadherin, which plays a direct impact on mucosal healing [42,43] . 5-ASA can also modulate transcriptional regulation of proteins,including junctional adhesion molecules (JAMs), claudins and epithelial cytoskeletal proteins [44] . TNF-α treatment led to internalization and disruption of junctional proteins like occludin (TJs), E-cadherin (AJs) and desmoglein-2 (desomosomes). With 5-ASA pretreatment, membranous localization of proteins were maintained [44] .
It has been studied that treated Caco-2 monolayers with TNF-α markedly increased the permeability of cell membrane and decreased claudin-1, occludin, and ZO-1 expression, accompanied by rearrangement of cytoskeletal F-actin [45][46][47][48] . Recently, it was found that some IBD-associated loci could regulate the expression of E-cadherin and the stability of AJshence, it was con rmed genetically that both of them played a role in intestinal barrier function [49][50][51][52] . In our study, we found that anti-TNF-α and 5-ASA increased the expression of the intestinal mucosal TJ protein, including E-cadherin, occludin and ZO-1, which further con rmed that TNF-α c reduce TJ proteins and recombine the cytoskeleton, thus destroying the intestinal epithelial barrier.
Actin-involved cytoskeleton contraction is closely related to MLCK according to the researches. The phosphorylated MLC by MLCK regulates the contraction of smooth and non-smooth muscle to induce cytoskeleton rearrangement, TJ disruption and opening of intercellular space, which eventually promotes the intercellular permeability [7,8] .The degree of phosphorylation of MLC depends on the activity of MLCK [53] . And MLCK could induce occludin endocytosis during A/R injury [54] . TNF-α cincrease the TJ permeability by an apoptosis-independent way [55] , owing to MLCK-related [23,[56][57][58][59] redistribution of TJ [60] and NF-kB-related down-regulation of ZO-1. Also TNF-α could upregulate distribution and expression of NF-κB p65 [61][62][63] , and NF-κB could combine with the promoter region of MLCK gene ,which increases transcription of MLCK [63] . 5-ASA regulates intestinal epithelial homeostasis by inhibiting ERK1/2,Wnt/β-catenin,NF-kB pathways and inducing cell cycle arrest [64] . Also 5-ASA Pretreatment could alleviate increase of nuclear p65 by TNF-α [44] . Blair SA et al have found that the expression of MLCK in IECs of 26 patients with IBD was increased by immuno uorescence assay [17] . However, intestinal mucosal permeability was not simultaneously measured so that the correlation between MLCK and intestinal mucosal barrier function was not proved. In our study, the expression, distribution and activity of MLCK protein in IECs were also measured. Compared with normal group, the expression of MLCK in DSS group was increased and the activity was enhanced. The intervention of anti-TNF-α and 5-ASA could decrease the expression of MLCK and the activity of MLCK (P < 0.050), which could improve the permeability of intestinal mucosa in colitis mice.
Overall, the present study showed innovative in evaluating the effect of anti-TNF-α and 5-ASA via TGFβ/Smad7 signaling pathway in experimental colitis. And we found that anti-TNF-α and 5-ASA both showed a signi cant effect on intestinal epithelial permeability in experimental colitis. The mechanism is partly due to the increase of TGF-β expression or the decrease of Smad7 expression which could inhibit epithelial MLCK expression and activity, leading to a reduction of the intestinal mucosal permeability in UC. This study may provide new evidence for treating IBD by upregulating TGF-β expression or downregulating Smad7 expression.