One hundred and twenty male Sprague-Dawley (SD) rats aged 4 wk and weighing 125-150 g were provided by the Animal Center of Zhejiang Chinese Medical University. The animals were housed in groups of five per cage under controlled illumination (12:12 h light/dark cycle, lights on/off: 6 h/18 h), humidity (60%) and temperature (22 °C ± 2 °C) for one week to acclimatize to the environment. The animals were then randomly divided into two groups (60 in each group): Control group (the rats were able to eat regular food and drinking water) and the model group (the rats were able to eat regular food and drinking water containing 100 μg/mL MNNG); each group was then randomly divided into three groups (20 rats in each group): C/M15, C/M25 and C/M40 (15, 25 and 40 represent the number of feeding weeks). The Animal Care and Use Committee of the Zhejiang Chinese Medical University approved the protocol.

Rats were fed normal food and drinking water or drinking water with MNNG. Rats in C/M15 were sacrified at the end of the 15^th week in both the control group (C15) and the model group (M15) and the gastric tissue was collected immediately and stored at -80 °C or was perfused with cold PBS on ice and then placed in a tube with 4% paraformaldehyde to obtain various measurements as follows. Similarly, rats in C/M25 or C/M40 were sacrified at the end of the 25^th week or 40^th week and gastric tissue was collected. The experimental protocol is described in Figure 1.

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Figure 1 The experimental protocol. VEGF: Vascular endothelial growth factor; STAT3: Signal transducer and activator of transcription 3.

The rats were killed at the end of the 15^th week, 25^th week and 40^th week respectively. The stomachs were separated immediately and were opened along the long axis on ice to record the following conditions: Edema, hyperemia, erosion, ulcer or mass, and photographs were taken by two pathologists who were unaware of the treatment groups. The stomach tissue was then divided into two parts, one was stored at -80 °C and the other was placed in tubes with 4% paraformaldehyde (PFA).

Gastric tissue was perfused with PBS followed by 4% PFA in 0.1 mol/L phosphate buffer and cryoprotected in 30% sucrose in PBS overnight at 4 °C. Samples were embedded in paraffin wax, and 5 μm specimens were stained with hematoxylin and eosin. The results of histology were divided into four types according to the following histological features: Chronic superficial gastritis, chronic atrophic gastritis, dysplasia and tumor.

Gastritis was defined as inflammation of the stomach lining associated with mucosal injury. Atrophic gastritis was characterized by chronic inflammatory processes of gastric mucosa leading to the loss of appropriate glands[19] and gastric dysplasia was diagnosed according to the classification of gastrointestinal tract epithelial neoplasia by the World Congress of Gastroenterology in Vienna in 1998[20]; gastric tumor was diagnosed by the presence of tumor cells.

The expression of p-STAT3 and VEGF in gastric mucosa in the different groups was evaluated using immunohistochemistry. Specimens were cut into 1-cm blocks transversely oriented to the hippocampal long axis. Blocks were placed in buffered PFA (Sigma, St Louis, MO, United States). After 48 h, the specimens were paraffin-embedded for immunohistochemistry as described by Jung et al[21] and Meng et al[22]. Briefly, endogenous peroxidase activity was blocked using 3% H₂O₂ in methanol for 15 min. The sections were washed, and stained with a rabbit polyclonal antibody against p-STAT3 and VEGF (Santa Cruz Biotechnology, TX, United States) diluted 1:50. Primary antibody binding was detected using the Bond Polymer Refine Detection Kit (Leica, Wetzlar, Germany). The sections were counterstained with hematoxylin, dehydrated and mounted.

Scoring of immunohistochemical results was performed according to the semi-quantitative method as described by Meng et al[22]. Briefly, the percentage of immunopositive cells was scored using a four-point system: 0 points, < 5% of positive cells; 1 point, 5%-25% of positive cells; 2 points, 26%-50% of positive cells; 3 points, 51%-75% of positive cells; 4 points, > 75% of positive cells. The staining intensity was scored similarly, with 0 points for negative staining, 1 point for weak staining (light yellow), 2 points for moderate staining (brown), and 3 points for strong staining (dark brown). The immunoreactivity score for each lesion was calculated as (the score for the percentage of immunopositive cells + the score for the staining intensity)/2. Based on the immunoreactivity scores, immunohistochemical staining was considered negative (< 0.5), weakly positive (0.5-1.5), or strongly positive (> 1.5).

Frozen gastric tissue samples (whole layer) were homogenized in homogenization buffer (50 mmol/L Tris-HCl, pH 7.2) containing Na3VO4 and a protease inhibitor cocktail (Sigma-Aldrich) using an OmniTH homogenizer (Omni International, Marietta, GA, United States). After sonication, the homogenate was centrifuged at 2000 rpm for 5 min. The resulting supernatants were collected as total proteins and protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad Inc., Hercules, CA, United States)[23].

Western blots were performed as previously described by Li et al[24] with minor modifications. Briefly, the proteins were separated on 12.5% SDS polyacrylamide gels and blotted onto nitrocellulose membranes. These membranes were incubated in PBST-milk (0.1% Tween-20 and 5% milk), followed by primary antibodies (2 h) for p-STAT3 and VEGF and β-actin (Santa Cruz Biotechnology Inc., TX, United States). The blots were then washed with PBST 3 times, and subsequently incubated for 1 h with corresponding fluorescently labeled secondary antibodies (Rockland Immunochemicals, Inc., Gilbertsville, PA, United States) diluted in PBST-milk. The blots were washed 3 times and the fluorescence intensity of the detected protein bands was quantified using the Quantity One system (Bio-Rad Inc., Hercules, CA, United States).

Statistical analyses were performed using SigmaStat 17.0 (SPSS, Chicago, IL, United States). Data are reported as means ± SE. The Student’s t-test was used to analyze the difference in measurements between the ES group and sham-ES group. Analysis of variance (ANOVA) was used for multiple comparisons. A P value of < 0.05 was considered statistically significant.

Administration of MNNG induced significant changes in macroscopy and microscopy in the model group compared with the control group. Little hyperemia and sporadic erosion were observed in gastric tissue at the end of the 15^th week (A15 group); and ulcer, reduction in mucosal folds, gray mucosa and even some mucosal hyperplasia in addition to hyperemia and erosion were observed at the end of the 25^th week (A25 group); obvious masses were observed at the end of the 40^th week (A40 group) (Figure 2A). Respective changes in microscopy were observed in these treatment groups. Inflammatory cells were observed in gastritis samples in the A15 group, loss of appropriate glands in some samples in the A25 group and A40 group and tumour cells were noted in some samples in the A40 group (Figure 2B). These changes were not observed in the control group.

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Figure 2 Administration of N-methyl-N'-nitro-N-nitrosoguanidine induced macroscopic changes: Gastritis, atrophic gastritis, dysplasia and gastric tumor in rats (A) and respective microscopic changes were observed by hematoxylin and eosin staining (B).

Only 20% of rats had gastritis (4/20) in the C15 group, while 42.1% of rats had gastritis (8/19), 10.53% of rats had atrophic gastritis (2/19) and 5.3% of rats had dysplasia (1/19) in the M15 group (Table 1). In the C25 group, 30% of rats had gastritis (6/20), 10% of rats had atrophic gastritis (2/20) and none of the rats had dysplasia or tumour; however, in the M25 group, 31.58% of rats had gastritis (6/19), 26.32% of rats had atrophic gastritis (5/19) and 15.79% of rats had dysplasia (3/19) although no rats had tumours (Table 1). At the end of the study, 16.67% of rats had gastritis (3/18) in the M40 group (vs 35% in the C40 group, 7/20), 27.78% of rats had atrophic gastritis (5/18) (vs 20% in the C40 group, 4/20) and 27.78% of rats had dysplasia (5/18) (vs 5% in the C40 group, 1/20). It was observed that 27.78% of rats had tumours (5/18) in the M40 group (vs 0% in the C40 group, 0/20) (Table 1).

The expression of p-STAT3 and VEGF in the model groups measured by immunohistochemistry increased significantly compared with the control groups. The expression of p-STAT3 in gastric tissue was mainly in the nucleus (Figure 3A), and the immunochemical score for p-STAT3 in tissues with gastritis, tissues with atrophy and tissues with dysplasia were significantly higher than those in the control groups (all P values < 0.05), and the expression of p-STAT3 in tissues with dysplasia was higher than that in tissues with gastritis and tissues with atrophy (P < 0.001, P < 0.001, respectively). The expression of p-STAT3 in tissues with tumor was substantially higher than that in tissues in the control group (P < 0.001); however, the difference in expression of p-STAT3 between tissues with gastritis and tissues with atrophy was not significant (P > 0.05) (Figure 4A).

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Figure 3 Expression of p-signal transducer and activator of transcription 3 and vascular endothelial growth factor in the model groups. A: The expression of p-signal transducer and activator of transcription 3 (STAT3) measured by immunohistochemistry. From a normal sample to samples with tumor induced by the administration of N-methyl-N'-nitro-N-nitrosoguanidine, the expression of p-STAT3 was more obvious; B: Similar results for the expression of vascular endothelial growth factor were observed.

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Figure 4 Immunohistochemical score of p-signal transducer and activator of transcription 3 and vascular endothelial growth factor. A: The immunohistochemical score of p-signal transducer and activator of transcription 3 (STAT3) was significantly increased by N-methyl-N'-nitro-N-nitrosoguanidine during the progression from gastritis to gastric tumor. The p-STAT3 score in precancerous lesions and tumor samples was significantly higher than that in the normal sample; and the p-STATS score in dysplasia samples was significantly higher than that in gastritis and atrophic gastritis, which was comparable to that in tumor samples; B: Similar results were observed for the vascular endothelial growth factor score (^aP < 0.05 vs normal group; ^cP < 0.05 vs gastritis or atrophic gastritis).

Similarly, the expression of VEGF, mainly in the cytoplasm (Figure 3B), was higher in precancerous lesions than in the control group (P < 0.05) (Figure 4B). Consistent with p-STAT3, the expression of VEGF in tissue with dysplasia was higher than that in tissue with gastritis, tissue with atrophy and the control group (P < 0.001, P < 0.001, respectively); the expression of VEGF in tissue with stromal tumor was substantially higher than tissue in the control group (P < 0.001) (Figure 4B), however, the difference between the tissue with gastritis and tissue with atrophy was not significant (P > 0.05).

To further determine whether the observed p-STAT3 and VEGF changes were consistent with the changes shown by immunohistochemistry, proteins were extracted from scraped freshly excised stomach tissue, and the samples were analyzed by SDS-PAGE and Western-blot using primary antibodies for p-STAT3 and VEGF. As shown in Figure 5: (A) both p-STAT3 and VEGF protein expression were significantly higher in tissue with gastritis, atrophy, dysplasia or tumor than in the control group (P < 0.05); (B) both p-STAT3 and VEGF protein expression were significantly higher in tissue with dysplasia or tumor compared with that in tissue with gastritis or atrophy (P < 0.05); and (C) there was no significant difference in p-STAT3 and VEGF protein expression between tissue with dysplasia and tumor (P > 0.05).

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Figure 5 Protein expression of p-signal transducer and activator of transcription 3 and vascular endothelial growth factor. A: The protein expression of p-signal transducer and activator of transcription (STAT3) in dysplasia and tumor samples was more obvious than that in the other samples; B: The protein expression of p-STAT3 in dysplasia and tumor samples was significantly higher than that in gastritis and atrophic gastritis samples, but the difference between the dysplasia and tumor samples was not significant. The same results were observed for gastritis and atrophic gastritis samples; C: Consistent results were found for the protein expression of vascular endothelial growth factor in samples (^aP < 0.05 vs normal group; ^cP < 0.05 vs gastritis or atrophic gastritis).

A strong correlation was observed between the expression of p-STAT3 and VEGF both in the immunohistochemistry and Western Blot results. In order to further examine the relationship between the expression of p-STAT3 and VEGF, we performed a relativity analysis and found that there was a strong positive correlation in the immunohistochemical score between the expression of p-STAT3 and VEGF (R² = 0.86, P < 0.001) as well as protein expression measured by Western Blot (R² = 0.90, P < 0.001), suggesting that p-STAT3 may regulate VEGF to promote the formation and development of gastric cancer (Figure 6).

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Figure 6 Protein expression of p-signal transducer and activator of transcription 3 and vascular endothelial growth factor. A: The protein expression of p-signal transducer and activator of transcription (STAT3) in dysplasia and tumor samples was more obvious than that in the other samples; B: The protein expression of p-STAT3 in dysplasia and tumor samples was significantly higher than that in gastritis and atrophic gastritis samples, but the difference between the dysplasia and tumor samples was not significant. The same results were observed for gastritis and atrophic gastritis samples; C: Consistent results were found for the protein expression of vascular endothelial growth factor in samples (^aP < 0.05 vs normal group; ^cP < 0.05 vs gastritis or atrophic gastritis).

Signal transducer and activator of transcription 3 (STAT3) has been shown to play a vital role in human gastric cancer angiogenesis. Vascular endothelial growth factor (VEGF) plays a very critical role in cancer metastasis due to its function in vascular formation. Several researchers have demonstrated that the expression of STAT3 is potentially associated with the expression of VEGF-C in various malignant diseases; Judd et al reported that constitutive STAT3 activation promoted VEGF expression and stimulated tumor angiogenesis. However, to the best of our knowledge, the role of STAT3 and VEGF in the formation of gastric precancerous lesions and gastric cancer induced by “N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) is still unknown.

The effects of STAT3 and VEGF on gastric cancer have been reported. The present study showed that STAT3 activation plays an important role in the changes in gastric mucosa from chronic gastritis to gastric tumor via the STAT/VEGF pathway.

To the best of our knowledge, this is the first report to show that p-STAT3 is also persistently activated during the progression from chronic gastritis to gastric carcinoma induced by the administration of MNNG in rats, and was positively associated with the expression of VEGF.

It was shown in this study that STAT3 is partially responsible for the progression from chronic gastritis to gastric carcinoma induced by MNNG and was significantly related to the expression of VEGF during this process. It is considered that STAT3 can induce an abnormal level of VEGF expression to promote the formation of gastric carcinoma.

Previous studies have established that STAT3 has close relationship with VEGF in cancers. However, this is the first time to report that p-STAT3 is also persistently activated from chronic gastritis to gastritis carcinoma induced by administration of MNNG in rats, which is positively associated with the expression of VEGF.