Therapeutic and preventative effects of ankaferd blood stopper in an experimental necrotizing enterocolitis model

Biomedicine & Pharmacotherapy 110 (2019) 105–110 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha Therapeutic and preventative effects of ankaferd blood stopper in an experimental necrotizing enterocolitis model T Mehmet Buyuktiryakia, , Cuneyt Taymana, Ismail Koyuncub, Ufuk Cakira, Tugba Taskin Turkmenogluc, Esra Cakird, Nilufer Okura ⁎ a Division of Neonatology, Health Sciences University, Zekai Tahir Burak Maternity Education and Research Hospital, 06230, Ankara, Turkey Department of Biochemistry, Harran University Faculty of Medicine, Sanlıurfa, Turkey c Department of Pathology, Health Sciences University, Dışkapı Yıldırım Beyazıt Training and Research Hospital, Ankara, Turkey d Department of Anesthesiology and Clinical of Critical Care, Health Sciences University, Ankara Numune Education and Research Hospital, Ankara, Turkey b A R T I C LE I N FO A B S T R A C T Keywords: Antioxidation Antiinflammation Necrotizing enterocolitis Premature Rat Necrotizing enterocolitis (NEC) is a major neonatal health problem that especially affects preterm infants and causes severe morbidity and mortality. Although its pathogenesis is not fully understood, important risk factors include prematurity, oxidative stress, inflammation, and apoptosis. Ankaferd Blood Stopper® (ABS) has antioxidant, antiinflammatory, antimicrobial, antiapoptotic, and wound healing accelerant properties. In this study, we aimed to investigate whether treatment with ABS reduced the severity of NEC in rat pups in an experimental NEC model. Thirty-six newborn Wistar albino rat pups were randomly assigned to the control, NEC + saline, or NEC + ABS groups. NEC was induced by intraperitoneal injection of lipopolysaccharide, feeding with hyperosmolar enteral formula, and exposure to hypoxia/hyperoxia and cold stress. ABS was administered intraperitoneally to the pups in the NEC + ABS group daily starting on day 1 of the study at a dose of 2 ml/kg by diluting 2 ml with saline at a ratio of 1:3. All pups were sacrificed on day 4. The terminal ileum including the proximal colon was removed for histopathological and immunohistochemical examination and biochemical analysis. Macroscopic assessment and intestinal injury scores were lower in NEC + ABS group compared to the NEC + saline group (p < 0.05). Immunohistochemical evaluations of caspase-3, -8, and -9 revealed significantly reduced apoptosis in the NEC + ABS group compared to the NEC + saline group (p = 0.001). Total oxidant status, oxidative stress index, tumor necrosis factor α and interleukin-1β levels, and lipid, protein, and deoxyribonucleic acid oxidation products were significantly lower in the NEC + ABS group compared to NEC + saline group (p < 0.001 for all), while total antioxidant status, glutathione, and superoxide dismutase levels were higher in the NEC + ABS group (p < 0.001, p < 0.001, p = 0.01, respectively). ABS treatment has the potential to effectively reduce the severity of intestinal damage in NEC due to its antioxidant, antiinflammatory, and antiapoptotic properties. Therefore, NEC may be an alternative option for treatment. 1. Introduction Necrotizing enterocolitis (NEC) is a gastrointestinal emergency that especially affects preterm infants and is a major cause of serious morbidity and mortality [1]. The overall incidence of NEC is between 0.3 and 2.4 per 1000 live births, while its incidence among preterm babies with a birth weight below 1500 g is 6–10% [2,3]. Although the pathogenesis of NEC is not fully understood, many factors are involved and ultimately result in intestinal damage. Prematurity, formula feeding, hypoxia, ischemia, immature intestinal tract, and bacterial colonization are important factors in the pathogenesis [1–5]. In addition, inflammatory mediators such as tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), and oxygen-derived free radicals play important roles in the development of NEC [6–8]. Ankaferd Blood Stopper® (ABS) (Immun Pharmaceutical Cosmetics Co. Ltd., Istanbul, Turkey) is a folkloric herbal extract used as a hemostatic agent in traditional Turkish medicine [9]. ABS consists of a standardized mixture of Thymus vulgaris, Alpinia officinarum, Vitis vinifera, Glycyrrhiza glabra, and Urtica dioica plant extracts [10]. ABS influences inflammatory and hemostatic processes via its effects on Corresponding author. E-mail addresses: mbuyuktiryaki@yahoo.com (M. Buyuktiryaki), ctayman22@gmail.com (C. Tayman), ismailkoyuncu1@gmail.com (I. Koyuncu), drufukcakir@hotmail.com (U. Cakir), tugbataskin78@hotmail.com (T. Taskin Turkmenoglu), pavulonmouse@hotmail.com (E. Cakir), n.matur@hotmail.com (N. Okur). ⁎ https://doi.org/10.1016/j.biopha.2018.11.023 Received 11 October 2018; Received in revised form 6 November 2018; Accepted 6 November 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). Biomedicine & Pharmacotherapy 110 (2019) 105–110 M. Buyuktiryaki et al. endothelium, blood cells, angiogenesis, cellular proliferation, vascular dynamics, and cellular mediators [9–11]. ABS is used to treat external bleeding and control gastrointestinal hemorrhages resistant to conventional anti-hemorrhagic measures [9,10]. ABS has also been shown to have antiinflammatory, antioxidant, antimicrobial, and antineoplastic effects and to accelerate wound healing [12–19]. Despite advances in NEC diagnosis and treatment, serious complications such as short bowel syndrome and neurodevelopmental disorders may occur, and the mortality rate is reported as nearly 60% [3,5]. Therefore, there is an obvious need for new treatment options to prevent the development and/or progression of NEC. In this study, we aimed to investigate whether treatment with ABS reduced NEC severity in an experimental neonatal rat model of NEC. 2.4. Histopathological, immunohistochemical, and macroscopic evaluation On day 4, all rat pups were killed under deep anesthesia induced with intraperitoneal injection of ketamine/xylazine (100/10 mg/kg). For each pup, the abdominal cavity was opened and the intestines were macroscopically evaluated for evidence of NEC such as color change, edema, bleeding, ileal distension, pneumatosis intestinalis, necrosis, and perforation, using a new scoring system [22]. A 2-cm specimen of terminal ileum including the proximal colon was removed and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for blind histopathological evaluation by the same pathologist. The remaining ileum and proximal colon were washed with saline and stored at −80 °C for biochemical analysis. The intestinal tissues were embedded in paraffin, cut into 4–5 μm sections, and stained with hematoxylin-eosin. Histological findings were scored between 0 and 4 using a grading system (grade 0: normal, grade 1: mild focal injury limited to villus tips, grade 2: partial or complete loss of villi, grade 3: necrosis extending to the submucosa, grade 4: transmural necrosis) [23]. For immunohistochemical analysis, caspase-3 (1:100; [CPP32] Ab-4 [rabbit PAP], 1 ml, Labvision [Thermo], RB-1197-P), caspase-8 (500 μl, Abcam, ab4052), and caspase-9 (LAP Ab-4) analyses were performed by staining with appropriately diluted primary antibodies. 2. Materials and methods 2.1. Animals and experimental design This experimental study was conducted after obtaining approval from the Experimental Animals Ethics Committee of Ankara Training and Research Hospital (Ankara, Turkey) (animal ethics committee certificate date and no: 24.05.2016-0032/427) and in accordance with the US National Institutes of Health (Washington, DC) guidelines for the care and use of laboratory animals. Thirty-six pups born to 4 Wistar albino rats were randomly divided into 3 groups. Pups in the control group (n = 12) remained with their mothers and continued to feed ad libitum on breast milk. Those in the NEC + saline group (n = 12) were subjected to the NEC procedure and intraperitoneal injection of saline solution. The NEC + ABS group (n = 12) was subjected to the NEC procedure and treated with intraperitoneal ABS. Pups in the NEC + saline and NEC + ABS groups were separated from their mothers immediately after birth to avoid the protective effect of breast milk and were housed in a humidified incubator at 37 °C. 2.5. Biochemical analysis Intestinal tissue samples were homogenized in saline (1 g in 85 ml) using a homogenizer (IKA T18 basic Ultraturrax, Germany) and centrifuged at 4000 x g (NF 800 R, Nüve) for 20 min. The supernatants were used for analysis. All measurements were made using a spectrophotometer (UV-1700, Shimadzu, Japan). Protein levels were measured with the Lowry method [24]. Superoxide dismutase (SOD) activity was determined using a method described by Sun et al. [25] and glutathione (GSH) level was measured as described by Koyuncu et al. [26]. Tissue total antioxidant status (TAS) and total oxidant status (TOS) were measured with an autoanalyzer (Cobas Integra 800, Roche) using commercially available kits (Rel Assay Diagnostics kit; Mega Medical, Gaziantep, Turkey) by a method developed by Erel et al. [27]. Oxidative stress index (OSI) was calculated using the formula OSI = TOS/ TAS. Tissue lipid hydroperoxide (LPO) levels were measured using commercially available kits (LPO Assay Kit, Item No. 705003, Cayman Chemical Company, Ann Arbor, USA). Levels of 8-Hydroxydeoxyguanosine (8-OHdG), and indicator of oxidative-dependent DNA damage, were measured using OXISLECT Oxidative DNA damage ELISA Kit (Cell Biolabs, San Diego, CA). Spectrophotometric measurement of advanced oxidation protein products (AOPP) was done using a method described by Witko et al. [28]. Tissue caspase-3 levels were measured with a rat CASP3 ELISA kit (BioSource Europe S.A., Nivelles, Belgium). TNF-α and IL-1β levels were measured in duplicate using ELISA kits (BioSource Europe S.A., Nivelles, Belgium) as per the manufacturer's instructions. 2.2. NEC procedure and ABS administration On day 1 of the study, NEC was induced in pups in the NEC + Saline and NEC + ABS groups by intraperitoneal injection of 1 mg/kg lipopolysaccharide (Escherichia coli serotype 0111:B4, Sigma-Aldrich Chemical, Germany) in saline [20]. The same volume of saline was given by intraperitoneal injection to pups in the control group. The pups in the NEC groups were fed special rodent formula (15 g Similac 60/40 [Ross Pediatrics, Columbus, Ohio]) prepared in 75 ml of canine milk (Beaphar-Bogena, BV Sedel, Netherlands). Feeding was initiated at 0.2 ml every 3 h and increased by 0.1 ml per day as tolerated. The pups were also subjected to 100% carbon dioxide (CO2) inhalation for 10 min, 97% oxygen (O2) for 5 min, and +4 °C cold exposure for 5 min twice daily for 3 days. ABS was administered intraperitoneally to the pups in the NEC + ABS group daily from day 1 to day 4 of the study at a dose of 2 ml/kg by diluting 2 ml with saline at a ratio of 1:3. This dose was determined based on previous studies in adult rats [21]. The NEC + placebo group was administered 2 ml/kg saline by intraperitoneal injection. 2.6. Statistical analysis SPSS software (version 16.0, IL, USA) was used for statistical analysis. Data were expressed as median and interquartile range (IQR) or mean ± standard deviation (SD). Biochemical and pathological parameters were analyzed using appropriate post hoc tests including analysis of variance (ANOVA) and multiple comparison tests (LSD). Pathological scores of the NEC + Saline and NEC + ABS groups were analyzed using Kruskal-Wallis test or one-way ANOVA. Comparisons between two groups were performed using the nonparametric MannWhitney U test for independent samples. Differences with p values < 0.05 were accepted as statistically significant. 2.3. Clinical sickness score and follow-up The pups were weighed each day using a scale with 0.01 g sensitivity and their weight was recorded. An observer blind to the study evaluated the pups at each feeding session during the study using a modified neonatal rat clinical sickness score (0=best, 12=worst) including findings such as appearance, natural activity, response to touch, and color [22]. Pup deaths were recorded daily for all groups. 106 Biomedicine & Pharmacotherapy 110 (2019) 105–110 M. Buyuktiryaki et al. Table 1 Representing the weight changes, clinical sickness score, macroscopic evaluation, histopathological evaluation, and mortality of the groups. Birth weight (g)* Weight on the fourth day (g)* Death† Clinical sickness score on the fourth day† Macroscopic assessment scoring† Intestinal injury scoring† Caspase-3-positive cell number / unit area† Caspase-8-positive cell number / unit area† Caspase-9- positive cell number / unit area† Control (n = 12) NEC + Saline (n = 12) NEC + ABS (n = 12) p 5.49 ± 0.35 10.18 ± 0.82 0 0 0 0 2 (2) 1 (1) 1 (2) 5.55 ± 0.21 7.35 ± 0.35 3 (%) 7 (1) 5.5 (2.25) 3.5 (1) 49 (20) 71.5 (27.75) 55.5 (40.25) 5.45 ± 0.24 9.4 ± 0.66 2 (%) 4 (1) 3.0 (1) 2 (1) 22 (6) 16 (6.5) 9 (3.5) 0.66a, 0.71b, 0.31c < 0.001, 0.03b 0.53 < 0.001 0.01 0.001 0.001 0.001 0.001 *Mean ± standard deviation † median (interquartile range). ABS, Ankaferd Blood Stopper; NEC, Necrotizing enterocolitis. p: Comparison of all 3 groups; pa: Comparison of control with NEC + Saline; pb: Comparison of control with NEC + ABS; pc: Comparison of NEC + saline with NEC + ABS. 3. Results 3.1. Histopathological findings During the study, 3 pups in the NEC + saline group and 2 pups in the NEC + ABS group died, while no pups in the control group died (p = 0.53). There was no significant difference between the groups in terms of mean birth weight. At the end of the study, mean body weight was 10.18 ± 0.82 g in the control group, 7.35 ± 0.35 g in the NEC + saline group, and 9.4 ± 0.66 g in the NEC + ABS group. Final mean body weight in the NEC + ABS group was significantly lower compared to the control group (p = 0.03) and significantly higher compared to the NEC + saline group (p < 0.001) (Table 1). Clinical sickness score at the end of the study was significantly better in the NEC + ABS group compared to the NEC + saline group (p < 0.001) (Table 1, Fig. 1). Macroscopic evaluation of the intestines was performed for each group using a macroscopic scoring system (0=best, 6=worst). No evidence of macroscopic changes associated with NEC was found in the control group. Signs of NEC were less severe in the NEC + ABS group compared to the NEC + saline group (p = 0.01) (Table 1, Fig. 1). The 5 pups that died during the study were excluded; postmortem evaluation revealed intestinal necrosis and perforation. In histopathological evaluation, the severity of intestinal damage was classified from grade 0 to grade 4. No histological changes were detected in the control group. Significantly less intestinal damage was detected in the NEC + ABS group compared to the NEC + saline group (p = 0.001) (Table 1, Figs. 1 and 2). In all groups, caspase activity was used to detect apoptotic changes in intestinal epithelial cells. There were significantly fewer cells positive for caspase-3, -8, and -9 in the NEC + ABS group compared to the Fig. 1. Clinical sickness score, macroscopic assessment scoring and intestinal injury scoring of pups in the study groups. *p < 0.05 was considered significant. NEC: Necrotizing enterocolitis, ABS: Ankaferd Blood Stopper. 107 Biomedicine & Pharmacotherapy 110 (2019) 105–110 M. Buyuktiryaki et al. Fig. 2. Images showing histopathological changes in the ileum of rat pups in each group (hematoxylin-eosin staining; 200 μm): (A) Normal ileum tissue, control group; (B) Necrosis extending to the submucosa and significant loss of villus structure, NEC + saline group; (C) Mild focal injury limited to the villus tips, NEC + ABS group. Fig. 3. (I) Caspase-3 immunohistochemical staining (200 μm) (II) Caspase-8 immunohistochemical staining (400 μm) (III) Caspase-9 immunohistochemical staining (200 μm). (A) Mild positive staining in control group; (B) More extensive positive staining in NEC + saline group; (C) Less positive staining in NEC + ABS group compared to the NEC + saline group. compared to the control group (p < 0.001). In addition to GSH and SOD levels, TAS was also significantly higher in the NEC + ABS group compared to the NEC + saline group (p < 0.05). Consistent with these results, lipid hydroperoxide, AOPP, and 8-OHdG levels were lower in the NEC + ABS group compared to the NEC + saline group (p < 0.001). TNF-α and IL-1β levels were lower in the NEC + ABS group compared to the NEC + saline group (p < 0.001) but higher compared to the control group (p < 0.05). NEC + saline group (p = 0.001) (Table 1, Fig. 3). 3.2. Biochemical analysis Biochemical analyses conducted on the intestinal tissues of the groups are shown in Table 2. TOS and OSI, which indicate increased oxidant status, were significantly lower in the NEC + ABS group compared to the NEC + saline group (p < 0.001) but were higher Table 2 Biochemical analysis of groups. TAS (mmol Trolox equivalent/g protein) * TOS (μmol H2O2 equivalent/g protein) * OSI (Arbitrary Unite) * GSH (nmol/g) * SOD (U/g protein) * AOPP (ng/mg protein) * Lipid hydroperoxide (nmol/l) * 8-OHdG (ng/ml) * TNF-α (pg/mg protein) † IL-1β (pg/mg protein) * Caspase-3 (ng/g protein) * Control (n = 12) NEC + Saline (n = 12) NEC + ABS (n = 12) p 5.66 ± 0.92 4.67 ± 1.04 0.82 ± 0.23 12.25 ± 2.35 2.2 ± 0.6 2.68 ± 0.43 0.48 ± 0.082 1.79 ± 0.43 5.6(1.2) 3.6 ± 1.2 8.3 ± 2.3 1.87 ± 0.46 31.79 ± 5.67 15.53 ± 6.73 3.85 ± 0.67 1.1 ± 0.7 14.47 ± 2.03 1.3 ± 0.32 6.78 ± 2.02 16.5(4.3) 9.7 ± 2.3 38.3 ± 5.6 4.49 ± 1.0 16.44 ± 3.14 3.66 ± 1.13 7.75 ± 0.66 1.8 ± 0.5 6.71 ± 1.36 0.76 ± 0.012 4.22 ± 0.98 8.3(3.1) 4.8 ± 1.6 19.4 ± 3.5 < 0.001 < 0.001 < 0.001 < 0.001 0.02a, 0.04b, 0.01c < 0.001 < 0.001 < 0.001 < 0.001 < 0.001, 0.03b < 0.001 *Mean ± standard deviation † median (interquartile range). ABS, Ankaferd Blood Stopper; AOPP, Advanced oxidation protein products; GSH, Glutathione; IL interleukin, Interleukin-1β; NEC, Necrotizing enterocolitis; 8-OHdG, 8-Hydroxydeoxyguanosine; OSI, Oxidative stress index; TAS, Total antioxidant status; TNF-α, Tumor necrosis factor-α; TOS, Total oxidant status, SOD, Superoxide dismutase. p: Comparison of all 3 groups; pa: Comparison of control with NEC + Saline; pb: Comparison of control with NEC + ABS; pc: Comparison of NEC + Saline with NEC + ABS. 108 Biomedicine & Pharmacotherapy 110 (2019) 105–110 M. Buyuktiryaki et al. has been shown to reduce inflammation scores in rat distal colitis and colorectal anastomosis models [13,14]. Intestinal apoptosis is an important factor in the pathogenesis of NEC [7,40] and was shown to be the earliest histopathological change in the rat NEC model [40]. Preterm infants have a tendency for increased apoptosis in intestinal tissues [7]. Necrosis in epithelial cells with increased intestinal apoptosis contributes to intestinal mucosal barrier function impairment [41]. It is clear that reducing apoptosis will decrease the incidence and severity of NEC. In this study we evaluated caspase-3, -8, and -9 immunoreactivities and showed that ABS therapy reduced intestinal epithelial apoptosis in a rat NEC model. The antiapoptotic effect of ABS has also been demonstrated in previous studies [10,12]. We found in the present study that ABS therapy was associated with significantly lower macroscopic assessment scores and intestinal injury scores, which reflect its ability to preserve intestinal cells. ABS has also been associated with significant improvement in histopathological macroscopic and microscopic scores in rat models of colorectal anastomosis and intestinal obstruction [13,14,18]. The five herbal extracts comprising ABS imbue the mixture with unique properties. Each ingredient adds antioxidant, antiinflammatory, antimicrobial, or wound healing properties to the mixture or further enhances these effects [9–19]. All of our findings support that ABS treatment resulted in positive outcomes in the neonatal rat NEC model. New treatment options are also needed to prevent the occurrence and progression of NEC, which is a common cause of serious morbidity and mortality in premature infants. This study revealed that ABS may be an effective treatment option. The present study has some limitations. Firstly, we did not do cultures or perform other microbiological studies using the intestinal tissue and blood of the rat pups. This would have enabled us to evaluate the antimicrobial activity of ABS. Secondly, we evaluated apoptosis with an immunohistochemical study of caspase-3, -8, and -9 but did not perform TUNEL staining. This staining eliminates speculation regarding apoptosis and provides more accurate results. 4. Discussion In this study we evaluated the effects of ABS in an experimental neonatal rat model of NEC. Our findings showed that ABS decreased oxidative stress by increasing antioxidant activity and thus reduced DNA and protein oxidation and lipid peroxidation. ABS was also associated with significantly lower TNF-α and IL-1β levels, significant improvement in histopathological findings, and reduced apoptosis. In brief, our findings demonstrate that ABS protects against intestinal damage due to its antioxidant and antiinflammatory properties. Prematurity is considered the most important factor in the development of NEC [1–3]. Preterm babies are particularly susceptible to ischemic and hypoxic injuries because their intestinal microcirculation has not fully matured [2,2,3]. Ischemia/hypoxia in intestinal tissue stimulates the proinflammatory process, which causes the release of cytokines and reactive oxygen species (ROS) and the migration and activation of neutrophils [1,2,5,29]. Reoxygenation after hypoxia also leads to an increase in ROS formation [30]. Through the oxidation of membrane lipids, DNA, and proteins, ROS have a major role in cellular injury and subsequent tissue damage and development of NEC [2,5,17,30,31]. TAS, TOS, and OSI are important indicators of global changes in oxidation status [27]. The NEC + saline group in our study exhibited greater oxidative stress with increased TOS and OSI and decreased TAS, as well as higher levels of AOPP, 8-OHdG, and lipid hydroperoxide levels. Other studies of experimentally induced NEC in rat pups have also demonstrated increased TOS and OSI with decreased TAS, and an associated increase in lipid peroxidation [6,32]. It has also been shown that higher TOS and OSI were associated with more severe NEC in preterm infants [33]. In the present study we demonstrated that administering ABS to rat pups with experimentally induced NEC resulted in increased TAS and decreased TOS, OSI, and levels of the cellular oxidation products AOPP, 8-OHdG, and lipid hydroperoxide. The antioxidant effects of ABS have been demonstrated in several experimental studies of induced gastrointestinal mucosal damage as well as an in vitro study [12,13,17,18]. Reactive oxygen radicals are neutralized by enzymes like SOD and glutathione peroxidase (GSH-Px), which protect the organism from oxidative damage [26,32,34]. In our study, SOD and GSH activities were significantly reduced in the NEC + saline group. In the ABStreated group, SOD and GSH activities were significantly higher compared to the NEC group but still lower than in the control group. In a study by Koçak et al. [13] using a distal colitis model, the SOD level was higher in the ABS treatment group compared to the salicylate therapy group, but there was no significant difference between them. Ours is the first study to demonstrate a significant rise in SOD and GSH levels with ABS treatment. Insufficient antioxidant activity and insufficient response to oxidative stress increase the risk of oxidative damage to the intestine in preterm infants [1,2,5,35]. Therefore, it is important to enhance the antioxidant capacity of preterm infants. Our results suggest that ABS can limit the oxidative damage that occurs in NEC due to its antioxidant properties. Intestinal inflammation is an important contributing factor in the pathogenesis of NEC [5–7]. Activation of pro-inflammatory cytokines such as TNF-α and IL-1β has been shown to play a major role in the onset and progression of NEC [5–7,32,34,36]. TNF-α plays a key role in triggering a series of inflammatory events. It promotes inflammation by stimulating the production of itself and other cytokines, thereby increasing mucosal damage [7,37,38]. In addition, TNF-α is the main regulator of ROS production and increases apoptosis in intestinal epithelial cells [7,39]. In our study, we showed that TNF-α and IL-1β increased significantly in rats with NEC and significantly decreased in the ABS-treated group compared to the untreated NEC group. Our results indicate that ABS has strong anti-inflammatory properties that reduce pro-inflammatory cytokine activation. In a study by Şen et al. [18], ABS therapy was shown to significantly decrease TNF-α, IL-1β, and IL-6 levels in a rat model of intestinal obstruction. In addition, ABS therapy 5. Conclusion To our knowledge, this is the first study to investigate the effects of ABS on an experimental rat model of NEC. Our study demonstrated that ABS treatment exerted antiinflammatory effects (decreased levels of TNF-ɑ and IL-6) as well as antiapoptotic effects (decreased number of cells positive for caspase-3, -8, and -9 and caspase-3 tissue levels). The antioxidant effects of ABS were demonstrated by the increase TAS, GSH, and SOD levels and decrease in TOS and OSI. In addition, the decreases in lipid hydroperoxide, AOPP and 8-OHdG, which are lipid, protein, and DNA oxidation products, were further evidence of the antioxidant effects of ABS. In conclusion, our results show that ABS had a protective effect against intestinal damage in an experimental neonatal rat NEC model due to its strong antioxidant, antiinflammatory, and antiapoptotic properties. Therefore, ABS may provide a new and effective treatment option for the prevention and treatment of NEC. Further studies are needed to determine its impact on the development and course of NEC in preterm infants. Compliance with ethical standards The authors declare no conflicts of interest. This research was not supported by any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author contributions MB, CT, UC, EC, IK, TTT and NO designed the research; MB, CT, NO, UC, EC conducted the research and performed the experiments. MB, CT, NO analyzed the data made statistical analyses. IK performed biochemical analyzes, TTT performed pathological examinations. MB, CT, 109 Biomedicine & Pharmacotherapy 110 (2019) 105–110 M. Buyuktiryaki et al. NO, UC wrote the paper with revision. MB and CT had primary responsibility for final content. intestinal obstruction, Int. J. Clin. Exp. Med. 7 (2014) 2677–2686. [19] N. Akkoc, M. Akcelik, I.C. Haznedaroglu, In vitro anti-bacterial activities of Ankaferd medicinal plant extract, Turk. J. Med. Sci. 29 (2009) 410–415. [20] S. Guo, R. Al-Sadi, H.M. Said, T.Y. 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