Polydeoxyribonucleotide Exerts Therapeutic Effect by Increasing VEGF and Inhibiting Inflammatory Cytokines in Ischemic Colitis Rats

Ischemic colitis is resulted from an inadequate blood supply to a segment or entire colon. Polydeoxyribonucleotide (PDRN), extracted from salmon sperm, has been reported to exert anti-inflammatory and anti-ischemic effects through the adenosine A2A receptor (A2AR). We investigated whether PDRN possesses therapeutic effectiveness on ischemic colitis rats. Ischemic colitis was induced by selective devascularization. The skin temperature on the ischemic colitis-induced region was determined. To assess the colonic damage score and collagen deposition, colonic tissue sections were stained with hematoxylin and eosin (H&E), and Masson trichrome staining was performed. Western blot analysis for A2AR, vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, Bax, Bcl-2, and extracellular signal-regulated kinase 1/2 (ERK1/2) was performed. Skin temperature was increased and mucosal damage and collagen deposition were observed in the affected colonic tissues in the ischemic colitis rats. Expressions of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and inflammatory mediator (COX-2) were upregulated in the ischemic colitis rats. Apoptosis was increased by decreasing the ratio of Bcl-2 to Bax and by suppressing the phosphorylated form of ERK1/2 expression in the ischemic colitis rats. Treatment with PDRN alleviated mucosal damage reduced the expressions of inflammatory cytokines and COX-2 and inhibited apoptosis in the ischemic colitis rats. PDRN treatment more enhanced the expressions of A2AR and VEGF in the ischemic colitis rats. PDRN showed therapeutic effectiveness on ischemic colitis by increasing VEGF expression and inhibiting inflammatory cytokines and COX-2 through enhancing A2AR expression.


Introduction
Ischemic colitis, the most common ischemic pathology of the gastrointestinal tract, generally results from an inadequate blood supply to a segment of the colon or the entire colon and leading to colonic inflammation and necrosis [1]. Although the incidence of ischemic colitis in the general population is likely underestimated because many cases are misdiagnosed as inflammatory bowel disease, the annual incidence is estimated at 4.5 to 44 cases per 100,000 persons [2,3]. Ischemic colitis frequently occurs with old people and female predominance, but it is not necessarily limited to older people and is more likely to develop ischemic colitis in young people with smoking and hyperuricemia [4]. e clinical treatment for ischemic colitis varies widely depending on the cause and degree of ischemia. Although there are various etiologies of ischemic colitis, which are unidentified in most cases, the important underlying mechanism is ischemia [1]. e colon is susceptible to ischemia due to its anatomical and physiological features, including relatively low blood flow and less-developed microvasculature plexus compared to the rest of the gastrointestinal system [5]. Moreover, in cases of hypotension, compensatory homeostatic mechanisms redirect blood flow to the brain at the expense of the visceral circulation [6]. Although ischemia is regarded as a self-limiting process of acute inflammation, the gut, as the origin and/or target of numerous inflammatory cytokines, plays an important role in local injury, and it is involved in systemic inflammation related to multiple-organ dysfunction or failure [7]. Likewise, during the onset of ischemic colitis, various factors promote inflammation of colonic endothelial cells, which increase tissue damage [8]. In particular, colonic and intestinal endothelial cell environment possesses both isoforms of cyclooxygenase (COX), and their induction has been demonstrated to occur in response to different inflammatory cytokines, such as interleukin-1α (IL-1α), IL-1β, and tumor necrosis factor-α (TNF-α). COX-2 is induced by inflammatory stimuli and synthesizes prostaglandins, which mediate the inflammatory process and tissue damage [9,10].
Angiogenesis is a pivotal process in all types of wound healing, including gastric and colonic mucosal healing [11,12]. It is regulated by many proangiogenic factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor, and endothelial growth factor. Of the many growth factors, VEGF is the most potent stimulus for angiogenesis and it plays an important role in colonic mucosal healing [11].
Apoptosis is controlled by the intrinsic pathway or extrinsic pathway or both. e extrinsic pathway is activated by ligand-induced cell surface receptors, such as tumor necrosis factor receptor (TNFR) and Fas. e intrinsic mitochondrial apoptosis pathway is induced by activation of apoptosis-related proteins, including Bax and Bcl-2, or other signaling pathways, including the mitogen-activated protein kinase (MAPK), phospholipase Cc-1 (PLC-c1), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways [13]. Extracellular signal-regulated kinase 1/2 (ERK1/2) is one of the MAPK families and modulates cell growth, differentiation, and survival [14,15]. Apoptosis in intestinal epithelial cells is implicated in the occurrence and procession of inflammatory bowel diseases [16].
Polydeoxyribonucleotide (PDRN) is a mixture of nucleotides with molecular weights of 50-1500 kDa that originates from the human placenta or salmon sperm [17]. PDRN facilitates skin regeneration by activating adenosine A 2A receptor (A 2A R) [17]. A 2A R is involved in the anti-inflammatory pathway and in the VEGF production [17,18]. PDRN inhibits apoptosis and exerts anti-inflammatory effect by suppressing the production of inflammatory cytokines [12,18].
us, enhancing VEGF production and suppressing inflammation are the therapeutic strategies for the treatment of ischemic colitis. In the present study, we investigated whether PDRN possesses therapeutic effectiveness on ischemic colitis rats.

Experimental Animals. Seven-week-old male Sprague
Dawley rats weighing 180 ± 5 g (n � 50) were used in this study. All experimental procedures were performed in accordance with the Guidelines for the Care and Use of Animals of the National Institutes of Health (NIH). is study was approved by the Institutional Animal Care and Use Committee of Kyung Hee University (KHUASP[SE]-14-038). Experimental animals were randomly divided into 5 groups (n � 10 in each group): sham-operated group, ischemic colitis-induced group, ischemic colitis-induced and 4 mg/kg PDRN-treated group, ischemic colitis-induced and 8 mg/kg PDRN-treated group, and ischemic colitis-induced and 16 mg/kg PDRN-treated group. Animals were provided food and water ad libitum and maintained at 24 ± 2°C with 60% humidity under a 12 h light/dark photoperiod (lights on at 7:00 a.m.).

Induction of Ischemic Colitis and Treatments.
Ischemic colitis was induced by selective devascularization, using a modified technique described by Irkorucu et al. [19]. e rats were anesthetized with Zoletil 50 ® (10 mg/kg, i.p.; Virbac Laboratories, Carros, France) and laparotomized. Subsequently, the rats underwent selective devascularization of a 4 cm segment of the descending colon by marginal vessel ligation at 4 points, with all of the vasa recta in between. e midline incision was sutured, and the rats were returned to their cages with free access to food and water for recovery following surgery. A sham laparotomy was performed on rats in the sham-operated group. e rats were treated with PDRN (Rejuvenex ™ ; Pharma Research Products, Seongnam, Korea) 48 h after induction of ischemic colitis. e timing of PDRN treatment in this study was performed considering the clinical and pathological progress of ischemic colitis [6,20]. PDRN diluted in 0.9% saline (500 µL) was injected intraperitoneally at respective dose (4, 8, and 16 mg/kg of body weight) daily for 21 consecutive days. To confirm the involvement of adenosine A 2A receptor in PDRN, 16 mg/kg 3,7-dimethyl-1-propargylxanthine (DMPX; Sigma Chemical Co., St. Louis, MO, USA) was applied. e rats in the sham-operated group and in the ischemic colitis-induced group received an equivalent volume of 0.9% saline intraperitoneally for the same duration.

Measurement of Skin Temperature on the Ischemic Colitis-Induced Region.
e skin temperature on the ischemic colitis-induced region was determined weekly using an infrared digital thermometer (MT6; Raytek Co., Santa Cruz, CA, USA).

Macroscopic Analysis and Tissue Preparation.
Immediately before sacrifice, the rats were anesthetized with an intraperitoneal injection of Zoletil 50 ® (10 mg/kg), and the laparotomy was reperformed. For macroscopic evaluation, the devascularized segment of the colon was exposed and photographed at the same angle and distance. Morphological damage score was evaluated with the Wallace macroscopic score (Table 1) [21].
Immediately after photographing, the rats were sacrificed, and the colon was excised. Excised colonic tissues were immediately frozen at − 80°C for western blot analysis and were fixed with a freshly prepared solution of 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 24 h for histological staining. Following dehydration with 70, 80, 90, and 100% ethanol, the colonic tissue samples were embedded in paraffin and sectioned (5 µm thickness) using a paraffin microtome (Shandon Finesse 325; ermo Electron Corp., Somerset, NJ, USA). e paraffin-embedded colonic tissue sections were mounted onto gelatin-coated slides and dried in an oven at 37°C overnight.

Hematoxylin and Eosin (H&E) Staining.
For morphologic examination, the colonic tissue sections were stained with hematoxylin and eosin (H&E) and evaluated under a light microscope (Olympus, Tokyo, Japan), according to the previously described method [15,16]. e sections were stained with Mayer's hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 30 sec, washed, and stained with eosin (Sigma-Aldrich) for an additional 3 sec. Following dehydration, the slides were mounted using Permount ® (Fisher Scientific, Fair Lawn, NJ, USA). Ischemic colonic damage was evaluated with the Wallace microscopic score (Table 1) [21].

Masson Trichrome Staining.
To detect collagen fibers in colonic tissues, Masson trichrome staining was performed, according to the previously described method [22]. e sections were deparaffinized and rehydrated with xylene and 100, 95, and 70% ethanol. After washing, the sections were fixed with Bouin's solution (Sigma-Aldrich) for 1 h at 56°C and stained with Weigert's iron hematoxylin (Sigma-Aldrich) for 10 min. After rinsing with running tap water, the sections were stained with Biebrich scarlet-acid fuchsin (Sigma-Aldrich) and then differentiated by incubation in phosphomolybdic-phosphotungstic acid solution (Sigma-Aldrich) for 10-15 min. Without rinsing, the sections were stained with aniline blue solution (Sigma-Aldrich) for 5 min and a light green solution for 1 min, successively. After rinsing, the sections were differentiated in glacial acetic solution for 5 min, dehydrated quickly with 95% ethanol, and cleared with xylene. e slides were mounted with Permount ® (Fisher Scientific). After Masson's trichrome staining, the collagen fibers, nuclei, and background were stained blue, black, and red, respectively. e fibrosis analysis was conducted according to the previously described method [23]. e collagen/area ratio was analyzed also for 3 areas on each slide by Image-Pro ® plus computer-assisted image analysis system (Media Cybernetics Inc., Silver Spring, MD, USA) attached to a light microscope (Olympus), and a mean ratio was defined by three investigators separately.

Western Blot
Analysis. According to the previously described method [12,24], western blot analysis was performed.
All of the experimental procedures for western blot were performed under standard laboratory conditions and at room temperature, except for the membrane transfer, which

Data
Analysis. e data were analyzed with one-way ANOVA and Duncan's post hoc test. All values are expressed as the mean ± standard error of the mean (SEM). P value less than 0.05 was considered significant.

Skin
Temperature. Skin temperature on the ischemic colitis-induced region is presented in Figure 1. Induction of ischemic colitis significantly increased the skin temperature of the lesion compared to that in sham rats, and the increased skin temperature was maintained for the first week of treatment with PDRN (P < 0.05). However, after 2 weeks, PDRN treatment significantly and dose-dependently decreased the skin temperature of the lesion, and the high dose of PDRN (16 mg/kg) most effectively reduced skin temperature (P < 0.05).

Morphological and Histological Changes.
e appearance of the devascularized colon at 3 weeks after induction of ischemic colitis is shown in Figure 2 (left column). Hyperemia and edema were observed in the ischemic colitisinduced rats. PDRN treatment for 3 weeks ameliorated these morphological changes. Colonic mucosa in the sham-operated rats showed a normal appearance with intact epithelium. In contrast, mucosa in the ischemic colitis-induced rats showed mucosal damage with losses of goblet cells and abnormal crypts. PDRN treatment remarkably alleviated the mucosal damage induced by selective devascularization of the colon (Figure 2, middle column). Collagen deposition in the colonic tissue was increased, especially in the mucosal layer, by induction of ischemic colitis. PDRN treatment reduced collagen deposition in the colonic tissue ( Figure 2, right column).
ese changes led to elevated colonic damage score and collagen (P < 0.05). However, PDRN treatment resulted in the dose-dependent decrease in colonic damage score and collagen (P < 0.05).

A 2A R and VEGF Expressions.
Induction of ischemic colitis increased A 2A R (45 kDa) and VEGF (21 kDa) expressions in the colonic tissue (P < 0.05). Treatment with PDRN further enhanced A 2A R and VEGF expressions (P < 0.05) (Figure 3).

Discussion
Inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, are frequently upregulated in the animal models of experimental colitis [25,26]. Increased level of TNF-α was observed in the serum of patients with ulcerative colitis and Crohn's disease [27,28]. IL-6 induces TNF-α and IL-1β production, and IL-6 also increases the expression of adhesion proteins, including intercellular adhesion molecule-1 (ICAM-1), involved in the activation and migration of inflammatory cells to the intestine [29]. IL-6 was elevated during intestinal ischemia and further increased during reperfusion [7]. Increased levels of TNF-α and IL-1β in the ischemic intestine caused the progress of intestinal injury [30]. As a result, double inhibition of TNF-α and IL-1β can effectively ameliorate postischemic deterioration of the intestine. PDRN reduced the expressions of inflammatory cytokines and altered the expression of apoptotic markers [26]. Specific COX-2 inhibitors ameliorated the symptoms of inflammation [31]. In the present study, treatment with PDRN effectively suppressed ischemic colitis-induced COX-2, TNF-α, IL-1β, and IL-6 expressions in the colonic tissues (Figure 4). e present results showed that PDRN potently inhibited inflammation in the ischemic colitis rats.
Adenosine suppresses TNF-α release from macrophages, primarily through A 2A R [32]. Activation of A 2A R has a robust anti-inflammatory effect, and A 2A R is involved in the repair of experimental colitis [33,34]. PDRN potentiated gastric healing by activation of A 2A R in gastric ulcer rats [24]. In the present study, induction of ischemic colitis increased A 2A R expression and PDRN treatment further increased A 2A R expression in colonic tissue (Figure 3, left). e present results showed that the effects of PDRN appeared through the enhancing of A 2A R expression.
Fibrosis is considered as a consequence of chronic inflammation in inflammatory bowel disease [35]. eiss et al. [36] suggested that TNF-α is associated with intestinal fibrosis, since TNF-α induces collagen accumulation and proliferation in intestinal myofibroblasts. Clinically, ischemic colitis is classified as full-thickness transmural colitis and partial-thickness ischemic colitis. e first type is associated with gangrene and multiorgan failure, while the latter is confined to the mucosa and submucosa [1]. In the present study, mucosal damage induced by selective devascularization was observed, which was effectively prevented by PDRN treatment. Administration of PDRN decreased collagen deposition in colonic tissue (Figure 2). e present results showed that PDRN alleviated mucosal damage and decreased collagen deposition in the ischemic colitis rats.
In addition, body temperature is a marker of severity in colitis and inflammation [37], and an increase in skin temperature is an indicator of inflammation. Pallio et al. [26] reported that PDRN improved the features and clinical symptoms of colitis, such as fever, weight loss, and histological alterations. In the present study, PDRN treatment reduced the colitis-induced increase of skin temperature (Figure 1). e present results showed that PDRN relieved the symptoms of ischemic colitis.
Two ischemic factors, VEGF and hypoxia-inducible factor-1α (HIF-1α), were constitutively expressed in the human colon tissue and overexpressed in the ischemic colitis lesion [38]. In ischemic crises, VEGF and HIF-1α were immediately induced and participated in the prevention against the expansion of tissue damage as well as for the repairing damaged tissue [38]. Under ischemic conditions, activation of A 2A R stimulated VEGF production in macrophages [39]. PDRN facilitated blood flow via stimulation of VEGF in an experimental model of ischemic skin flaps. Increased level of VEGF contributed to angiogenesis and symptom improvement [40,41]. PDRN maintained high VEGF level during angiogenesis and restoration of blood flow through activation of A 2A R [18]. us, PDRN can be considered to possess anti-ischemic effect [33]. Our study also showed that induction of ischemic colitis increased VEGF expression and PDRN treatment further increased VEGF expression in colonic tissue (Figure 3, right). e present results showed that PDRN facilitated vascularization in the ischemic colitis rats.
Apoptosis was increased in ischemic colitis rats along with inflammation and oxidative damage [8]. Excessive apoptosis damages mucosal integrity and barrier function, leading to mucous epithelial injury, ulcerative formation, inflammatory cell infiltration, and other conditions related to colitis [13,42]. Pallio et al. [26] reported that PDRN treatment affected Bax and Bcl-2 expressions in experimental colitis by reducing the numbers of apoptotic and necrotic cells in all tissue layers. In their study, TNF-α induced either apoptosis or necrosis depending on cell types, environmental conditions, and the magnitude of the cellular insults [26]. Increase of Bcl-2 to Bax ratio represents suppression of apoptosis [43,44]. In addition, caspase-3, which is the most widely studied caspase, is a key executor of apoptosis [45].
In the present study, induction of ischemic colitis affected the expressions of Bcl-2 and Bax, resulting in decrement of the ratio of antiapoptotic protein Bcl-2 to proapoptotic protein Bax. However, PDRN treatment increased Bcl-2 and suppressed Bax, resulting in enhancement of the ratio of Bcl-2 to Bax ( Figure 5, left). Furthermore, our present study showed that PDRN treatment suppressed the ischemic colitis-induced increase in caspase-3 expression ( Figure 5, right-upper).
e present results showed that PDRN suppressed apoptosis in the ischemic colitis rats. e protective effect of ERK activation is mediated by antiapoptotic members of the Bcl-2 family, including Bcl-2 and Bcl-xL [14,15]. Activation of ERK inhibits caspase-8 cleavage and translocation of Bax and consequently suppresses the release of cytochrome c from mitochondria [46]. TNF-α-induced apoptosis in polyamine-depleted IEC-6 cells, a nontransformed intestinal crypt epithelial cell line, was prevented by ERK1/2 activation [47]. ERK1/2 activation is implicated in the facilitation of regeneration [44]. In the present study, PDRN treatment enhanced phosphorylation of ERK1/2 in the colonic tissues of ischemic colitis rats ( Figure 5, right-lower), which might contribute to inhibiting apoptosis.
e present results showed that the inhibitory effect of PDRN on apoptosis appeared through phosphorylation of ERK1/2.

Conclusions
PDRN treatment reduced the morphological score and colonic damage score. is promising therapeutic effect of PDRN on colonic damage may be attributable to its ability to enhance A 2A R expression. PDRN also improved gastric ulcer healing by suppressing inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 through A 2A R. In addition, the present study additionally observed a relationship between DMPX, a specific adenosine A 2A receptor antagonist, and PDRN in a model of ischemic colitis. ese findings strongly support that PDRN may be efficacious in the treatment. e present results suggest that PDRN possesses therapeutic efficacy for ischemic colitis by increasing VEGF expression and inhibiting inflammatory cytokines through enhancing A 2A R expression.

Data Availability
All data generated or analyzed during the present study are included in this article.

Conflicts of Interest
e authors declare no conflicts of interest.