All animal procedures were in accordance with the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985), authorized by our Local Institution (protocol #03/2013), and were in accordance with the ARRIVE Guidelines (McGrath et al., 2010). A total of 56 male Sprague-Dawley rats (14 weeks old, 250–300 g) were purchased from Charles River Laboratories (Calco, Milan, Italy). Animals were maintained in plastic cages under standard environmental conditions and feed ad libitum in the Animal Facility of the Department of Clinical and Experimental Medicine of the University of Messina, Messina, Italy. Animals were allowed to acclimatize for 1 week before the beginning of the experiments.

PDRN was a kind gift of Mastelli S.R.L. (Sanremo, Italy) and was prepared fresh daily in a 0.9% NaCl solution. The dose and route of administration were chosen according to previously published reports (Galeano et al., 2008; Altavilla et al., 2011; Bitto et al., 2011; Polito et al., 2012). DNBS was purchased from Sigma-Aldrich (Milan, Italy) and dissolved in 50% ethanol. DSS (MW = 36,000–50,000), was purchased from MP Biomedicals (Solon, OH, USA) and prepared daily in fresh drinking water at 8%. 3,7-Dimethyl-1-propargylxanthine (DMPX), an antagonist of A_2A receptor, was purchased from Sigma-Aldrich and was prepared fresh daily in dimethyl sulfoxide (DMSO) and then 1:10 in saline. The dose and route of administration were chosen according to previously published reports (Munzar et al., 2002; Arolfo et al., 2004; Yao et al., 2006; Thorsell et al., 2007; Balasubramanyan and Sharma, 2008)

Colitis was induced in 21 fasted rats under light anesthesia by a single intra-colonic instillation of DNBS (25 mg in 0.8 ml 50% ethanol) through a catheter that was inserted into the colon (for 8 cm) via the anus up to the splenic flexure. Animals were then kept for 15 min in Trendelenburg position to avoid reflux and after 6 h were randomized to receive the vehicle (1 ml/kg; n = 7), PDRN (8 mg/kg, n = 7), or PDRN+DMPX; (10 mg/kg; n = 7) intraperitoneally. The treatment was administered daily until the day of sacrifice. Control animals (n = 7) received a single intra-colonic instillation of 0.8 ml saline solution. At day 7, rats were sacrificed, the abdomen was opened by a midline incision and the descending colon was removed, opened along the anti-mesenteric border, rinsed and cut into two equal pieces, one stored for histological assessments, the other for measurement of biochemical markers. Blood was collected by heart puncture, centrifuged, and kept frozen until analysis.

Colitis was induced in 21 rats by oral administration of 8% DSS dissolved in drinking water from day 0 to day 5, as reported by Onishi et al. (2015). After 24 h, the animals were randomized to receive either PDRN (n = 7), or PDRN+DMPX (n = 7), or vehicle (n = 7) as above for the duration of the study. Control animals (n = 7) received standard drinking water. At day 5, animals were sacrificed, blood and colon samples were collected and stored as described above.

In both models the body weight, food intake, stool consistency, rectal bleeding, or the presence of blood in the stool were monitored daily for the duration of the study. Body weight and food intake were recorded every day between 9:00 and 10:00 a.m. from the day of colitis induction to the end of the experiment. Body weight results were expressed as raw data and compared with food intake, by using the following formula: food intake divided by body weight in grams and multiplied by 100. In the DSS model water consumption, from a graduated bottle was recorded every day between 9:00 and 10:00 a.m. from day 0 to the end of the experiment.

Colon length was measured and damage was scored by two independent observers, as previously described (Wallace et al., 1992), according to the following criteria: 0 (no damage), 1 (localized hyperemia without ulcers), 2 (linear ulcers with no significant inflammation), 3 (linear ulcers with inflammation at one site), 4 (two or more major sites of inflammation and ulceration extending 1 cm along the length of the colon), and 5–8 (one point is added for each centimeter of ulceration beyond an initial 2 cm).

For light microscopy, colon tissues were fixed in 10% buffered formalin for 24 h, then specimens were embedded in paraffin, sectioned at 5 μm thickness, stained with hematoxylin and eosin (H&E), and observed with a Leica microscope (Leica Microsystems, Milan, Italy). Assessment of tissue changes was carried out by two observers blinded to the experimental protocol. The following morphological criteria were considered: score 0 (no damage), score 1 (mild: focal epithelial edema and necrosis), score 2 (moderate: diffuse swelling and necrosis of the goblet cells), score 3 (severe: necrosis and neutrophil infiltrate in the submucosa), score 4 (very severe: widespread necrosis with massive neutrophil infiltrate and hemorrhage) as previously reported (Rahimian et al., 2010).

Paraffin-embedded tissues were sectioned (5 μm), rehydrated, and antigen retrieval was performed by using 0.05 M sodium citrate buffer (pH 6.0) in a microwave for 5 min. Tissues were treated with 1% hydrogen peroxide to block endogenous peroxidase activity, and with normal horse serum (Vector Laboratories, Burlingame, CA, USA) to prevent non-specific staining. Primary antibodies against either Bax (Abcam, Cambridge, UK) or Bcl-2 (Cell Signaling Technologies Danvers, MA, USA) were used and the slides were kept overnight at 4°C in a humid box. The slides were then washed in phosphate buffer sodium (PBS), the appropriate secondary antibody was added, and the ABC system (Vectastain Elite ABC kit, Vector Laboratories) was used to detect antibody localization. The location of the reaction was visualized with diaminobenzidine tetra-hydrochloride (Sigma-Aldrich, Milan, Italy). Slides were counterstained with hematoxylin, dehydrated, and mounted with coverslips. As a part of the histologic evaluation, all slides were de-identified with regards to treatment group and evaluated by a pathologist at 5× to 40× magnification with a Leica microscope (Leica Microsystems, Milan, Italy).

Myeloperoxidase (MPO), a marker of polymorphonuclear leukocyte accumulation, was determined as previously described (Mullane et al., 1985). Equal amounts of colon tissue were homogenized mechanically with the MICCRA D-1 homogenizer (Miccra Gmbh, Müllheim, Germany), in a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7.0). Lysates were then centrifuged for 30 min at 15,000 rpm at 4°C. An aliquot of the supernatant was allowed to react with a solution of 1.6 mM tetra-methyl-benzidine and 0.1 mM H₂O₂. The absorbance was measured with a spectrophotometer at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 μmol hydrogen peroxide/min at 37°C and was expressed in units per gram of tissue.

The levels of malondialdehyde (MDA) in the colon were determined as an indicator of lipid peroxidation (Ohkawa et al., 1979). Equal amounts of colon tissue were homogenized mechanically with the MICCRA D-1 homogenizer (Miccra Gmbh, Müllheim, Germany), in 1.15% KCl solution, a 0.1 ml aliquot of the homogenate was added to a reaction mixture containing 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid, 1.5 ml of 0.8% thiobarbituric acid and 700 ml distilled water. Samples were boiled for 1 h at 95°C and centrifuged at 3000×g for 10 min. The absorbance of the supernatant was measured by spectrophotometer at 650 nm.

Serum samples were assayed in duplicate using commercially available enzyme linked immunosorbent assay (ELISA) kits for Interleukin 1 beta (IL-1β) and TNF-α (Abcam, Cambridge, UK). The absorbance was read at 405 nm and the results were obtained interpolating the measurements with the respective standard curves. For each sample, the mean of the duplicates was used and expressed in pg/ml.

All quantitative data are expressed as mean ± SEM for each group and compared by one-way or two-way ANOVA for non-parametric variables with Tukey’s post-test for intergroup comparisons. Statistical significance was set at p < 0.05. Graphs were drawn using GraphPad Prism software version 5.0 for Windows (GraphPad Software Inc., La Jolla, CA, USA).

Colitis was successfully induced in rats as early as 6 h after DNBS treatment as demonstrated by the appearance of diarrhea followed by significant loss in body weight from day 3. PDRN-treated animals demonstrated an initial slight weight reduction (not significant) followed by a recovery by the end of the experiment (p < 0.0001 vs animals treated with DNBS+drug vehicle) with the appearance of solid feces after 3 days (Figures 1A,B). The concomitant administration of DMPX prevented weight and appetite loss in the DNBS+PDRN+DMPX group. Furthermore, at the end of the study, this group still had diarrhea, suggesting that DMPX completely blocked the effectiveness of PDRN in these animals (Figures 1A,B).

DSS-administered animals showed diarrhea and rectal bleeding after 24 h, while weight reduction was observed starting from day 3 (p < 0.0001 vs control group at the end of experiment). Treatment with PDRN resulted in reduced bleeding and improved food intake and body weight (p < 0.0001 vs DSS+drug vehicle at the end of the experiment; Figures 1C,D). DMPX abolished the positive effect of PDRN on food intake and body weight in DSS administered animals. Rectal bleeding was still present in these animals at the end of the study, indicating that DMPX fully antagonized the effects of PDRN (Figures 1C,D).

At the end of the experiment, the colon of animals in the DNBS+drug vehicle group appeared ulcerated, edematous, and hyperemic compared to control group (p < 0.0001; Figures 2A,B). In the group treated with PDRN a significant reduction in the extent and severity of colon injury was observed (p < 0.0001 vs DNBS+drug vehicle group; Figures 2A,B). As shown in Figure 2D, DNBS also caused a significant shortening of the colon, as compared to control animals (p < 0.0001). Treatment with PDRN significantly improved this condition. The positive effects of PDRN administration on macroscopic damage were blunted by DMPX (Figures 2A–D).

In the DSS+drug vehicle group, as well as in the DSS+PDRN+DMPX group, the presence of non-formed feces and blood was observed at the end of the study. However, the animals receiving PDRN demonstrated normal colon appearance in contrast to the animals that did not receive PDRN (Figure 2C). As shown in Figure 2E, DSS caused a significant reduction in colonic length as compared to control animals (p < 0.0001). Treatment with PDRN significantly improved this condition. No difference was observed in water consumption among groups during the study period.

A normal appearance of the colonic mucosa with intact epithelium was observed in the control group (Figures 3A,E). DNBS administration resulted in epithelial necrosis, massive infiltration of neutrophils and macrophages into the mucosal and submucosal layers, thickening of the colon wall, loss of goblet cells and edema (Figures 3B,E). The administration of PDRN resulted in: (i) a significant reduction in the extent and severity of epithelial and mucosal alterations; (ii) regeneration of the epithelium with preservation of crypts and goblet cells; and (iii) reduced infiltration of inflammatory cells (Figures 3C,E). Blockade of the adenosine receptor with DMPX counteracted the beneficial effects of PDRN (Figures 3D,E).

In the DSS model, the histological analysis showed a flattening of the mucosa with a reduced presence of mucosal glands, a massive inflammatory infiltrate and edema in the submucosal layer (Figures 4B,E) as compared to control animals (Figures 4A,E). Treatment with PDRN significantly reduced the extent and severity of the histological alteration associated with DSS administration and stimulated regeneration of the epithelium, crypts, and goblet cells (Figures 4C,E). DMPX administration abrogated the protective effects of PDRN on histological features (Figures 4D,E).

Tissues taken from control rats of both experimental models demonstrated, as expected, very little staining for Bax (Figure 5A). Sections obtained from the DNBS+drug vehicle group exhibited a diffuse positive staining for Bax (Figure 5B), demonstrating the activation of apoptosis in the epithelial and submucosal layers. PDRN administration markedly reduced the degree of Bax positive staining, almost restoring a normal appearance of the colon (Figure 5C), while DMPX co-administration abrogated the protective effect of PDRN (Figure 5D). The Bcl-2 staining in the control group from both experiments was highly positive (Figures 6A and 8A). DNBS administration almost abrogated the presence of Bcl-2 due to the massive necrosis of the tissue (Figure 6B). PDRN treatment restored the presence of Bcl-2 (Figure 6C) in the newly formed mucosal layer, however, when co-administered with DMPX the beneficial effects were no longer observed (Figure 6D).

In the DSS model, the immunostaining for Bax revealed wide expression of the pro-apoptotic molecule in DSS animals compared to controls (Figures 7A,B) that was reduced to basal levels by PDRN treatment (Figure 7C). A strong reduction in Bcl-2 staining was observed in the DSS+drug vehicle group (Figure 8B). The administration of PDRN was able to restore Bcl-2 positivity in the regenerated tissue (Figure 8C). The co-administration of DMPX blunted the effects of PDRN on Bax and Bcl-2 expression (Figures 7D and 8D).

MDA levels and MPO activity were increased in the groups of animals that received DNBS+drug vehicle and DSS+drug vehicle (Figures 9A–D). The administration of PDRN markedly reduced both lipid peroxidation and the accumulation of polymorphonuclear granulocytes, in both experimental models (Figures 9A–D). DMPX, abolished the protective effects of PDRN on both MDA level and MPO activity (Figures 9A–D).

Circulating levels of the proinflammatory cytokines IL-1β and TNF-α were measured 7 days after DNBS administration. As shown in Figures 10A,B, a significant increase in both IL-1β and TNF-α was found in serum from DNBS+drug vehicle group compared to control animals (p < 0.0001). A_2A stimulation reduced the serum levels of IL-1β and TNF-α significantly (p < 0.0001 vs DNBS+drug vehicle group). In the DSS model, IL-1β and TNF-α were similarly upregulated (Figures 10C,D), and PDRN reduced their expression. Again, PDRN failed to elicit an inhibitory effect on cytokine levels in the presence of DMPX (Figures 10A–D).