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Introduction

Various traumas on a body can cause gastrointestinal tract erosion and mucosal epithelium damage which lead to gastrointestinal tract bleeding and ulcer perforation and finally aggravate the origin of disease. The gastrointestinal mucosal epithelium is a fundamental barrier that provides protection against the outside environment. It is important to protect the mucosal epithelium from damage. The cytoprotective functions in protecting gastrointestinal tract against ongoing damage may be accomplished in both the early phase of epithelial repair known as restitution and in the subsequent, protracted phase of epithelial renewal (1–3). Restitution, the ability of epithelial cells to spread and migrate across the basement membrane to cover shallow defects, is the key initial step in repair of mucosal injury and can achieve restoration of mucosal continuity over broad areas of damage within hours (4,5). It is well established that the integrity of the gastrointestinal mucosa and other epithelium are maintained by a number of secreted factors, but many studies suggest that one important mechanism involves the secretion of the members of a protease-resistant protein family known as the trefoil factor family (TFF) (6). TFF, a recently recognized family of protease-resistant small peptides, is expressed in a regional specific pattern throughout the normal gastrointestinal tract. This family, comprising the intestinal trefoil factor (ITF) and the gastric peptides SP and pS2, plays a critical role in epithelial restitution and proliferation within the mammalian gastrointestinal tract (7). The members of this family share an array of structural features including, most notably, a motif of six cysteine residues termed a trefoil or a P domain, which is distinct from those found in other peptide families. They are rapidly upregulated at the margins of mucosal injury, and they are believed to promote epithelial cell proliferation and migration (8,9). ITF is a predominant factor of TFF and has been demonstrated in in vitro and in vivo studies to play an important role in mucosal homeostasis of the gastrointestinal mucosa (10,11). Though ITF could maintain gastric mucosal integrity, the underlying mechanisms controlling this process remain unclear. It is necessary to explore the mechanisms of ITF that regulate the proliferation, migration and maintenance integrity of gastric mucosal epithelial cells.

It is well known that phosphatidylinositol 3′-kinase (PI3K) is a vital regulatory protein responsible for maintaining cell viability and cell survival. PI3K phosphorylates phosphoinositides at their 3′-position in turn, activates downstream effector molecules. Akt/protein kinase B is a primary downstream target of PI3K intermediates and major component in cell survival. Akt is activated by translocation to plasma membrane when the PI3K-generated 3-phosphoinositides bind to its pleckstrin homology domain. PI3K/Akt signaling pathway plays an essential role in regulating cell proliferation, differentiation, apoptosis and migration (12,13). In addition to its metabolic actions, PI3K/Akt signaling pathway has been shown to preserve epithelial integrity during inflammation (14). Numerous studies have indicated that growth factors participate in many important physiological and pathologic processes in gastrointestinal diseases via regulating downstream signaling pathways which could mediate cell survival, cell apoptosis, cell migration and immune response. Evidence has shown that ITF (TFF3) as a growth factor could act on the epidermal growth factor receptor (EGFR), which then activates several downstream signaling pathways, including MAPK and PI3K/Akt signaling pathway (15). The above studies also demonstrated that ITF exerts antidepressant-like effects that might be mediated by the PI3K/Akt signaling pathway in the basolateral amygdala. These studies suggested that PI3K/Akt signaling may participate in the regulation of ITF on the cell physiological and pathologic processes. Based on these observations, we hypothesized that ITF could activate the PI3K/Akt signaling to regulate the proliferation, migration and maintain epithelial integrity of GES-1 in vitro.

The present study examined the effects of ITF on the proliferation, migration and epithelial integrity of GES-1 cells in vitro. To investigate the underlying mechanisms, we evaluated the role of the PI3K/Akt signaling pathway in these physiological processes regulated by TFF3. Thus, we investigated the protective effect of ITF on the gastric mucosa epithelium after damage aiming to lay the foundations of clinical application of ITF as a new type of gastric mucosal protective agent.

Materials and methods

Cell culture

GES-1 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in high glucose-Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Thermo Scientific, San Jose, CA, USA) with 10% FBS (Gibco, Paisley, UK; Invitrogen, Carlsbad, CA, USA), 1% L-glutamine, and a 1% solution of penicillin and streptomycin seeded at a density of 2×106 cells/ml onto uncoated flasks, and cultured in a humidified incubator at 37°C in 5% CO. When GES-1 cells reached 80% confluence, they were routinely passaged using 0.25% trypsin and were diluted 1:2 at each passage. Cells treated with appropriate concentration of LPS, ITF and LY294002 were used in the following experiments.

Cell viability assay

Cell viability was evaluated by the CCK-8 (Cell Counting Kit-8) test. Cells were placed on a 6-well culture plate at 2×106 cells/ml in 2 ml culture medium. After incubation for 12 h, the wells were treated with ITF (100 ng/ml, PeproTech, Inc., Rocky Hill, NJ, USA), LY294002 (15 μM, Cell Signaling Technology, Inc., Danvers, MA, USA) or LPS (10 μg/ml, Sigma-Aldrich, Inc., MO, USA) separately, then treated cells were incubated at 37°C in an atmosphere of 95% air and 5% CO for 24, 48 and 72 h. After treatment for different periods, cells in each group were plated on a 96-well culture plate (n=8) at 2×104 cells/well in 100 μl culture medium, after incubated for 12 h, the culture medium was removed and 100 μl serum-free medium was added with 10 μl CCK-8 solution to each well of a culture plate. After incubation for 4 h, absorbance was measured at optical density (OD) of 450 nm with a multi- detection micro plate reader (VersaMax, USA).

Cell migration assay

The migration ability of GES-1 cells was determined by their ability to cross the 8-μm pores of a migration chamber that consists of trans-wells fitted with Millipore membranes (6.5-mm filters; Costar). Cells were suspended in serum-free culture medium at a concentration of 4×105 cells/ml and then added to the upper chamber (at 4×104 cells/well). Simultaneously, 0.5 ml of culture medium with 10% FBS containing ITF (100 ng/ml) or LY294002 (15 μM) was added to the lower compartment. The cells were allowed to migrate in a humidified CO incubator at 37°C for 12 h. After incubation, cells that had entered the lower surface of the filter membrane were fixed with 90% ethanol for 30 min at room temperature, washed three times with distilled water, and stained with 0.1% crystal violet in 0.1 M borate and 2% ethanol for 30 min at room temperature. Cells remaining on the upper surface of the filter membrane were gently scraped off with a cotton swab. Images of penetrated cells were captured by a photomicroscope (BX51; Olympus). Cell migration ability was quantified in a blinded manner by counting the number of the penetrated cells on the lower surface of the membrane with five fields (x100 magnification) per chamber. Experiments were performed three times in duplicates.

Western blot analysis

The dose and time response of Akt activation by ITF was determined. From our initial observations, Akt activation occurred within minutes. On the basis of this, all further studies evaluated Akt with time-points ranging between 5 and 240 min. GES-1 cells were first treated with increasing doses of ITF (0–500 ng/ml) for 30 min. Next, the optimum dose (100 ng/ml) was used to determine the time-dependent response of Akt activation. Cell proteins were obtained from cultured or treated GES-1 cells in each group. Western blot analysis of cellular lysates was performed as previously described (22). Briefly, the cells were washed with ice-cold PBS and lysed in a modified RIPA buffer containing 1 mM DTT, 1 mM PMSF, complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA) for 30 min. The whole lysates were then centrifuged at 12,000 g for 30 min at 4°C, and the protein concentration in the supernatant was determined using BCA assays. Supernatants were mixed with an equal volume of 2X loading buffer and boiled for 10 min. The isolated protein samples were loaded at 30 μg on 12% SDS-polyacrylamide gel to perform electrophoresis. The separated proteins were then transferred to the polyvinylidene fluoride (PVDF) membranes using standard procedures. For immunoblotting, the membranes were incubated at 37°C for 1 h in blocking buffer (PBS, 0.1% Tween-20, 1% BSA and 5% non-fat milk). The primary antibodies were added to the membranes and incubated at 4°C overnight. After three washes in PBS, the membranes were incubated with 1:10,000 diluted secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG, Boster, Wuhan, China) at 37°C for 1 h. After additional washing with TBST, the target proteins on the blot membrane were visualized using the ECL system. The Odyssey Scanning System (LI-COR Inc., USA) was used for image capture. Equal loading of proteins was confirmed by visualization of β-actin. Band intensities were quantified by densitometry using Image J Software (version 1.41). The primary antibodies were employed as follows: rabbit anti-Akt, rabbit anti-pAkt (Cell Signaling Technology Inc.), rabbit anti-occludin and mouse anti-β-actin (Abcam Inc., Cambridge, MA, USA).

FDA and PI staining for morphologic evaluation

The integrity of cell membrane was detected using FDA/PI staining. Cells were placed on a 6-well culture plate at 1×106 cells/ml in 2 ml culture medium. After 12 h, cells were treated with LPS (10 μg/ml), ITF (100 ng/ml) and LY294002 (15 μM) for 24 h, and then plated at a density of 2×105 cells/well onto 96-well plates, stained with 5 μg/ml PI and 4 μg/ml FDA, and observed under a fluorescent microscope (Nikon Eclipse TE2000-S, Japan).

Flow cytometric assay

Cell apoptosis was measured by Annexin V-FITC and propidium iodide (PI) staining through flow cytometry (Becton-Dickinson, San Jose, CA, USA). The cells were transplanted to 6-well culture plates at a density of 2×106 cells/ml, after 12 h, the cells were treated with LPS (10 μg/ml), ITF (100 ng/ml) and LY294002 (15 μM), then cells were incubated at 37°C in an atmosphere of 95% air and 5% CO for 48 h. By centrifugation at 1,200 rpm for 6 min the cells were collected. After the cells were washed twice with cold PBS, they were re-suspended in 500 μl binding buffer at a concentration of 1×106 cells/ml. Each cell sample was then stained with 5 μl Annexin V-FITC and 5 μl PI according to the manufacturer’s instructions. Treated cells were incubated in the dark at 37°C for 15 min. Samples were acquired on a FACScan flow cytometer and analyzed.

Immunofluorescent staining

For studying the protective effect of ITF on maintaining integrity of GES-1 cells and investigating the regulation of Akt signaling pathway, 10 μg/ml LPS was added into cultured GES-1 cells. After treatment for 4 h, 100 ng/ml ITF was added, then treated GES-1 cells were cultured for 48 h. Immunofluorescence analysis of GES-1 cells was performed as described previously (16). Briefly, cells were first fixed with 4% paraformaldehyde for 10 min. To block unspecific binding sites, the cells were incubated with PBS containing 2% BSA for 1 h at 37°C. After blocking of the non-specific staining, the cells were incubated with the primary antibody (rabbit anti-occludin, Abcam, Inc., CA, USA) at a dilution of 1:200 at 4°C overnight. After three washes with PBST (PBS with 0.2% Triton X-100), GES-1 cells were incubated with a secondary antibody (goat anti-rabbit/mouse Alexa Fluor 594 or 488, Invitrogen Life Technologies, Gaithersburg, MD, USA) at a 1:400 dilution in 2% BSA for 1 h at 37°C in dark. Then cells were stained with 10 μg/ml 4′,6′-diamidino-2-phenylindole (DAPI, Biyuntian, Inc., Nantong, Jiangsu, China) for 10 min to identify cellular nuclei. The images were captured using a confocal fluorescence microscope (Olympus, Tokyo, Japan).

Statistical analysis

Experimental results were expressed as mean ± standard deviation (mean ± SD). Statistical analyses were performed using one-way ANOVA techniques in Microsoft Excel 2003 using SPSS software. This was followed by a Student Newman-Keuls’ post-test. Differences between treatment groups were considered significant at P<0.05, and highly at P<0.01.

Results

ITF promotes GES-1 cell proliferation and migration

In this study, we first used 100 and 500 ng/ml ITF to treat the cultured GES-1 cells in vitro. The microphotographs of GES-1 cells after treatment with ITF are presented in Fig. 1A. We found that different concentration of ITF promoted the proliferation of GES-1 cells after treatment for 48 h. Cell viability of GES-1 cells treated with 100 and 500 ng/ml ITF for 24, 48 and 72 h was then detected by CCK-8 assay (Fig. 1B). The results of CCK-8 indicated that cell viability was significantly increased in GES-1 cells treated with ITF for 48 or 72 h (P<0.01) compared with control. Transwell migration assay was used to investigate the effect of ITF on the migration of GES-1 cells after treatment with different concentrations of ITF. As displayed in Fig. 1C and D, ITF improved the migration of treated GES-1 cells compared with control (P<0.01). Furthermore, 500 and 100 ng/ml ITF promoted the cell migration by 2.3-fold (P<0.01) and 1.6-fold (P<0.01) as compared to the control. These results indicated that ITF could promote the proliferation and migration of GES-1 cells after treatment with ITF in vitro.

Figure 1 Effects of ITF on the proliferation and migration of GES-1 cells. ITF promotes GES-1 cells proliferation and migration in vitro. (A) Phase-contrast images of treated GES-1 cells. (B) Cell proliferation of treated GES-1 cells detected by CCK-8 (n=6). (C) Transwell migration analysis of GES-1 cells treated with different concentration of ITF. The images are representative of three independent experiments. (D) The number of penetrated cells was quantified using a microscope at ×100 magnification. Values are mean ± SD from three experiments. Asterisk denotes a response that is significantly different from the control (*P<0.05 and **P<0.01).

ITF activates the Akt signaling pathway

To investigate the underlying mechanisms of ITF in the processes of cell proliferation and migration, we next investigated the functional role of Akt signaling pathway in these regulation processes of ITF. Akt as a vital regulatory factor responsible for maintaining cell viability and cell survival, we first measured the pAkt expression level in GES-1 cells in response to increasing doses of ITF. Western blot illustrated that treatment of GES-1 cells with ITF resulted in an increase in protein expression of pAkt (Fig. 2A). Increasing concentrations of ITF proportionally induced the expression level of pAkt with maximum stimulation at 4.5-fold (P<0.01) as compared to the control at a concentration of 100 ng/ml (Fig. 2A). Next, we detected and evaluated the time course of pAkt induction by ITF. As illustrated in Fig. 2B, using a concentration of 100 ng/ml ITF, we found that ITF induced a peak in pAkt expression level within 30 min of exposure. The protein expression level of pAkt in treated GES-1 cells reached a peak at 15 min and increased 3.3-fold (P<0.01) compared to control. The pAkt expression level in treated GES-1 cells could be maintained for 60 min, and we did not find additional modulation in Akt kinase function following evaluation for extended time-points (Fig. 2B). The results of western blotting in Fig. 2A and B show that ITF was able to induce a dose- and time-dependent increase in Akt kinase activity.

Figure 2 Western blot analysis of the expression of pAkt in the treated GES-1 cells. ITF induced a dose- and time-dependent increase in the expression of pAkt in the treated GES-1 cells. The PI3K inhibitor LY294002 resulted in a substantial reduction of Akt expression following treatment with ITF. (A) Western blot analysis of pAkt expression in the GES-1 cells treated with different concentration of ITF. (B) Western blot analysis of pAkt expression in the GES-1 cells treated with ITF or LY294002. (C) Western blot analysis of pAkt expression in the GES-1 cells treated with ITF for different time. Responses were quantified by densitometry and normalized to the expression of β-actin. Densitometry data are shown as mean ± SD (*P<0.05 vs. control group, **P<0.01 vs. control group). n=6 at each group.

We expected that ITF-mediated PI3K activity would result in the activation of Akt kinase activity. To confirm this, we evaluated the ability of ITF to induce pAkt expression in the presence of the PI3K inhibitor LY-249002. As shown in Fig. 2C, inhibition of PI3K by LY-294002 resulted in decreased induction of pAkt expression level following treatment with ITF in GES-1 cells compared with control. All the western blot results indicated that ITF activated the Akt signaling pathway to regulate the physiological process of GES-1 cells. In the following experiments, we used LY294002 to regulate the Akt signaling pathway to evaluate the cell proliferation and migration of GES-1 cells.

Activated Akt signaling is required for ITF-induced GES-1 cell proliferation and migration

ITF have been reported to promote cell proliferation and migration of epithelial cells in some studies (7,17,18), and our experiments indicated that ITF promote proliferation and migration of GES-1 cell in vitro. To verify whether Akt signaling pathway participated in this stimulatory effect, CCK-8 assays and transwell migration assays were performed. As shown in Fig. 3A and B, the results illustrated that ITF promote the proliferation of GES-1 cells, which is decreased significantly compared with ITF group (P<0.01) by LY294002 inhibiting Akt signaling pathway. LY24002 inhibited Akt signaling to reduce the cell viability induced by ITF, which suggested that Akt signaling might be an essential downstream mediator in ITF-regulated cell proliferation.

Figure 3 ITF promotes the proliferation and migration of GES-1 cells via activating the Akt signaling pathway. ITF promoted GES-1 cell proliferation and migration in vitro which is attenuated by LY294002 via inhibiting the Akt signaling pathway. (A) Phase-contrast images of GES-1 cells treated with ITF or LY294002. (B) Cell proliferation of treated GES-1 cells detected by CCK-8. LY294002 decreased the cell viability which was induced by ITF (n=6). (C) Transwell migration analysis of GES-1 cells treated with ITF or LY294002. LY294002 inhibited the Akt signaling to reduce the penetrated cell number after GES-1 cell treatment with ITF. The images are representative of three independent experiments. (D) The number of penetrated cells was quantified using a microscope at ×100 magnification. Values are mean ± SD from three experiments. Asterisk denotes a response that is significantly different from the control (*P<0.05 and **P<0.01).

The results of transwell migration assays showed that ITF (100 ng/ml) promoted the cell migration by 1.7-fold (P<0.01) as compared to the control. In contrast, the motility was inhibited by LY29002 significantly (P<0.01) as compared to ITF group (Fig. 3C and D). This result illustrated that the cell migration induced by ITF was dramatically attenuated because of Akt signaling blockade. Therefore, the results of cell proliferation and migration demonstrated that ITF regulated the cell viability of GES-1 cells via activating the Akt signaling pathway.

ITF activates the Akt signaling pathway to protect GES-1 cells from LPS induced epithelium injury

In order to investigate the protective effects of ITF on the cell survival and epithelium integrity in GES-1 cells, 10 μg/ml LPS was used to induce epithelium injury of GES-1 cells. GES-1 cells exposed to 10 μg/ml LPS for 24 h were stained with FDA (green) and PI (red). As illustrated in Fig. 4A, necrotic cells were obviously increased after LPS treated compared with control. In contrast, ITF decreased the number of necrotic cells in LPS+ITF group, however, LY294002 inhibited the protection of ITF and increased the number of necrotic cells in LPS+ITF+ly294002 group. Apoptosis was detected in cultured GES-1 cells after 48-h exposure to LPS (Fig. 4B and C). As shown in Fig. 4B and C, apoptosis of GES-1 cells was significantly increased (P<0.01) after LPS treatment. Treatment with ITF after the addition of LPS significantly decreased (P<0.01) the incidence of apoptosis by approximately one-half (8.9±1.2%). Addition of the PI3K inhibitor LY-249002 after LPS treatment increased the number of apoptotic cells to 18% of the total population, even in the presence of ITF (18.6±2.2%). On the basis of these results, we demonstrated that the PI3K/Akt signaling pathway contributes to ITF protection against LPS-induced necrosis and apoptosis.

Figure 4 ITF activates the Akt signaling pathway to protect GES-1 cells from LPS-induced necrosis and apoptosis. LPS induced necrosis and apoptosis of GES-1 cells which is attenuated by ITF treated. However, LY294002 inhibited Akt signaling to lead the loss of protective effect of ITF. (A) Fluorescent images of treated GES-1 cells following FDA-PI staining. Cells exposed to LPS for 24 h stained with FDA (green) and PI (red). Viable cells were stained green and necrotic cells were stained red. Scale bar, 200 μm. (B) Flow cytometric analysis for quantification of apoptotic cells. Cell apoptosis percentages were evaluated by flow cytometry. Cells were stained with FITC-coupled Annexin V and propidium iodide (PI). ITF reduced the percent of apoptotic cells treated with LPS. Conversely, LY294002 blocked the protection of ITF on GES-1 cells treated with LPS. The results are shown as a percentage of apoptotic cells (Annexin V-positive) and are representative of three independent experiments with similar results. (C) Percentage of apoptotic GES-1 cells. Values are means ± SD. Asterisk denotes a response that is significantly different from the control (*P<0.05 and **P<0.01).

ITF is a potent protection factor specific to the epithelium that promotes mucosal epithelial cell survival, accelerates wound closure, and maintains epithelium integrity. In the following experiments, we detected the epithelial tight junction (occludin and ZO-1) and specific epithelium markers (CK-18 and CK-19) in GES-1 cells after treatment with LPS. The immunofluorescence results (as shown in Fig. 5) indicated that LPS induced mucosal epithelium injury and tight junction damage, which led the expression of occludin (Fig. 5A) and ZO-1 (Fig. 5B) decreased. The expression level of specific epithelium markers CK-18 (Fig. 5C) and CK-19 (Fig. 5D) was also decreased in GES-1 cells. On the contrary, ITF prevented the epithelium injury from LPS and preserved epithelial integrity of GES-1 cells. However, addition of the PI3K/Akt signaling inhibitor LY294002 resulted in the loss of protective effects of ITF on epithelium integrity.

Figure 5 ITF preserved the gastric mucosal epithelium integrity via activating Akt signaling pathway. Immunofluorescence staining data indicated that Akt signaling pathway plays an essential role in protective effect of ITF on GES-1 cells after treatment with LPS. Activation of Akt signaling by ITF preserved mucosal epithelium integrity and attenuated the epithelium damage induced by LPS. Expression of tight junction proteins (occludin and ZO-1) and specific epithelium markers (CK-18 and CK-19) were detected in this experiment. (A) Immunofluorescent analysis of occludin. (B) Immunofluorescent analysis of ZO-1. (C) Immunofluorescent analysis of CK-18. (D) Immunofluorescent analysis of CK-19. Original magnification, ×600 (scale bar, 50 μm).

Western blot analysis was performed for quantitative analysis of tight junction and expression of specific epithelium markers in treated GES-1 cells for 48 h. The results showed a similar result as immunofluorescence data supporting the protection of ITF on preserving epithelium integrity in GES-1 cells (Fig. 6) via activating the Akt signaling pathway. As illustrated in Fig. 6, the western blot results indicated that LPS decreased the expression of occludin, ZO-1, CK-18 and CK-19 in LPS group significantly (P<0.01), which is reversed via ITF treatment in LPS+ITF group. However, LY294002 decreased the expression levels of these proteins in LPS+ITF+LY294002 group via inhibiting the Akt signaling even in the presence of ITF. Therefore, immunofluorescence staining and western blot data confirmed that ITF maintained mucosal epithelium integrity against LPS-induced injury via activating the Akt signaling. It indicated that Akt signaling pathway plays a critical role in the physiological and pathological process of gastric mucosal epithelium regulated by ITF.

Figure 6 The expression levels of occludin, ZO-1, CK-18 and CK-19 in GES-1 cells were evaluated in whole cell lysates by western blot analysis. The data indicated that ITF protected the tight junction from LPS-induced injury and increased the epithelium marker expression compared with LPS treated group. LY294002 prevented the protective effect of ITF on LPS-treated GES-1 cells via inhibiting the Akt signaling pathway in vitro. (A) Western blot analysis of occludin, ZO-1, CK-18 and CK-19 expression. (B) The protein bands were quantified by densitometry and normalized to the expression of β-actin. Densitometry data are shown as mean ± SD (*P<0.05 vs. control group, **P<0.01 vs. control group). n=6 at each group.

Discussion

The trefoil factor family (TFF) peptides are small regulatory proteins consisting of three members. ITF (TFF3) is a major component in the goblet cells in small and large intestines (19), especially a typical secretary peptide of the normal human antral and pyloric gastric mucosa. Recent studies have demonstrated that ITF was able to attenuate the gastrointestinal mucosal injury caused by various injury factors and promotes the repair of damaged mucosa epithelium. ITF plays an essential role in inducing mucosal epithelial healing (7,20), cell migration (21) and maintaining normal gastrointestinal mucosal epithelium integrity (22,23). However, we know little as yet about the detailed mechanism underlying the regulation of ITF on the physiological and pathological process of gastric mucosal epithelium.

Rapid proliferation and migration of mucosal epithelial cells is the key mechanism for resurfacing epithelial defects after various forms of mucosal epithelium injury. In the present study, we explored the effects of ITF on the proliferation and migration of GES-1 cells. Firstly, we used ITF to treat the cultured GES-1 cells to detect the cell viability and migration in vitro. After GES-1 cells were treated with different concentration of TTF, we found that different concentration of ITF is sufficient to promote the proliferation and migration of GES-1 cells. The higher the concentration of ITF, the more cells proliferated and migrated. These results of ITF effects on the cell proliferation and migration were similar with previous studies (24–26). In our present study, we additionally found that ITF could preserve mucosal integrity resistance to LPS-induced injury.

Although some studies have demonstrated that TFF family including ITF, regulate cell migration and cell survival via TGF-β signaling pathway, MAPK/ERK signaling pathway, β-catenin signaling pathway or EGF signaling pathway (8,17,27–31) in some cell types, the defined underlying mechanism of protective effects of ITF on the physiological and pathological process of GES-1 cells needs to be clarified.

Many studies have indicated that ITF regulates multiple downstream pathways in remodeling of gastrointestinal mucosal tissues. The effects of ITF are transmitted to signaling cascades by still unknown adaptor proteins. Despite the absence of an identified cell surface receptor for ITF, ITF can act through the EGFR to activate several downstream effector pathways, including ERK1/2, Jun N-terminal kinase, and PI3K signal (15,32). In the present study, the results of western blotting (Fig. 2) suggested that the expression of pAk which is the essential components of PI3K/Akt signaling was upregulated in GES-1 cells treated with ITF. In addition, these data indicated that ITF induces a dose- and time-dependent increase in Akt kinase activity of treated GES-1 cells. The downstream regulatory signaling pathway of ITF, the PI3K/Akt signaling pathway as an intracellular signal transducer plays a vital role in regulating cell survival, proliferation, migration and apoptosis (12,13,32–36). In this study, the results indicated that PI3K/Akt signaling pathway participate in the regulation of ITF in GES-1 cell proliferation and migration in vitro. Using LY294002, the inhibitor of the PI3K/Akt signaling pathway, we found that the cell proliferation and migration induced by ITF was attenuated significantly. LY294002 inhibited the PI3K/Akt signaling to reduce GES-1 cell proliferation and migration, which suggested that Akt signaling as an essential signal regulated the effects of ITF on the proliferation and migration processes in vitro.

The gastrointestinal mucosal epithelium is a fundamental barrier providing protection against the outside stress environment. Interestingly, several in vitro and in vivo studies have demonstrated that trefoil peptides can also protect the intestinal epithelium from a variety of noxious agents, including bacterial toxins, chemicals, and drugs (10,37–40). It is unknown whether ITF could act to exert cytoprotective effects on the gastric epithelium integrity. Based on the above results, we used LPS to act as a damage factor to induce the GES-1 cell injury. After LPS treatment, we found that LPS induced necrosis and apoptosis in GES-1 cells. However, the addition of ITF as a protective factor protected the cells from necrosis and apoptosis. In contrast, LY294002 inhibited the protective effects via inhibiting the PI3K/Akt signaling pathway. Our results obtained from GES-1 cells indicated that ITF provided protection but only when PI3K/Akt signaling is active. In the presence of PI3K inhibition, ITF lost the ability to protect cells from necrosis and apoptosis. The results indicated that Akt signaling provides an essential function in the GES-1 cells as a central survival pathway during the ITF protection process. We also detected the epithelium tight junction (occludin and ZO-1) and epithelium marker (CK-18 and CK-19) expression in treated GES-1 cells using immunofluorescence and western blot analysis. Our results demonstrated that LPS led to epithelium tight junction damage and reduced the epithelium marker expression. Using ITF treatment, we found that ITF prevents ongoing damage induced by LPS. However, LY294002 inhibited the protection of ITF in GES-1 cells. Our results suggested that ITF is a potent survival factor for the gastric mucosal epithelium integrity and provides protection against many external stimuli. On the basis of our above investigation, we believe that protection afforded by ITF is at least in part related to activation of the PI3K/Akt signaling pathway and possibly others. Activation of this survival pathway helps to protect gastric mucosal epithelium barrier function during inflammatory stress and facilitates wound repair when the barrier is compromised.

The results in this study suggested that Akt signaling is a critical downstream effector of ITF in the protection of gastric mucosal epithelium. It is supported by the following observations: i) Akt kinase activity was upregulated by ITF in the GES-1 cell line. ii) ITF-induced cell proliferation and migration was abolished when Akt signaling was inhibited. iii) ITF maintained mucosal epithelium integrity via activating the Akt signaling.

In conclusion, our results revealed that ITF regulated GES-1 cell proliferation, migration and maintained epithelium integrity via triggering the PI3K/Akt response. As a downstream mediator of ITF, activated Akt signaling pathway contributed to the protection process of ITF. The present study supports the notion that ITF can promote cell proliferation, migration and preserve epithelium integrity. In addition, it suggests that protection of ITF in gastric mucosal epithelium is mediated in part through activation of the PI3K/Akt signaling pathway. Therefore, the PI3K/Akt axis may serve as a therapeutic target to preserve mucosal epithelial integrity and provide a useful strategy to block damage thereby limiting gastric disease such as gastric ulcer in humans.

Acknowledgements

This study was supported by a grant from the Major Projects Foundation of Nanjing Military Region (12Z32), the Medical Science Foundation for young cultivation project of PLA (13QNP038) and the Natural Science Foundation of Jinling Hospital (2013023 and 2014004).

References