Journal of Pediatric Surgery 50 (2015) 1119–1124 Contents lists available at ScienceDirect Journal of Pediatric Surgery j ourna l homepage: www.e lsev ie r .com/ locate / jpedsurg Protective effects of dexpanthenol in an experimental model of necrotizing enterocolitis☆,☆☆ Ahmet Karadag a, Ramazan Ozdemir a,⁎, Ahmet Kurt b, Hakan Parlakpinar c, Alaadin Polat d, Nigar Vardi e, Elif Taslidere e, Abdurrahman Karaman f a Division of Neonatology, Department of Pediatrics, Inonu University School of Medicine, Malatya, Turkey b Department of Pediatrics, Inonu University School of Medicine, Malatya, Turkey c Department of Pharmacology, Inonu University School of Medicine, Malatya, Turkey d Department of Physiology, Inonu University School of Medicine, Malatya, Turkey e Department of Histology and Embryology, Inonu University School of Medicine, Malatya, Turkey f Department of Pediatric Surgery, Inonu University School of Medicine, Malatya, Turkey a b s t r a c ta r t i c l e i n f o ☆ Conflict of interest: The authors have no conflicts of to disclose. ☆☆ Funding source: This study was supported by Ino Scientific Research Projects (Project No.: 2013/124). ⁎ Corresponding author at: Division of Neonatology, D University School of Medicine, Turgut Ozal Medical Ce Tel.: +90 422 3410660; fax: +90 422 3410736. E-mail address: ramazanoz@yahoo.com.tr (R. Ozdem http://dx.doi.org/10.1016/j.jpedsurg.2014.10.053 0022-3468/© 2015 Elsevier Inc. All rights reserved. Article history: Received 2 July 2014 Received in revised form 10 October 2014 Accepted 10 October 2014 Key words: Necrotizing enterocolitis Dexpanthenol Pantothenic acid Antioxidant Reactive oxygen species Background/purpose: In pathogenesis of necrotizing enterocolitis (NEC), both oxidative stress and inflammation are considerable risk factors. The study was designed to evaluate whether administration of dexpanthenol (Dxp) is able to attenuate intestinal injury through the antioxidant and antiinflammatory mechanisms in a neo- natal rat model of NEC. Methods: Forty newborn pups divided into four groups were included in the study: control, control + Dxp, NEC, and NEC+Dxp. NECwas induced by hyperosmolar formula and additionally the pupswere exposed to hypoxia/ hyperoxia and cold stress. They were sacrificed on postnatal day four, and their intestinal tissues were analyzed biochemically and histopathologically. Results: Dxp caused a significant decrease in intestinal damage as determined by the histological score, villus height and number of goblet cells in NEC groups (p b 0.0001). Tissue malondialdehyde, total oxidant status, and oxidative stress indexes levels were higher in the NEC group than in the control and control + Dxp groups (p b 0.001). These values were reduced in the pups treated with Dxp (p ≤ 0.004). Superoxide dismutase, gluta- thione peroxidase, and reduced glutathione activities were significantly reduced in the NEC group compared to the others (p b 0.005). Treatmentwith Dxp significantly reduced elevations in tissue homogenate levels of tumor necrosis factor-α and interleukin-1β in the NEC + Dxp group (p = 0.002 and p = 0.01, respectively). Conclusions: Dexpanthenol seems to have antiinflammatory and antioxidant properties. Prophylaxis with Dxp has a potential to reduce the severity of intestinal damage in NEC in the animals. © 2015 Elsevier Inc. All rights reserved. Necrotizing enterocolitis (NEC) remains a major cause of morbidity andmortality among premature infants born at less than 1500 g. Recent data suggest 7% of these infants develop NEC, and 20–30% of them do not survive [1]. Despite many advances in the management of the criti- cally ill neonates, the exact etiology, attempts for the prevention and the best treatment strategy for NEC have been elusive [2]. The pathogenesis of NEC is multifactorial, and was poorly under- stood. The premium theory in demand is local intestinal inflammation initiated by perinatal stress. Intestinal bacteria and their products ad- here to the epithelium, breach the immature and fragile intestinal interest relevant to this article nu University Department of epartment of Pediatrics, Inonu nter, 44280, Malatya, Turkey. ir). mucosal barrier, and activate nuclear factor-κB in lamina propria immunocytes, causing them to secrete proinflammatory mediators and cytokines, chemokines, platelet-activating factor, and nitric oxide. Inflammatory mediators including tumor necrosis factor-α (TNF-α), and interleukins (IL-6, IL-8, IL-10, IL-12, and IL-18) were also proposed to have significant roles in the pathogenesis [3]. TNF-α, secreted pre- dominantly by polymorphonuclear neutrophils, is a proinflammatory cytokine to induce apoptosis. The pivotal role of TNF-α in the pathogen- esis of NEC has been well documented [4]. Interleukin-1β (IL-1β) is another well-recognized proinflammatory cytokine, and several studies have shown that it is associated with immune activation in NEC [5]. On the other hand, reactive oxygen species (ROS) have also been reported to play an important role in NEC pathogenesis [6,7]. Miller et al. demon- strated that ROS make a substantial contribution to intestinal injury in an experimental model of NEC, and that this injury was eliminated by the addition of superoxide dismutase (SOD) [8]. Recently, we also dem- onstrated that all-trans-retinoic acid, a derivate of vitamin A, and etanercept (a TNF-α inhibitor) therapy reduce the severity of NEC in http://crossmark.crossref.org/dialog/?doi=10.1016/j.jpedsurg.2014.10.053&domain=pdf http://dx.doi.org/10.1016/j.jpedsurg.2014.10.053 mailto:ramazanoz@yahoo.com.tr http://dx.doi.org/10.1016/j.jpedsurg.2014.10.053 http://www.sciencedirect.com/science/journal/ 1120 A. Karadag et al. / Journal of Pediatric Surgery 50 (2015) 1119–1124 pups via their antiinflammatory and antioxidant properties [9,10]. The use of several antiinflammatory and antioxidants reduces the NEC severity in experimental models that were shown, as well [11,12]. Thus, we have assumed that preventing the generation of free radicals and/or inflammatory mediators, or neutralization of these factors might have a reducing effect on the severity of NEC. Dexpanthenol (Dxp), an alcoholic analogue of pantothenic acid (PA), is oxidized to PA within tissues, which is known to protect against cell damage produced by ROS. PA that supports cellular antioxidant systems, including glutathione, glutathione peroxidase (GPx), catalase (CAT), SOD, and the other enzymatic reactions that prepare the host to encounter physiopathological conditionsmediated by ROS, is well docu- mented. PA and its derivatives increase the level of reduced glutathione, coenzyme-A and adenosine-5′-triphosphate synthesis within the cell [13–15]. All of themplay amajor role in cellular defense and in the repair systems against oxidative stress and the inflammatory response [16,17]. Given these characteristics of Dxp, using Dxp that might prevent or reduce inflammatory responses is suggested. Therefore, in this histo- pathological and biochemical study it was aimed to evaluate whether Dxp administration could attenuate intestinal injury, and prevent NEC in a neonatal rat model. 1. Materials and methods 1.1. Animals and experimental design After the ethics committee approval, the present study was per- formed at the Animal Laboratory of Inonu University, School of Medi- cine, according to the Guidelines for the Care and Use of Laboratory Animals of the US National Institutes of Health (Washington, DC). The pregnant rats were kept in identical cages, and were left to feed with regular laboratory chow and water. Forty newborn pups from Wistar-albino pregnant time-mated rats were divided randomly into four groups in thefirst day of life. Group1 (n=10)was assigned as con- trol which were only nursed by their mothers; Group 2 (n = 10) was also nursed by their mothers, but received Dxp. Group 3 (n = 10) was subjected to NEC procedure, and treated with intraperitoneal physio- logical saline. Group 4 (n = 10) was also assigned to NEC procedure and treated with Dxp. To prevent from protective effect of mother's milk, Groups 3 and 4 were immediately separated from the mothers, and kept at 37 °C in a humidified incubator. These animals were fed orally using a plastic sheath of 24-gauge polyflon Venocath with 0.2 mL of special rodent formula prepared with 15 g Similac 60/40 (Ross Pediatrics, Columbus, Ohio, USA) and 75 mL of puppy-canine milk replacement (Beaphar BV, Raalte, Netherlands). 1.2. NEC procedure and Dxp application Hypoxiawas accomplishedbyplacing the pups in an airtight plexiglass chamber that was perfused with 100% CO2 for 10 min. At the end of this period, the animals became cyanotic and began gasping. After the hypoxia procedure, the animals were exposed to+4 °C cold for 5min, and 97% O2 for 5 min twice a day to induce NEC. The procedure was applied to the pups for three days [9,11]. The pups were weighed daily. NEC-induced pups in Group 3were given physiologic saline (0.2mL) by intraperitoneal (IP) injection. Those in Groups 2 and 4 were treated with Dxp (Bepanthene ampul®, 500 mg, Bayer Corp., Istanbul, Turkey) and were administered once a day by IP injection at a dose of 500 mg/kg of body weight, starting from birth until postnatal day four. The dosage of Dxp was chosen based on the previous studies which have been reported to causemarked antioxidative effects in rats [18,19]. 1.3. Tissue preparation All rats were sacrificed on the 4th day of experiment. For each rat, the abdomen was opened, and the intestines were inspected for macroscopic evidence of NEC such as intestinal discoloration, edema, fragility, weakness of tissue integrity, ileal distension, intestinal hemor- rhage, pneumatosis intestinalis, perforation, and necrosis. Three centi- meters of the terminal ileum, including the caecum, was harvested for biochemical and histological evaluations. Tissue specimens were flushed with cold saline solution, and half of the distal intestine was fixed in 10% buffered formalin for histological evaluation. The other half was frozen in liquid nitrogen, and then stored at −80 °C for biochemical examination. 1.4. Biochemical analysis On the day of analysis, phosphate buffer (pH 7.4) was added to the frozen tissues, which shortly after were homogenized on an ice cube using a homogenizer in order the mechanical process and the heat not to contribute to the oxidation of the tissue. The supernatant was used for the entire assay. The protein content of tissue homogenates was de- termined as described by Lowry et al. [20] bymeans of the standard bo- vine serum albumin. The malondialdehyde (MDA, one of the specific markers and end product of lipid peroxidation [LPO]) concentrations of the homogenates were determined spectrophotometrically [21] by determination of the presence of thiobarbituric acid reactive substances (TBARS). The amount of lipid peroxides was calculated as TBARS of LPO. Another oxidation marker, SOD activity, was assayed using the nitroblue tetrazolium method of Sun et al. [22]. GPx activity was assayed using the method described by Paglia and Valentine [23]. The GSH content in kidney tissue as nonprotein sulfhydryls was an- alyzed following a previously described method [24]. Tissue total an- tioxidant status (TAS), total oxidant status (TOS), and oxidative stability index (OSI) markers were assayed as described previously [25] using commercial assay kits (Rel Assay Diagnostics, Gaziantep, Turkey). Intestinal tissue levels of TNF-α and IL-1β were analyzed in duplicate with a commercially available enzyme-linked immunosor- bent assay (ELISA) kit (Hangzhou Eastbiopharm Co., Ltd., Hangzhou, China) according to the manufacturer's instructions. 1.5. Histological evaluation Intestinal tissues were fixed in 10% formalin, andwere embedded in paraffin. Sections of tissue were cut at 5 μm, mounted on slides, stained with hematoxylin-eosin (HE) and periodic acid Schiff (PAS). The sec- tions were examined by a Leica DFC 280 light microscope. Intestinal in- jurywas classified as follows: Grade 0, no injury; Grade 1, for swelling of the surface epithelial cell less than 25% of total villous; Grade 2, for swelling of the surface epithelial cell 25–75% of total villous; Grade 3, for swelling of the surface epithelial cell more than 75% and Grade 4, for loss of villi. The median height of the intestinal villi was measured from 100 villi per each of the groups. Goblet cells were counted under 40× objective magnification using Leica Q Win Image Analysis System (Leica Micros Imaging Solution Ltd. Cambridge, UK). 1.6. Statistical analysis Statistical analysis was carried out using SPSS® for Microsoft Windows. Data are expressed as medians (minimum to maximum). We analyzed differences among groups using the Kruskal-Wallis test. Posthoc comparisons among groupswith significant values were evalu- ated with the Bonferroni-corrected Mann-Whitney U tests. Statistical significance was defined as p b 0.05. 2. Results One rat per NEC and NEC + Dxp groups died throughout the study. Table 1 Comparison of biochemical evaluations for each group. Groups Control (n: 10) Control + Dxp (n: 10) NEC (n: 9) NEC + Dxp (n: 9) MDA, nmol/g protein 16.5 (13.5–20.1) 17.2 (12.3–18.7) 27.8 (22.8–33.8)a,b 18.1 (15.4–21.1)c SOD, U/g protein 1.9 (1.2–2.1) 2.1 (1.3–2.5) 1.1 (0.9–1.5)a,b 1.8 (1.2–2.1)c GPX, U/g protein 218 (156–387) 220 (124–337) 121 (65–204)a,b 191 (144–293)c GSH, μmol/g protein 14.6 (11.8–19.1) 19.1 (13.8–25.9)a 11 (8.4–15)a,b 18.7 (12.8–22.1)c TOS, μmol H2O2/L 5.6 (3–7.1) 4.9 (4.2–8.2) 9.3 (6.1–11.5)a,b 4.6 (3.4–9.8)c TAC, mmol Trolox Eq/L 0.84 (0.73–0.93) 0.88 (0.74–0.93) 0.88 (0.62–0.94) 0.89 (0.82–0.99) OSI, arbitrary unit 6.6 (3.5–10.1) 5.7 (4.7–10.8) 11.3 (7.1–15.6)a,b 5.6 (3.5–9.9)c TNF-α, μg/g protein 1.3 (1–2.8) 1.8 (1.4–2.1) 2.6 (2–7.2)a,b 1.7 (1.1–2.4)c IL-1β, ng/g protein 11.2 (9.2–19.2) 15.1 (10–16.9) 18.9 (14–28.6)a,b 13.3 (9.9–23.4)c Dxp, dexpanthenol; NEC, necrotizing enterocolitis; MDA,malondialdehyde; SOD, superoxide dismutase; GPx, glutathione peroxidase; GSH, reduced glutathione; TOS, total oxidant status; TAC, total antioxidant capacity; OSI, oxidative stress indexes; TNF-α, tumor necrosis factor α; IL-1β, interleukins 1β. a Significantly different from the control group, b Significantly different from the control + Dxp group, c Significantly different from the NEC group. 1121A. Karadag et al. / Journal of Pediatric Surgery 50 (2015) 1119–1124 2.1. Biochemical findings TissueMDA, TOS, andOSI levels were higher in Group 3 pups than in Groups 1 and 2 (p b 0.001), suggesting increased LPO and protein oxida- tion. These values were reduced in the pups treated with Dxp (p b 0.0001, p = 0.004, and p = 0.001, respectively). Tissue SOD, GPx, GSH and TAC levels were evaluated as antioxidant enzyme activities. SOD, GPx, and GSH activities were significantly re- duced in the NEC group compared to the other groups (p b 0.005; NEC vs other groups [for three activities]). On the other hand, GSH level was higher in the control + Dxp group than in the control group (p=0.01). Therewere no significant differences among four groups ac- cording to TAC level. As inflammatory mediators, TNF-α and IL-1β were analyzed. The median intestinal TNF-α and IL-1β levels were higher in Group 3 pups than in Groups 1 and 2 (p b 0.001). Treatment with Dxp significantly re- duced elevations in TNF-α and IL-1β in Group 4 pups (p = 0.002 and p = 0.01, respectively). All biochemical values are summarized in Table 1 and Fig. 1. 2.2. Histological findings The gross findings of NEC procedure including intestinal edema, dis- coloration, fragility and weakness of tissue integrity were observed in the NEC group. Less severe findings such as edema, necrosis and mini- mal weakness of tissue integrity were observed in the control + Dxp group. Intestinal histological appearance and mucosal integrity were Fig. 1. Comparison of biochemica observed intact in the control group (Fig. 2-A). Dxp alone treated groupwas similar to that of the control group (Fig. 2-B). The PAS (+) re- action showed a magenta staining where goblet cells were present among surface epithelial cells of villi. Numerous goblet cells were seen on the surface epithelium of villi in the control and control + Dxp groups (Fig. 2-C,D). Morphological damage was observed toward from swelling of the epithelial cells (Fig. 3-A) to loss of villi (Fig. 3-B) in the NEC group. The histological score of NEC group was found to be significantly increased compared to the control group (p b 0.0001). On the other hand, al- though intestinal damage was recognized as alleviated in NEC + Dxp group, the lesions did not completely improve. Degenerative alterations such as swelling of the surface epithelial cells in some areas were still present in this group. Median villus height was found to be significantly increased in NEC + Dxp group compared with NEC group (p b 0.0001) (Fig. 3-C). Another remarkable finding in the NEC group was reduction in the number of goblet cells. Goblet cellswere nearly lost in some areas, too (Fig. 4-A). On the other hand, in NEC+Dxp group, numbers of gob- let cells were found to be significantly increased compared with NEC group (p b 0.0001) (Fig. 4-B). The results of semiquantitative histologi- cal grade, morphometric measure of villi and the number of goblet cells in all groups were shown in Table 2. 3. Discussion In the present study the effects of Dxp in an experimental model of NEC in newborn rats were evaluated. The study findings showed that l evaluations for each group. Fig. 2.Control (A) and control+Dxp (B) groups. The villi are long and normal. H-E; ×20. Control (C) and control+Dxp (D) groups. PAS (+) staining goblet cells are seen on the surface of villus epithelium (arrows). PAS; ×40. 1122 A. Karadag et al. / Journal of Pediatric Surgery 50 (2015) 1119–1124 Dxp attenuates intestinal injury, and decreases inflammation. We have also shown that Dxp therapy decreases the LPO and diminishes the ox- idation, while increasing antioxidant status in this model. ROS have been linked to the development of NEC in premature in- fants [26]. ROS cause cellular injury via several mechanisms including Fig. 3. NEC group. (A) Swelling of the epithelial cell (arrows). (B) Loss of villi. H-E; ×20. (C) Th epithelium of villi is still evident (asterisk) H-E; ×20. peroxidation of membrane lipids as well as oxidation of proteins and DNA, determined by MDA, TOS, and OSI, leading to tissue damage and NEC [27–29]. In the present study, it was demonstrated that significant- ly higher levels of intestinal MDA, TOS, and OSI showed off in the NEC group than in the control and control + Dxp groups (p b 0.001), e some of villi show nearly normal histological appearance (arrows) but swelled surface image of Fig.�2 image of Fig.�3 Fig. 4.NEC+ Dxp group. (A) Notice the number of goblet cells severely decreased compare to control group (arrows). (B) Swelling of surface epithelium and preservation of goblet cells (arrows). PAS; ×40. 1123A. Karadag et al. / Journal of Pediatric Surgery 50 (2015) 1119–1124 which suggests an increase in LPO. However, we found similar TAC levels in all four groups. Recently, Aydemir et al. [29] demonstrated that higher TOS and OSI levels in pretermneonateswith NECwere asso- ciated with development of severe NEC. Thus, the suggestion was that antioxidant intervention might be an important part of the medical therapy of NEC in the future. Dxp is oxidized enzymatically to PA, which is widely distributed in tissues. PA protects against cell damage produced by ROS. It has been demonstrated that PA supports cellular antioxidant systems, including GSH, GPx, CAT, SOD, and the other enzymatic reactions that prepare the host to encounter physiopathological conditions mediated by ROS [13–15,30]. In a viable intestinal tissue, reactive radicals are neutralized by the action of enzymes such as SOD, GPx, and GSH. In the presence of intestinal pathology, loss of intestinal integrity leads to a limited antiox- idant enzyme capacity in neonates, which is considered an important predisposing factor for intestinal tissue injury in NEC [31]. In the present study, intestinal SOD, GPx, and GSH activitieswere significantly reduced in the NEC group (p b 0.005). However, rats treated with Dxp had nor- mal or near-normal enzyme activities. On the other hand, GSH level was higher in the control + Dxp group than in the control group (p=0.01). Previously, it was reported that PA provides an increase GSH synthesis in cells [13,30]. We hypothesize that Dxp treatment impeded antioxi- dant consumption and increased the use of these enzymes via an anti- oxidant effect and increasing the reduced glutathione in cells. We also found that Dxp treatment reduced the intestinal MDA, TOS, and OSI el- evation significantly down to the levels of that of the control. This obser- vation demonstrated the protective role of Dxp in the intestinal mucosa damaged during the NEC procedure by inhibiting ROS-induced LPO and scavenging ROS. The literature displays Dxp as a significant attenuator of serum LPO [30]. Recently, Altintas et al. [18] studied Dxp on I/R in- duced renal injury in a ratmodel, and reported beneficial effects related to the reduction in oxidative stress. In another study, it was tested in bleomycin induced pulmonary fibrosis in a rat model, and a significant protection of Dxp from lung fibrosis by reduced oxidative stress was pointed out [19]. In a testicular ischemia-reperfusion study, Etensel et al found it to be useful in reducing LPO, which translates into Table 2 The results of semiquantitative histological assessment and morphometric analysis. Groups Control (n: 10) Control + Histological score 0 (0–1) 1 (0–2 Villus height, μm 237.6 (135–390) 222.5 (111 Number of the goblet cells 121 (95–135) 111 (98– a Significant increase (p b 0.0001), vs. control group. b Significant increase (p b 0.0001), vs. control + Dxp group. c Significant decrease (p b 0.0001), vs. NEC group. d Significant decrease (p b 0.0001), vs. control group. e Significant decrease (p b 0.0001), vs. control + Dxp group. f Significant increase (p b 0.0001), vs. NEC group. protection against the sequences of oxidative stress that may amplify the inflammatory response [32]. Cytokines are thought to play a central role in intestinal inflamma- tion and injury during NEC by inducing exaggerated inflammatory re- sponses, leading to significant intestinal injury in premature neonates [3,12]. It is widely accepted that elevated cytokines are principal modu- lators of the release of ROS during NEC [33]. In the present study, it is shown that TNF-α and IL-1β are increased in the NEC rats, and reduced in the NEC+Dxp rats.We postulate that the inhibitory effect of Dxp on TNF-α and IL-1β levels might have been a consequence of possible modulation of Dxp on antioxidant defenses via ROS inhibition. Further- more, the antiinflammatory effect of Dxp has been ascribed to the inhi- bition of the release of TNF-α, IL-1β (like myeloperoxidase) from human polymorphonuclear neutrophils [16]. However, this hypothesis needs further investigation. The histological score of NEC + Dxp group was found to be signifi- cantly reduced compared to the NEC group. On the other hand, median villus height and number of goblet cells in NEC + Dxp group were sig- nificantly higher than in NEC group. These findings obviously suggest a possible “Dxp shield” against newborn's NEC. 4. Conclusion To the best of our knowledge, this is the first study to show the ben- eficial effects of Dxp treatment in a neonatal rat model of NEC. This study emphasizes the potential of Dxp as an antioxidant and antiinflammatory agent in the prophylaxis of NEC in premature infants. Thus, Dxpmay be considered a new and potentially effective therapy in the prevention and treatment of NEC. Acknowledgments Weare grateful to Prof. Dr. SaimYologlu, Department of Biostatistics, Inonu University, for his polite help in the statistical analysis. We are also grateful to Inonu University Department of Scientific Research Pro- jects (Project No.: 2013/124), for it was financial support. Dxp (n: 10) NEC (n: 9) NEC + Dxp (n: 9) ) 7 (5–9)a,b 4 (4–6)c –365) 167.9 (92–292)d,e 189.4 (99–329)f 132) 44.5 (31–65)d,e 64.5 (55–81)f image of Fig.�4 1124 A. Karadag et al. / Journal of Pediatric Surgery 50 (2015) 1119–1124 References [1] Neu J, Walker WA. Necrotizing enterocolitis. N Engl J Med 2011;364:255–64. [2] Raval MV, Hall NJ, Pierro A, et al. Evidence-based prevention and surgical treatment of necrotizing enterocolitis — a review of randomized controlled trials. Semin Pediatr Surg 2013;22:117–21. [3] De Plaen IG. Inflammatory signaling in necrotizing enterocolitis. Clin Perinatol 2013; 40:109–24. [4] Caplan MS, Sun XM, Hseuh W, et al. Role of platelet activating factor and tumor ne- crosis factor-alpha in neonatal necrotizing enterocolitis. J Pediatr 1990;116:960–4. [5] Van Haver ER, Sangild PT, Oste M, et al. Diet-dependent mucosal colonization and interleukin-1beta responses in preterm pigs susceptible to necrotizing enterocolitis. J Pediatr Gastroenterol Nutr 2009;49:90–8. [6] Clark DA, Fornabaio DM, McNeill H, et al. Contribution of oxygen-derived free radi- cals to experimental necrotizing enterocolitis. Am J Pathol 1988;130:537–42. [7] Zhou Y,WangQ,Mark Evers B, et al. Oxidative stress-induced intestinal epithelial cell apoptosis ismediated by p38MAPK. BiochemBiophys Res Commun2006;350:860–5. [8] Miller MJ, McNeill H, Mullane KM, et al. SOD prevents damage and attenuates eicos- anoid release in a rabbit model of necrotizing enterocolitis. Am J Physiol 1988;255: 556–65. [9] Ozdemir R, Yurttutan S, Sari FN, et al. All-trans-retinoic acid attenuates intestinal in- jury in a neonatal ratmodel of necrotizing enterocolitis. Neonatology2013;104:22–7. [10] Yurttutan S, Ozdemir R, Canpolat FE, et al. Beneficial effects of etanercept on exper- imental necrotizing enterocolitis. Pediatr Surg Int 2014;30:71–7. [11] Guven A, Uysal B, Gundogdu G, et al. Melatonin ameliorates necrotizing enterocolitis in a neonatal rat model. J Pediatr Surg 2011;46:2101–7. [12] Travadi J, Patole S, Charles A, et al. Pentoxifylline reduces the incidence and severity of necrotizing enterocolitis in a neonatal rat model. Pediatr Res 2006;60:185–9. [13] Slyshenkov VS, Dymkowska D, Wojtczak L. Pantothenic acid and pantothenol in- crease biosynthesis of glutathione by boosting cell energetics. FEBS Lett 2004;569: 169–72. [14] Slyshenkov VS, Omelyanchik SN, Moiseenok AG, et al. Pantothenol protects rats against some deleterious effects of gamma radiation. Free Radic Biol Med 1998;24: 894–9. [15] Slyshenkov VS, Piwocka K, Sikora E, et al. Pantothenic acid protects jurkat cells against ultraviolet light-induced apoptosis. Free Radic Biol Med 2001;30:1303–10. [16] Slyshenkov VS, Rakowska M, Moiseenok AG, et al. Pantothenic acid and its deriva- tives protect Ehrlich ascites tumor cells against lipid peroxidation. Free Radic Biol Med 1995;19:767–72. [17] Slyshenkov VS, Rakowska M, Wojtczak L. Protective effect of pantothenic acid and related compounds against permeabilization of Ehrlich ascites tumour cells by digi- tonin. Acta Biochim Pol 1996;43:407–10. [18] Altintas R, Parlakpinar H, Beytur A, et al. Protective effect of dexpanthenol on ischemia-reperfusion-induced renal injury in rats. Kidney Blood Press Res 2012; 36:220–30. [19] Ermis H, Parlakpinar H, Gulbas G, et al. Protective effect of dexpanthenol on bleomycin-induced pulmonary fibrosis in rats. Naunyn Schmiedebergs Arch Pharmacol 2013;386:1103–10. [20] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75. [21] Uchiyama M, Mihara M. Determination of malonaldehyde precursor in tissues by tiobarbituric acid test. Anal Biochem 1978;34:271–8. [22] Sun Y, Oberley L, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500. [23] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characteriza- tion of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–70. [24] Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7. [25] Harma M, Harma M, Erel O. Increased oxidative stress in patients with hydatidiform mole. Swiss Med Wkly 2003;133:563–6. [26] Lee JW, Davis JM. Future applications of antioxidants in premature infants. Curr Opin Pediatr 2011;23:161–6. [27] Slater TF. Free-radical mechanisms in tissue injury. Biochem J 1984;222:1–15. [28] Yurttutan S, Ozdemir R, Canpolat FE, et al. Protective effects of colchicine in an ex- perimental model of necrotizing enterocolitis in neonatal rats. J Surg Res 2013; 183:156–62. [29] Aydemir C, Dilli D, Uras N, et al. Total oxidant status and oxidative stress are in- creased in infants with necrotizing enterocolitis. J Pediatr Surg 2011;46:2096–100. [30] Wojtczak L, Slyshenkov VS. Protection by pantothenic acid against apoptosis and cell damage by oxygen free radicals-the role of glutathione. Biofactors 2003;17:61–73. [31] Hsueh W, Caplan MS, Qu XW, et al. Neonatal necrotizing enterocolitis: Clinical con- siderations and pathogenetic concepts. Pediatr Dev Pathol 2003;6:6–23. [32] Etensel B, Ozkisacik S, Ozkara E, et al. Dexpanthenol attenuates lipid peroxidation and testicular damage at experimental ischemia and reperfusion injury. Pediatr Surg Int 2007;23:177–81. [33] Baregamian N, Song J, Bailey CE, et al. Tumor necrosis factor-alpha and apoptosis signal-regulating kinase 1 control reactive oxygen species release, mitochondrial autophagy, and c-Jun N-terminal kinase/p38 phosphorylation during necrotizing enterocolitis. Oxid Med Cell Longev 2009;2:297–306. http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0005 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0010 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0010 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0010 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0015 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0015 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0170 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0170 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0025 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0025 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0025 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0030 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0030 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0035 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0035 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0040 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0040 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0040 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0045 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0045 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0050 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0050 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0055 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0055 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0060 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0060 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0065 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0065 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0065 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0070 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0070 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0070 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0075 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0075 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0080 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0080 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0080 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0085 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0085 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0085 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0090 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0090 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0090 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0095 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0095 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0095 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0100 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0100 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0105 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0105 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0110 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0110 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0115 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0115 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0120 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0125 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0125 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0130 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0130 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0135 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0140 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0140 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0140 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0145 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0145 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0150 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0150 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0155 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0155 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0160 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0160 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0160 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0165 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0165 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0165 http://refhub.elsevier.com/S0022-3468(14)00704-0/rf0165 Protective effects of dexpanthenol in an experimental model of necrotizing enterocolitis 1. Materials and methods 1.1. Animals and experimental design 1.2. NEC procedure and Dxp application 1.3. Tissue preparation 1.4. Biochemical analysis 1.5. Histological evaluation 1.6. Statistical analysis 2. Results 2.1. Biochemical findings 2.2. Histological findings 3. Discussion 4. Conclusion Acknowledgments References