GLYPROLINES EXERT PROTECTIVE AND REPAIR-PROMOTING EFFECTS
IN THE RAT STOMACH: POTENTIAL ROLE OF THE CYTOKINE GRO/CINC-1

The glyproline peptide family includes the simplest proline-containing peptides, Pro-Gly, Gly-Pro, Pro-Gly-Pro (PGP) and the respective peptides with hydroxylated proline residues. Glyprolines have two common putative sources: 1) synthesis and catabolism of collagen and elastin, 2) intestinal absorption of proline-containing di- and tripeptide products of digestion (1). Glyprolines have a broad spectrum of biological activity including gastroprotection (2-6).

Gastric ulceration and the associated acute inflammation are caused by an imbalance between aggressive factors and protective mechanisms in the stomach (7-10). The gastric mucosal barrier is a complex and dynamic system that includes epithelial, immune and vascular elements (11). The functional integrity of the gastric mucosa and its secretory gland units are maintained in part by the constant renewal of the epithelium (12). An imbalance of cell proliferation and apoptosis are implicated in the pathogenesis of gastric mucosal ulceration (13).

Epithelial cells located at mucosal surface can secrete chemoattractant and pro-inflammatory cytokines in response to bacterial infections and inflammation. Cytokines play a vital role in maintaining gastrointestinal mucosal homeostasis. The epithelial cytokine response to pathogenic agents depends on various factors, including the site of infection and the specific pathogen. Thus, the epithelial cells act as an early warning system for immune and inflammatory events (14).

In the present study, we investigated the effects of two glyprolines, PGP and N-acetyl PGP (AcPGP), on the formation and healing of acetic acid-induced gastric ulcers in rats. In addition we examined the expression of the cytokine GRO/CINC-1 in these experiments, since in preliminary studies, of a wide spectrum of cytokines examined (including IL-1β, IL-6, IL-12, IL-17, TNF-α, TGFα, TGFβ), GRO/CINC-1 was the most consistently expressed in gastric epithelium after induction of ulceration with acetic acid.

MATERIALS AND METHODS

Animal studies

All of the protocols involving animals were approved by the Institutional Animal Care and Use Committee of Josai University, where the studies were performed.

Male, Wistar rats (200 – 300 g) were obtained from Sankyo Lab. Service Co. (Tokyo, Japan). The rats were kept in standard plastic and stainless wire cages, five per cage, under a 12:12 h light/dark schedule at an ambient temperature of 23 ± 2°C and were supplied with food and water ad libitum. The rats were acclimated to the testing conditions for 7 days prior to the experiment, and were then divided into four groups: sham-operated, or acetic acid-induced ulceration followed by treatment with distilled water, PGP or AcPGP. Ulcers were induced using a modification (4) of the method of Okabe et al. (15). The glyprolines were intranasally administered at a dose of 3.7 µmol/kg, either on days 1 – 3 days or on days 4 – 6 after acetic acid application or sham operation, and were sacrificed one day after the final treatment. This dose was selected on the basis of previous studies with these glyprolines (2-3). All of the protocols involving animals were approved by the Institutional Animal Care and Use Committee of Josai University, where the studies were performed.

Evaluation of gastric ulcers

The area of ulcers was blindly measured (in mm2). Sections of the gastric wall of each rat were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and embedded using a paraffin tissue-processing machine. Slices of the stomach were made at a thickness of 6 µm and stained with hematoxylin and eosin (H&E) for blind histological evaluation. The number of rats in each group was 5.

Preparation and cultivation of rat gastric epithelial cells

Rat gastric epithelial cells were isolated as previously described (16). One day after application of acetic acid to the serosal wall, the rats were anesthetized and the stomach was removed, everted, inflated with Hanks’ balanced salt solution (HBSS), and then immersed in DispaseR II solution for 1 h at 37°C. Gastric epithelial cells were dislodged by gentle pipetting of the everted surface in HBSS containing 0.1% BSA and 0.5 mM EGTA. The cells were collected by centrifugation at 50 × g for 3 min. Precipitated cells were re-suspended and washed twice with culture medium (Dulbecco’s Modified Eagle Medium mixed with Ham F-12, 1:1) containing gentamicin sulfate (100 µg/ml) and fungizone (1 µg/ml). The cells (~1 × 106) were then suspended in culture medium containing 10% FBS, and inoculated onto 35 mm plastic dishes covered with collagen gel. The cells were incubated for 3 days at 37°C in a CO2 incubator. They formed numerous small islets by the 3rd day of cultivation. The cultured gastric epithelial cells were stained for PAS and lectin to identify gastric surface mucosal cells (sections of gastric mucosa were also stained as a control for the staining of isolated cells). Cultured gastric epithelial cells were washed with PBS and fixed in 4% PFA. For lectin staining, the cultured cells were treated with 0.1 % Triton X-100 for 10 minutes, and washed with PBS. The cells were incubated with 2 µg/ml FITC (fluorescein isothiocyanate) for 30 min. For PAS staining, the cultured cells were treated with 0.5% periodic acid for 10 minutes, carefully washed with distilled water. The cells were immersed for 30 min in Schiff’s reagent, and after that washed with sulfurous acid solution. The slides were examined under an Olympus BX51 microscope (Japan).

Reverse transcription - polymerase chain reaction (RT-PCR)

Extraction of RNA was performed using the acid guanidinium thiocyanate-phenol-chloroform method. The dried pellet of total RNA was dissolved in diethyl pyrocarbonate-treated water containing RNase inhibitor. According to a conventional method, followed by reverse transcription using oligo (dT) primers, amplification was performed by PCR. PCR products were separated by 2% agarose gel containing ethidium bromide, and visualized under UV light. Primer sequences of PCR and product size are shown in Table 1.

Table 1. Sequence of primers and product size

Real time PCR

SYBR® Premix Ex TaqTM was used for real time PCR. Analysis was carried out by ABI PRISM® 7500 real-time PCR System (Applied Biosystem Inc.).

Measurement of GRO/CINC-1

On the 3rd day of the gastric surface epithelial cell cultivation, glyprolines (PGP, AcPGP), LPS or vehicle were added. Supernatants were collected after 24 and 48 h. Concentrations of GRO/CINC-1 were determined by ELISA. All data were corrected by quantitative value of DNA in each dish.

Materials

Rat (growth-regulated gene product/cytokine-induced neutrophil chemoattractant (GRO/CINC-1) assay kit, SYBRR Premix Ex TaqTM, lipopolysaccharide (LPS, E.coli), bovine serum albumin (BSA), DispaseR II and Ulex Europaeus Agglutinin-I, FITC Conjugate (FITC-UEA-I) were purchased from Immuno-Biological Laboratories Co., Ltd (Gunma, Japan), Takara Bio Inc. (Shiga, Japan), List Biological Laboratories (Campbell, California, USA), Sigma Chemical Co. (St. Louis, MO, USA) and Godo Shusei Co. (Tokyo, Japan) and Vector Laboratories Inc. (Burlingame, CA, USA), respectively. PGP and AcPGP were synthesized in the Institute of Molecular Genetics RAS (Moscow, Russia).

Statistical analysis

Statistical evaluation was carried out using the two-way Factorial ANOVA and the Mann-Whitney U test, or ANOVA and Dunnett’s Multiple Comparison test. Data are show as mean ± S.E.M. and P < 0.05 was considered significant.

RESULTS

Macroscopic and histological evaluation of gastric damage after glyproline treatment

Effects of PGP and AcPGP on the development and healing of acetic acid ulcer are shown in Fig. 1. On the 4th day after the application of acetic acid on the gastric serosa, the mean area of ulcer in the control group was 92.4 ± 5.5 mm2. Treatment with PGP or AcPGP significantly decreased the size of the ulcers to the same extent (by 76%). When examined on the 7th day after acetic acid application (glyproline treatment on the final 3 days), the mean ulcer area of the control group was 36.8 ± 9.5 mm2. Treatment with PGP or AcPGP significantly accelerated the healing of the ulcers (to 5.0 ± 3.5 mm2 and 12.5 ± 2.9 mm2, respectively). These data demonstrate that the peptides given via intranasal injection have profound protective and therapeutic effects in this ulcer model.

Fig. 1. Treatment with either glyproline (Pro-Gly-Pro, PGP; or N-acetyl-PGP, AcPGP) reduced gastric ulcer formation and promoted healing. The differences in ulcer area between Day 1 and Day 4 represent effects on ulcer formation. The differences in ulcer area between Day 4 and Day 7 represent effects on ulcer healing. Columns represent area of ulcers in mm2 (mean ± S.E.M). * P < 0.05, ΨP < 0.05 vs. the respective control group (n = 5 per group).

Histological examination of acetic acid-induced gastric ulcers in the control group showed extensive damage of the gastric mucosa, often with adhesions of the gastric wall to the liver and spleen. On day 4 there was extensive edema, severe leukocyte infiltration (predominantly neutrophils) and areas of hemorrhage in the zone of ulceration (Fig. 2). Conversely, almost no injury, except for slight edema in the submucosa of some rats, was seen in the sham group on day 4. The rats with ulcers that were treated with glyprolines exhibited less severe necrotic lesions, decreased submucosal oedema and reduced leukocyte infiltration of the gastric mucosa on the 4th day. The morphological pattern of inflammatory reaction in the PGP group on day 4 after serosal application of acetic acid was similar to the control group except for less pronounced edema. In the AcPGP group, less pronounced leukocyte infiltration and edema were evident as compared to the control group. A mild granulation tissue infiltrated mainly with fibroblasts and macrophages in the ulcer area adjacent to the serosa was typical on the 4th day. In both of the glyproline-treated groups there was preservation of the muscularis mucosae.

Fig. 2. Histological comparison of the effects of treatment with PGP or AcPGP on gastric ulcer formation (day 4 after serosal application of acetic acid). Sham: the mucous membrane and wall of the stomach appear normal, with only slight edema. Control: sections of marginal zone of the acetic acid-induced ulceration, extensive oedema, severe leukocyte infiltration with predominance of neutrophils and areas of hemorrhage in the zone of ulceration. In the PGP- and AcPGP-treated groups, the morphological pattern of inflammatory reaction on day 4 was similar to the control group, except for less pronounced oedema, and preservation of the muscularis mucosae. The AcPGP-treated group exhibited less pronounced leukocyte infiltration and oedema as found compared to the control group. A mild granulation tissue infiltrated mainly with fibroblasts and macrophages in the ulcer area adjacent to the serosa were typical on the 4th day. All sections were stained with H&E.

On the 7th day after ulcer induction, the area of ulceration in the control group was reduced by half, but the depth remained unchanged (Fig. 3). A mild granulation tissue containing mainly macrophages, fibroblasts, neutrophils and lymphocytes was formed. Treatment with either of the glyprolines on days 4 through 7 significantly accelerated healing of the ulcer. Dense irregular connective tissue with leukocytes filled the area of the stomach wall defect, mainly with fibroblasts in the PGP group. Scar tissue in AcPGP group was formed similarly to that in the PGP group. Once again, glyproline treatment resulted in preservation of the muscularis mucosae. Thus, the histological examination confirmed that PGP and AcPGP given intranasally exerted protective and therapeutic anti-ulcer effects.

Fig. 3. Histological comparison of the effects of treatment with PGP or AcPGP on gastric ulcer healing (day 7 after serosal application of acetic acid). Sham: slight oedema in submucosa of some animals. Control: No change in depth of injury as compared to day 4 (see Fig. 2). A mild granulation tissue containing mainly macrophages, fibroblasts, neutrophils and lymphocytes was formed. PGP-treated: dense, irregular connective tissue with leukocytes filled the area of the stomach wall defect, mainly with fibroblasts in the PGP-treated group. The muscularis mucosae was largely preserved. AcPGP-treated: Similar morphology to the tissues from rats treated with PGP. All sections were stained with H&E.

Effects of glyprolines on GRO/CINC-1 mRNA expression after acetic acid-induced ulceration

Expression of GRO/CINC-1 mRNA in the acetic acid-induced ulcers was significantly increased (9-fold) as compared to that in the sham group. Intranasal treatment with either PGP or AcPGP significantly suppressed GRO/CINC-1 mRNA expression on the 4th day after acetic acid application, but the effect was significantly greater with PGP (Fig. 4). On the 7th day after application of acetic acid, PGP treatment significantly reduced gastric GRO/CINC-1 mRNA expression, while treatment with AcPGP had no significant effect (Fig. 4).

Fig. 4.Changes of GRO/CINC-1 mRNA expression after acetic acid-induced ulceration. Panels A and B show as endpoint images the expression of GRO/CINC-1 and β-actin mRNA by RT-PCR at days 4 (panel A) and 7 (panel B) after ulcer induction (1, Sham; 2, Ulcer + Vehicle treatment; 3, Ulcer + PGP treatment; 4, Ulcer + AcPGP treatment). Panel C shows the quantitative determination of GRO/CINC-1 mRNA by qPCR from rat stomach homogenates on days 4 and 7 after serosal application of acetic acid, or sham operation, respectively (n = 5). Mean ± S.E.M., *P < 0.05 vs. Sham, ΨP < 0.05 vs. Vehicle, βP < 0.05 versus corresponding group treated with PGP.

Expression of mRNA and secretion of GRO/CINC-1 in cultured gastric epithelial cells

Lectin and PAS staining confirmed the identity of rat gastric epithelial cells (Fig. 5), which were isolated for determination of the effects of glyprolines on release of GRO/CINC-1 and on GRO/CINC-1 mRNA expression. There were no differences in the expression of GRO/CINC-1 mRNA between cells obtained from sham-operated rats versus those from rats with acetic acid-induced ulceration (data not shown). Treatment with the glyprolines (0.01 – 1 mM) had no effects on mRNA expression or release of GRO/CINC-1 from rat gastric epithelial cells. Thus, to confirm that these cells were capable of producing GRO/CINC-1, they were exposed to endotoxin (LPS; 1 µg/ml) as a positive control. As shown in Fig. 6A, exposure to LPS markedly increased GRO/CINC-1 mRNA expression at 1, 3 and 6 hours, but co-incubation with either of the two glyprolines did not significantly affect this expression. Moreover, GRO/CINC-1 secretion was significantly increased by exposure to LPS, but once again, neither of the glyprolines affected the level of secretion from cultured epithelial cells (Fig. 6B).

Fig. 5. Lectin and periodic acid-Schiff stain of cultured rat gastric epithelial cells and the gastric wall. Microphotographs of cultured epithelial cells (A, B) and wall of stomach (C, D) stained with FITC-UEA-1 (A, C) or PAS (B, D). The rat gastric surface epithelial cells formed small islets by the 3rd day of cultivation.

Fig. 6. Effects of PGP and AcPGP on LPS-induced release of GRO/CINC-1 and mRNA expression in cultured gastric epithelial cells. Rat gastric epithelial cells were treated with LPS and/or peptides. Panel A: Time-course of GRO/CINC-1 mRNA expression in response LPS (0.1 µg/ml) with or without the glyproline peptides (0.1 mM) for 1, 3 and 6 h (n = 3). Panel B: Release of GRO/CINC-1 in response to incubation for 12 h with LPS (0.1 µg/ml), with or without various concentrations of the glyproline peptides for 12 h (n = 5). Results are expressed as mean ± S.E.M. In each panel, all groups treated with LPS differed significantly from the “No LPS” group, but there were no significant differences among the LPS-treated groups.

DISCUSSION

Glyprolines have a broad spectrum of biological activities including protective and therapeutic effects in models of ethanol- or stress-induced damage (4, 17-19). PGP has previously been shown to exert anti-ulcer effects in several experimental models of ulceration and with various means of administration (4, 17-19). Less is known about the anti-ulcer effects of AcPGP. The results of the present study demonstrate that intranasal administration of PGP and AcPGP to rats significantly increased resistance against acetic acid-induced ulceration, as well as accelerating ulcer healing. Both glyprolines significantly reduced expression and release of the chemotaxin GRO/CINC-1, with PGP having more consistent and significant effects than AcPGP.

Some previous studies have shown that experimental gastric ulcer severity correlated with an increase in production or release of pro-inflammatory cytokines such as GRO/CINC-1, TNF-α and IL-1β (20-23). These cytokines are secreted by granulocytes, endothelial cells, fibroblasts, T cells, keratinocytes, monocytes/macrophages and epithelial cells (20-23). The cytokine-secreting ability of epithelial cells in response to inflammatory or pathogenic stimuli has been well established (24). However, there is less known regarding which cytokines are secreted from the gastric surface epithelial cells in rats. Moreover, it is unknown if glyprolines influence the expression and release of cytokines by gastric surface epithelial cells, which could contribute to their protective and pro-healing effects. In preliminary studies of a range of cytokines, we observed that GRO/CINC-1 was the most consistently expressed in gastric epithelium. We therefore performed in vitro and in vivo experiments aimed at determining if GRO/CINC-1 exerted beneficial (or detrimental) effects in terms of gastroprotection and/or enhancement of healing of gastric ulcers. Acetic acid-induced ulceration considerably enhanced expression and production of GRO/CINC-1 in gastric tissue. Treatment with glyprolines, significantly reduced the expression and release of this cytokine by gastric tissue. This clearly suggests that the beneficial effects of glyprolines in this model were dependent, to some extent, on GRO/CINC-1.

The migration of neutrophils, eosinophils, macrophages and lymphocytes into the inflamed tissue is regulated by cytokines and chemokines (25, 26). Among the major chemoattractants for neutrophils are the CXC chemokine family, such as IL-8 and growth-regulated oncogene (GRO)a, which also play an important role in the activation of those cells. In this connection, we focused on rat GRO/CINC-1, the rat orthologue of IL-8 (27). GRO/CINC-1 is secreted from gastric surface epithelial cells. Administration of glyprolines to the epithelial cells from normal and acetic acid ulceration stomachs did not change the expression or release of GRO/CINC-1 in vitro. It is possible that the isolation of the gastric epithelial cells resulted in a diminution of their responsiveness to the glyprolines. However, the gastric epithelial cells did retain the ability to respond to LPS. Administration of LPS at a dose of 0.1 µg/mL significantly increased GRO/CINC-1, a concentration in the range previously shown to significantly affect mucosal resistance to injury and gastric secretion (30, 31). It is also possible that in the context of ulcer healing, the epithelium may not have been the primary target for the beneficial effects of the glyprolines. Rather, preservation of subepithelial structures, as was observed with the muscularis mucosae in glyproline-treated rats, may have been more important to prevention of ulceration and promotion of healing. A role for GRO/CINC-1 in the pathogenesis of H. pylori-induced leukocyte activation in the stomach of Mongolian gerbils has also been suggested (32). Suzuki et al. (33) reported that GROα levels were markedly increased in H. pylori-positive human gastric tissue in a similar manner as the IL-8 levels. Their data suggested that GROα played a role of pathogenesis of H. pylori-associated gastric mucosal injury. The two glyprolines we tested in the present study, PGP and AcPGP, had no effect on LPS-induced GRO/CINC-1 mRNA expression or secretion in cultured gastric epithelial cells. In the acetic acid-induced model, ulceration of the stomach is initiated from the serosa side of the stomach. This is in contrast to the ulceration associated with H. pylori-associated ulceration or with use of nonsteroidal anti-inflammatory drugs, where the injury occurs from the mucosal side. However, there is a common role for neutrophils in mediating a significant portion of tissue injuries in all of these scenarios, and therefore the potential for a common role of neutrophil chemoattractants such as IL-8 and GRO/CINC-1.

The gastroprotective and repair-promoting effects of glyprolines may have been mediated in part through release of other angiogenic and reparative factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which have well-characterized abilities to promote healing in experimental ulcer models (35, 36). Indeed, PGP has been shown to increase expression of the VEGF family of genes, and to reduce tissue ischemia (37).

In conclusion, we have demonstrated the ability of two glyprolines, administered intranasally, to profoundly reduce the severity of gastric damage and to accelerate healing of gastric ulcers in rats. Inhibition of GRO/CINC-1 expression and secretion by the glyprolines may contribute to the beneficial effects of the two glyprolines that we tested. The beneficial effects of glyprolines in the stomach may be produced, in part, through reduction of neutrophil infiltration into the mucosa.

Acknowledgements: This research has been supported by the grant “JREX Fellowship Program” from the Foundation of Japanese-Russian Center. Dr. Wallace’s research is supported by a grant from the Canadian Institutes of Health Research.

Z.V. Bakaeva, A.D. Sangadzhieva, S. Tani, and T. Tanaka contributed equally to the work.

Conflict of interest: None declared.

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