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BY 4.0 license Open Access Published online by De Gruyter May 23, 2022

Sildenafil for the treatment of necrotizing enterocolitis: an experimental study

Nekrotizan enterokolit tedavisinde sildenafil: deneysel bir çalışma
Sevcan İpek ORCID logo, Hatice Güneş ORCID logo, Sadık Yurttutan ORCID logo, Fatma Tolun ORCID logo, Ülkü Kazancı ORCID logo, Tuncay Kuloğlu ORCID logo, Ufuk Gullu ORCID logo and Şükrü Güngör ORCID logo

Abstract

Backround

This study was conducted to evaluate whether sildenafil effectively treats necrotizing enterocolitis (NEC).

Material and Methods

38 rat pups were divided into 4 groups: control, sildenafil-control, NEC, and sildenafil-NEC (Sil-NEC). NEC was induced by hypoxia/reoxygenation and cold stress. The pups were treated by administering 1 mg/kg sildenafil by intraperitoneal injection once a day until the fourth postnatal day. The tissues were stained with hematoxylin/eosin staining and examined with the TUNEL test for apoptosis. The intestinal levels of malondialdehyde (MDA), interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS), caspase-3, and glutathione peroxidase (GSH-px) activity were quantified.

Results

TUNEL positivity (p=0.002) and intestinal damage grade (p<0.001) were found to be significantly lower in the Sil-NEC group. In addition, MDA, IL-1β, iNOS, caspase-3 levels, and GSH-px activity were also found to be significantly lower in the Sil-NEC group (p<0.001, p=0.004, p=0.011, p=0.026, p=0.002 respectively).

Conclusion

In this study, sildenafil has been shown to reduce intestinal damage and prevent the development of necrosis biochemically and histopathologically, with its antioxidant, anti-apoptotic, and anti-inflammatory effects, in the treatment of the experimental necrotizing enterocolitis model. This may suggest that sildenafil can be used to treat necrotizing enterocolitis, but further clinical studies are required.

Öz

Amaç

Bu çalışma, sildenafilin nekrotizan enterokolit (NEK) tedavisinde etkin olup olmadığını değerlendirmek için yapılmıştır.

Gereç ve Yöntem

38 yenidoğan sıçan yavrusu 4 gruba ayrıldı: kontrol, sildenafil-kontrol, NEK ve sildenafil-NEK (Sil-NEK). NEK, hipoksi/reoksijenasyon ve soğuk stres tarafından indüklendi. Yavrulara postnatal dördüncü güne kadar günde bir kez intraperitoneal enjeksiyon yoluyla 1 mg/kg sildenafil uygulandı. Dokular hematoksilen/eozin ile boyandı ve apoptoz için TUNEL testi ile incelendi. Malondialdehit (MDA), interlökin 1β (IL-1β), indüklenebilir nitrik oksit sentaz (iNOS), kaspaz-3 ve glutatyon peroksidaz (GSH-px) aktivitesinin bağırsak seviyeleri ölçüldü.

Bulgular

TUNEL pozitifliği (p=0,002) ve bağırsak hasar derecesi (p<0,001) Sil-NEK grubunda anlamlı olarak daha düşük bulundu. Ayrıca MDA, IL-1β, iNOS, kaspaz-3 seviyeleri ve GSH-px aktivitesi de Sil-NEK grubunda anlamlı olarak daha düşük saptandı (sırasıyla p<0,001, p=0,004, p=0,011, p=0,026, p=0,002).

Sonuç

Bu çalışmada deneysel olarak oluşturulan nekrotizan enterokolit tedavisinde sildenafilin antioksidan, antiapoptotik ve antiinflamatuar etkileri ile biyokimyasal ve histopatolojik olarak bağırsak hasarını azalttığı ve nekroz gelişimini engellediği gösterilmiştir. Bu, sildenafilin nekrotizan enterokoliti tedavi etmek için kullanılabileceğini düşündürebilir, ancak daha fazla klinik çalışmaya ihtiyaç vardır.

Introduction

Necrotizing enterocolitis (NEC) is a gastrointestinal emergency in neonates with high morbidity and mortality despite early diagnosis and aggressive treatment. Although it was first described in 1965, its pathogenesis is still not fully understood and an effective treatment has not been found, and so studies in this direction are ongoing [1].

Necrotizing enterocolitis is claimed to be multifactorial in many studies. More than 90% of affected infants are born before 32nd gestational week [2]. Exposure of the gastrointestinal tract to ischemia contributes to the development of NEC. Circulatory failure and intestinal ischemia are associated with NEC, especially in term infants. Reperfusion occurring in response to hypoxia after decreased intestinal blood flow has been thought to possibly contribute to intestinal damage. Increased basal intestinal vascular resistance after birth, an imbalance between mediators, such as nitric oxide and endothelin, perinatal asphyxia, recurrent apnea, hypotension, left–right shunt heart disease, patent ductus arteriosus, indomethacin therapy, umbilical artery catheterization, polycythemia, anemia, and blood transfusion all reduce mesenteric blood flow and increase the risk of necrotizing enterocolitis [3, 4]. In the early stage of life, enteral feeding can lead to ischemia, and immature bowel fails to provide adequate hemodynamic responses after feeding, causing necrotizing enterocolitis to develop. Regulating the microcirculation of the immature intestine can prevent the development of necrotizing enterocolitis [5]. In a rat pup model of NEC, Downard and colleagues found that the main mechanical event in NEC is vasoconstriction in the mesenteric arteriole [6]. Agents that improve mesenteric blood flow and cardiovascular function, such as dobutamine, have been shown to be useful in the treatment of NEC [6, 7].

Sildenafil, a phosphodiesterase-5 (PDE-5) inhibitor, causes vasodilation of the vascular smooth muscle bed by increasing the cyclic guanosine monophosphate (cGMP) level. In addition, intracellular cGMP accumulation has been shown to reduce inflammation by decreasing proinflammatory cytokines and oxidative damage in experimental models [8]. In many experimental studies, it has been suggested that sildenafil may be beneficial in the treatment of various diseases by thanks to its anti-inflammatory, antioxidant and antiapoptotic properties [9], [10], [11]. In an experimental study of intestinal ischemia, intraperitoneal administration of sildenafil was shown to reduce intestinal mucosal damage [9]. Thus, in this study, sildenafil was investigated to test whether it would be effective in the treatment of experimentally induced necrotizing enterocolitis.

Materials and methods

Animals

The pregnant Wistar albino rats used in this study were supplied by Kahramanmaraş Sütçü İmam University Medical Sciences Experimental Research Center. A total of 38 male rat pups were divided into 4 groups: control (n=10, control rat pups fed exclusively with breast milk ad libitum); Sil-control, control+sildenafil (n=10, control rat pups fed with breast milk ad libitum and administered sildenafil); NEC, necrotizing enterocolitis (n=8, necrotizing enterocolitis procedure performed and administered saline); and Sil-NEC, necrotizing enterocolitis+sildenafil (n=10, necrotizing enterocolitis procedure performed and administered sildenafil). The male rat pups in the NEC and Sil-NEC groups were weaned from their mothers immediately after birth and held in a moisturized incubator at 37 °C to ensure avoidance of the protective effects of breast milk. They were fed with 0.2 mL of specific rodent formula six times a day [12, 13].

Necrotizing enterocolitis procedure

Experimental necrotizing enterocolitis was established using the hypoxia/reoxygenation and cold injury method according to the procedure described by Guven et al. [14], [15], [16]. The pups were exposed to 100% CO2 in an airtight plexiglass box for 10 min and maintained in a hypoxic environment. At the end of this process, the pups were breathless and cyanotic. Necrotizing enterocolitis was induced by subjecting the animals to an environment with a temperature of 4 °C for 5 min followed by the administration of 97% O2 for 10 min two times per day. This process was performed for three days for each rat pup. The pups’ daily weights were measured and recorded.

Sildenafil administration

The rat pups in the Sil-NEC were administered an intraperitoneal injection of 1 mg/kg sildenafil once a day from birth until the postnatal fourth day [9]. The necrotizing enterocolitis group administered an intraperitoneal physiological saline alone.

Histopathological analysis

An experienced pathologist blinded to the study protocol performed histopathological tissue examinations. Intestinal specimens were fixed with 10% neutral-buffered formalin solution for light microscopy. The blocks of paraffin-embedded samples were cut into 4-µm-thick slices and stained with hematoxylin and eosin (H&E). Histopathological examination was graded as follows: grade 0 indicates normal tissue; grade 1 indicates mild disease with separation of the villous nucleus without other abnormalities; grade 2 indicates moderate disease with villous nucleus detachment, submucosal edema, and epithelial peeling; grade 3 indicates severe disease with villous, full-thickness necrosis, or epithelial peeling with loss of perforation; grade 4 indicates total intestinal injury, complete destruction of the mucosa, transmural necrosis, and pneumatosis intestinalis [17, 18]. If the intestinal damage scoring was ≥2, the rat pup was considered to have necrotizing enterocolitis.

TUNEL method

After mounting 4- to 6-μm-thick sections of paraffin blocks on polylyzed slides, an ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, USA) (Cat No: S7101) was used to detect apoptotic cells. The sections, deparaffinized with xylene, were passed through graded alcohol series and washed with phosphate-buffered saline (PBS). Tissues were incubated with 0.05% proteinase K for 15 min, and then incubated with 3% hydrogen peroxide for 10 min to prevent endogenous peroxidase activity. After washing the tissues with PBS, they were incubated with equilibration buffer for 6 min and left to incubate for 60 min with the working solution at 37 °C in a humid environment. The samples were then retained in stop/wash buffer for 10 min. Afterward, the apoptotic cells were observed by dropping diaminobenzidine substrate on tissues treated with anti-digoxigenin-peroxidase for 40 min. Tissue contrasted with Harris hematoxylin, were covered with entellan and examined under light microscope and photographed. An average 500 normal and apoptotic average 500 cells were counted in sections at a magnification of 10×. The apoptotic index (AI) was calculated by proportioning apoptotic cells to total (normal-apoptotic) cells.

Biochemical analysis

Whole tissue specimens were washed with saline solution and stored at −80 °C for further analysis. The tissues weighed before analysis were homogenized with 50 mM (pH 7.4) phosphate buffer at 14,000 rpm for 30 min. Then, the supernatants were separated by centrifugation at 10,000 g for 30 min at +4 °C. The lipid peroxidation level was measured by the Ohkawa method which is based on the spectrophotometric measurement of the color formed during the reaction of thiobarbituric acid with malondialdehyde (MDA) at 535 nm [19]. The MDA levels were expressed in mM/g protein. The Paglia and Valentine method was used to determine glutathione peroxidase (GSH-px) activity [20]. In this method, GSH-px catalyzes the oxidation of glutathione in the presence of hydrogen peroxide, and while converting oxidized glutathione to a reduced form, the GSH-px activity is determined by measuring the absorbance change that occurs by oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. The GSH-px activity was expressed in U/g protein for tissue. Protein was measured according to the method outlined by Lowry [21].

The rat interleukin (IL)-1β levels were quantified by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Rel Assay Diagnostics, Gaziantep, TURKEY cat no:RLDR0120). Its sensitivity was found to be 15.18 pg/mL, and the detection range was 30–9000 pg/mL. The intra-assay CV was <8%, and inter-assay CV was <10%. The results were expressed in pg/mL. The level of cysteinyl aspartate specific proteinase-3 (caspase-3) was quantified by ELISA method using a commercial kit (Rel Assay Diagnostics, Gaziantep, TURKEY cat no:RLDR0281). Its sensitivity was 0.022 g/mL, and the detection range was 0.05–10 ng/mL. The intra-assay CV was <8% and inter-assay CV was <10%. The results were expressed in ng/mL. The levels of inducible nitric oxide synthase (iNOS) were quantified by ELISA using a commercial kit (Rel Assay Diagnostics, Gaziantep, TURKEY cat no: RLDR0741). Its sensitivity was 0.23 g/mL, and the detection range was 0.5–200 ng/mL. The intra-assay CV was <8%, and inter-assay CV was <10%. The results were expressed in ng/mL. An automatic ELISA microplate reader (Thermo Scientific), and a computer program (Skanlt for Multiscan FC 2.5.1) was used for ELISA studies.

Statistical analysis

Statistical analyses were performed using SPSS, version 25.0 (IBM Corp., NY, USA). Normality of distribution was checked using the Shapiro–Wilk test. Normally distributed data were expressed as mean ± standard deviation and non-normally distributed data as median (min–max). Parametric variables were compared by means of the T-test and one-way ANOVA with post-hoc Scheffe-test. A p value <0.05 was considered statistically significant.

Results

All the rat pups survived until the postnatal fourth day. Although no difference in weight was found between the groups at the beginning of the study, the weights of rat pups were significantly lower in the NEC group than in the other groups at the end of the study (p<0.001, One-way ANOVA test with post hoc Scheffe test) (Table 1, Figure 1).

Table 1:

Comparison of weights and biochemical assessments of experimental rat pups.

Control n=10, Mean ± SD Sil-control n=9, Mean ± SD NEC n=8, Mean ± SD Sil-NEC n=9, Mean ± SD p*
Birth weight (g) 5.13 ± 0.478 5.1 ± 0.298 5.31 ± 0.488 5.16 ± 0.522 0.773
Last weight (g) 11.47 ± 0.751 11.14 ± 1.077 5.91 ± 0.814 9.24 ± 1.440 <0.001
MDA (nmol/mg protein) 0.95 ± 0.281 1.63 ± 0.812 6.08 ± 4.720 1.75 ± 1.405 <0.001
GSH-px (U/mg protein) 1.35 ± 0.757 4.72 ± 1.426 18.15 ± 15.800 8.31 ± 11.494 0.002
IL-1β (pg/μg protein) 4.99 ± 3.197 4.08 ± 1.420 20.63 ± 19.677 5.75 ± 6.483 0.004
iNOS (ng/μg protein) 0.16 ± 0.099 0.21 ± 0.166 0.70 ± 0.750 0.17 ± 0.121 0.011
Caspase-3 (ng/mg protein) 13.28 ± 12.679 7.64 ± 3.788 19.97 ± 12.502 13.05 ± 9.497 0.026

  1. Data are shown as statistics: *one-way ANOVA test, post hoc Scheffe. GSH-px, glutathione peroxidase; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; NEC, necrotizing enterocolitis; Sil, sildenafil.

Figure 1: 
Graphical representation of weights and biochemical assessments of experimental rat pups.

Figure 1:

Graphical representation of weights and biochemical assessments of experimental rat pups.

Histopathological findings

Intestinal damage was shown with microscopic examination after H&E staining. The H&E images of the tissue samples under light microscope are shown in Figure 2. Intestinal histological injury scores of each group are shown on Table 2 and Figure 3. Histopathological examination group could not be performed on two samples from the Sil-control group because they showed signs of were autolysis. All other tissue samples were examined. Mucosal integrity and histological appearance were labeled as normal in the control group (Figure 2A). In the Sil-control group, except for submucosal edema in 1 pup (intestinal injury score=1) and focal necrosis in 2 pups (intestinal injury score=1), the other specimens had normal mucosal integrity and histological appearance (Figure 2B). Hemorrhage, submucosal edema, ulceration and focal necrosis were observed in the NEC group (Figure 2C). In the NEC group, 1 pup exhibited grade 1 NEC, 3 pups exhibited grade 2 NEC, and 4 pups exhibited grade 3 NEC. Grade 4 NEC was not seen in any offspring. In the Sil-NEC group, intestinal damage was not observed in 4 pups (Figure 2D). Grade 1 NEC was seen in 5 pups and grade 2 NEC in 1 pup. There was a significant improvement in histopathological findings in the Sil-NEC group compared with the NEC group (p<0.001, One-way ANOVA, post-hoc Scheffe test).

Figure 2: 
Histological changes in the intestinal structures of the terminal ileum from each group stained with hematoxylin and eosin: (A) The control group showing normal mucosa with intact epithelial surface (Original magnification, ×200), (B) The Sil-control group showing normal intestinal appearance (Original magnification, ×100), (C) Histopathological changes in the intestinal architecture showing destruction of the epithelium and transmural necrosis in the NEC group (Original magnification, ×100) and (D) Histological sections of the Sil-NEC group showing significant improvement in histological damage (Original magnification, ×400). NEC, necrotizing enterocolitis.

Figure 2:

Histological changes in the intestinal structures of the terminal ileum from each group stained with hematoxylin and eosin: (A) The control group showing normal mucosa with intact epithelial surface (Original magnification, ×200), (B) The Sil-control group showing normal intestinal appearance (Original magnification, ×100), (C) Histopathological changes in the intestinal architecture showing destruction of the epithelium and transmural necrosis in the NEC group (Original magnification, ×100) and (D) Histological sections of the Sil-NEC group showing significant improvement in histological damage (Original magnification, ×400). NEC, necrotizing enterocolitis.

Table 2:

Intestinal histological injury scores of each group.

Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 NECa
Control (n=10) 10 (100) 0 0 0 0 0
Sil-control (n=9c) 8 (100) 0 0 0 0 0
NEC (n=8) 0 1 (12.5) 3 (37.5) 4 (50) 0 7b
Sil-NEC (n=10) 4 (40) 5 (50) 1 (10) 0 0 1

  1. NEC, necrotizing enterocolitis; Sil, sildenafil. Data expressed as n (%). aIntestinal histological injury scores ≥2 were considered as NEC. bp<0.001 (Sil-NEC vs. NEC) (one-way ANOVA, post hoc Scheffe). cA sample from the Sil-control group was autolyzed.

Figure 3: 
Graphical representation of intestinal histological injury scores of each group.

Figure 3:

Graphical representation of intestinal histological injury scores of each group.

Biochemical analysis

Biochemical analysis of all tissue samples was performed except for an autolyzed sample from the Sil- NEC group. The levels of MDA, GSH-px activity, IL-1β, inducible nitric oxide synthase (iNOS), and caspase-3 are shown on Table 1. The MDA level (nmol/mg protein) was 6.08 ± 4.72 in the NEC group, 0.95 ± 0.28 in the control group, 1.63 ± 0.81 in the Sil-control group, and 1.75 ± 1.41 in the Sil-NEC group (p<0.001). The GSH-px activity (U/mg protein) was determined to be 18.15 ± 15.800 in the NEC group, 1.35 ± 0.76 in the control group, 4.72 ± 1.43 in the Sil-control group, and 8.31 ± 11.49 in the Sil-NEC group (p=0.002). The IL-1β level (pg/μg protein) was 20.63 ± 19.68 in the NEC group, 4.99 ± 3.197 in the control group, 4.08 ± 1.42 in the Sil-control group, and 5.75 ± 6.48 in the Sil-NEC group (p=0.004). The iNOS level (ng/μg protein) was 0.70 ± 0.75 in the NEC group, 0.16 ± 0.10 in the control group, 0.21 ± 0.17 in the Sil-control group, and 0.17 ± 0.12 in the Sil-NEC group (p=0.011). The caspase-3 level (ng/mg protein) was 19.97 ± 12.50 in the NEC group, 13.28 ± 12.68 in the control group, 7.64 ± 3.79 in the Sil-control group and 13.05 ± 9.50 in the Sil-NEC group (p=0.026) (One-way ANOVA test, post-hoc Scheffe test).

Finndings of the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick end-labeling method

The results of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick end-labeling (TUNEL) test was performed to determine apoptotic cells with light microscopy, and TUNEL positivity revealed that the results were similar in the control group (Figure 4A) and the Sil-control group (Figure 4B) (p=0.589). TUNEL positivity was significantly higher in the NEC group (Figure 4C) compared with the control group (p=0.002) and significantly lower in the Sil-NEC group compared with the NEC group (Figure 4D) (p=0.002). AI (%) is shown on Table 3 and in Figure 5.

Figure 4: 
Histological sections stained with TUNEL: Similar apoptotic cells are seen in (A) the control and (B) the Sil-control group. Increased apoptotic cells are seen in (C) the NEC group and decreased apoptotic cells are seen in (D) the NEC group. Black arrows indicate the TUNEL-positive cells (scale bar represents 50 μm). NEC, necrotizing enterocolitis.

Figure 4:

Histological sections stained with TUNEL: Similar apoptotic cells are seen in (A) the control and (B) the Sil-control group. Increased apoptotic cells are seen in (C) the NEC group and decreased apoptotic cells are seen in (D) the NEC group. Black arrows indicate the TUNEL-positive cells (scale bar represents 50 μm). NEC, necrotizing enterocolitis.

Table 3:

Apoptotic index (%) of each group.

Apoptotic index (%), Mean (min–max)
Control 1.83 (1.00–3.00)
Sil-control 1.50 (1.00–3.00)
NEC 17.50 (13.00–22.00)a
Sil-NEC 2.83 (2.00–6.00)b

  1. Data are shown as median (min–max) or mean (min–max). aCompared to the Sil-control group (p=0.002). bCompared to the NEC group (p=0.002). NEC, necrotizing enterocolitis; Sil, Sildenafil.

Figure 5: 
Graphical representation of apoptotic index (%) of each group.

Figure 5:

Graphical representation of apoptotic index (%) of each group.

Discussion

In this study, the therapeutic effect of sildenafil was investigated in an experimental model of necrotizing enterocolitis in rat pups. Previously, experimental studies have shown that apoptosis increased at the onset of necrotizing enterocolitis [18, 22]. TUNEL positivity a marker of showing apoptosis was significantly lower in the Sil-NEC group compared with the NEC group. Also, intestinal damage was significantly lower in the Sil-NEC group in histopathological examination.

Mesenteric ischemia may cause necrotizing enterocolitis [23]. Free oxygen radicals, which are intensively generated by hypoxia/reoxygenation, harm intestinal tissue through tissue damage and apoptosis. Antioxidant systems exert a protective effect against this event by removing free oxygen radicals. Sildenafil has been found to prevent oxidative damage in other organs and tissues [24]. MDA is the end product of lipid peroxidation. It occurs when free oxygen radicals oxidize membrane lipids and is an indicator of oxidative stress [25]. GSH-px is an important factor of an antioxidant system [26]. In an experimental burn study conducted by Bostanci et al., it was shown that GSH-px activity and MDA levels were reduced in rats receiving sildenafil treatment after burn; thus, the authors of the study suggested a positive effect of sildenafil on wound healing in burns. In an experimental burn study Bostanci et al. demonstrated that sildenafil has a positive effect on wound healing in burns [27]. In another study, Marganis et al. reported that sildenafil decreased the level of colonic MDA in an experimentally created colitis model [28].

In this study, the levels of GSH-peroxidase and MDA the indicators of oxidative stress, were lower in the Sil-NEC group compared with the NEC group. This decrease in GSH-peroxidase and MDA levels in the Sil-NEC group is consistent with previous studies, suggesting that sildenafil may be beneficial in NEC thanks to its antioxidant effect.

Sildenafil prevents the breakdown of cGMP by inhibiting the PDE-5 enzyme. The PDE-5 receptors are in vascular smooth muscle, intestines, heart, platelets, placenta, and chondrocytes [29]. cGMP accumulating in vascular smooth muscle causes vasodilation. In addition, intracellular cGMP accumulation has been shown to reduce inflammation by reducing the amount of proinflammatory cytokines and mitigating oxidative damage in several experimental models [8]. Yazji et al. showed that when animals with necrotizing enterocolitis were treated with sildenafil, left in hypoxia for four days and fed with formula gavage, the severity of necrotizing enterocolitis was reduced [30]. Soydan et al. demonstrated the protective effect of sildenafil against intestinal damage after intestinal ischemia–reperfusion [31]. Moore et al. created ischemia–reperfusion damage in 12-week-old male mice and administered sildenafil intraperitoneally at various concentrations at follow-up. Consequently, they suggested that administering sildenafil following intestinal ischemia may limit intestinal mucosal damage [9]. In this study, sildenafil was found to be useful in treating necrotizing enterocolitis in the hypoxia/reoxygenation and cold model.

Inflammation plays a significant role in the development of necrotizing enterocolitis development. A previous study suggested that sildenafil caused vasodilation by decreasing cGMP degradation and by increasing the sensitivity of vascular smooth muscles to endogenous and exogenous NO [32]. NO also has a regulatory role in immune response and inflammation [33]. Proinflammatory cytokines such as IL-1β, IL-1α, IL-18, IL-6, TNF- α which are released in the early stages of inflammation have all been shown to increase in NEC [34, 35]. In an experimental study, Araujo et al. showed that sildenafil-treated group’s IL-1β level decreased; the authors credited the anti-inflammatory effect of sildenafil [36]. In this study, IL-1β was higher in the NEC group but it was significantly lower in the sildenafil-treated NEC group.

Phosphodiesterase and iNOS enzymes are expressed in macrophages, dendritic cells, T cells, and neutrophils. iNOS is a type of nitric oxide synthetase that increases in inflammatory states [37]. Kosutova et al., in an experimental study on acute lung injury, detected lower levels of iNOS and stated that inflammation was less severe in the sildenafil-administered group [33]. In the present study, iNOS had a lower level in the Sil-NEC group than in the NEC group. This indicates that sildenafil administration mitigated inflammation in the experimental NEC study with rat pups. The caspase-3 is known to be one of the markers of apoptosis. Jia et al., in their study on neonatal rats, found a decrease in the caspase-3 level with the administration of sildenafil in hypoxic myocardial cells [38]. In the present study, the caspase-3 level was lower in the Sil-NEC group compared with the NEC group. This is a significant finding in that - it indicates the antiapoptotic effect of sildenafil in the treatment of NEC.

Sildenafil has been widely used in treating pulmonary hypertension in neonates and has been shown to be effective and safe in meta-analyses. A retrospective study by Hornik et al., involving 232 infants who were born at 24–41 weeks of age and who received sildenafil treatment, showed no relationship between systemic hypotension and sildenafil use [39]. Cohen et al. reported that low-dose sildenafil was well tolerated in a retrospective study of pediatric patients with pulmonary hypertension [40]. The fact that sildenafil was well tolerated and safe in pediatric patients in these two studies made us think that it can be used as an option in the treatment of necrotizing enterocolitis, a highly fatal disease.

An important limitation of our study was that we could not show the development of grade 4 NEC in rat pups. Despite this, various degrees of intestinal damage were demonstrated histopathologically in pups who underwent NEC induction. In addition, in the biochemical analysis, MDA, GSH-px, IL-1β, iNOS, Caspase-3 were higher in the animals that developed NEC but significantly lower in the NEC group treated with sildenafil. This showed the therapeutic effect of sildenafil in necrotizing enterocolitis in our study.

This study was involved a small number of animals so as to meet the limitation imposed by the ethics committee. Another important limitation of this study was that sildenafil was administered simultaneously with the induction of necrotizing enterocolitis. Administering sildenafil after the development of NEC may be more helpful. Also, if we had carried out the steps of NEC induction separately, we could have shown exactly at which step sildenafil was more effective. These are the main limitations of our study. Therefore, clinical studies involving larger numbers of animals or humans are needed.

Conclusion

This study was conducted to test whether sildenafil, which has been proved to be safe in the aforementioned studies, may also be used for treating necrotizing enterocolitis. The results of the present study showed that sildenafil reduced intestinal damage induced by hypoxia/reoxygenation and cold injury with its antioxidant, anti-apoptotic and anti-inflammatory effects in an animal model of necrotizing enterocolitis.


Corresponding author: Sevcan İpek, Department of Pediatrics, Kahramanmaraş Sutcu Imam University, Medical Faculty, Avşar Mah. Batı Çevreyolu Blv. No:251/A, Onikişubat, 46040, Turkey, E-mail:

Funding source: The Scientific Research Projects Coordination Unit of Kahramanmaraş Sutcu Imam University

Award Identifier / Grant number: 2018/5-16 M

  1. Research funding: The Scientific Research Projects Coordination Unit of Kahramanmaraş Sutcu Imam University (grant number 2018/5-16 M).

  2. Author contribution: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  3. Conflict of interest: The authors declare that they have no conflict of interest.

References

1. van Heesewijk, AE, Rush, ML, Schmidt, B, Kirpalani, H, DeMauro, SB. Agreement between study designs: a systematic review comparing observational studies and randomized trials of surgical treatments for necrotizing enterocolitis. J Matern Fetal Neonatal Med 2020;33:1965–73. https://doi.org/10.1080/14767058.2018.1533948.Search in Google Scholar PubMed

2. Duchon, J, Barbian, ME, Denning, PW. Necrotizing enterocolitis. Clin Perinatol 2021;48:229–50. https://doi.org/10.1016/j.clp.2021.03.002.Search in Google Scholar PubMed

3. Knell, J, Han, SM, Jaksic, T, Modi, BP. Current status of necrotizing enterocolitis. Curr Probl Surg 2019;56:11–38. https://doi.org/10.1067/j.cpsurg.2018.11.005.Search in Google Scholar PubMed

4. Karadag, A, Ozdemir, R, Kurt, A, Parlakpinar, H, Polat, A, Vardi, N, et al.. Protective effects of dexpanthenol in an experimental model of necrotizing enterocolitis. J Pediatr Surg 2015;50:1119–24. https://doi.org/10.1016/j.jpedsurg.2014.10.053.Search in Google Scholar PubMed

5. Koike, Y, Li, B, Ganji, N, Zhu, H, Miyake, H, Chen, Y, et al.. Remote ischemic conditioning counteracts the intestinal damage of necrotizing enterocolitis by improving intestinal microcirculation. Nat Commun 2020;11:4950. https://doi.org/10.1038/s41467-020-18750-9.Search in Google Scholar PubMed PubMed Central

6. Zhang, HY, Wang, F, Feng, JX. Intestinal microcirculatory dysfunction and neonatal necrotizing enterocolitis. Chinese Med J 2013;126:1771–8.Search in Google Scholar

7. Gephart, SM, McGrath, JM, Effken, JA, Halpern, MD. Necrotizing enterocolitis risk: state of the science. Adv Neonatal Care 2012;12:77–87. quiz 8–9. https://doi.org/10.1097/anc.0b013e31824cee94.Search in Google Scholar

8. Szczypka, M, Obmińska-Mrukowicz, B. The effects of selective and nonselective phosphodiesterase inhibitors on phagocytic cells in mice. Immunopharmacol Immunotoxicol 2010;32:507–13. https://doi.org/10.3109/08923970903359850.Search in Google Scholar PubMed

9. Moore, HM, Drucker, NA, Hosfield, BD, Shelley, WC, Markel, TA. Sildenafil as a rescue agent following intestinal ischemia and reperfusion injury. J Surg Res 2020;246:512–8. https://doi.org/10.1016/j.jss.2019.09.037.Search in Google Scholar PubMed PubMed Central

10. Maziero Alves, G, Aires, R, de Souza Santos, V, Zambom Côco, L, Peters, B, de Leone Evangelista Monteiro Assis, A, et al.. Sildenafil attenuates nonsteroidal anti-inflammatory-induced gastric ulceration in mice via antioxidant and antigenotoxic mechanisms. Clin Exp Pharmacol Physiol 2021;48:401–11. https://doi.org/10.1111/1440-1681.13414.Search in Google Scholar PubMed

11. Zinni, M, Pansiot, J, Léger, PL, El Kamouh, M, Baud, O. Sildenafil-mediated neuroprotection from adult to neonatal brain injury: evidence, mechanisms, and future translation. Cells 2021;10:2766. https://doi.org/10.3390/cells10102766.Search in Google Scholar PubMed PubMed Central

12. Kumral, A, Yesilirmak, DC, Tugyan, K, Baskin, H, Tekman, I, Duman, N, et al.. Activated protein C reduces intestinal injury in an experimental model of necrotizing enterocolitis. J Pediatr Surg 2010;45:483–9. https://doi.org/10.1016/j.jpedsurg.2009.07.077.Search in Google Scholar

13. Ozdemir, R, Yurttutan, S, Sari, FN, Oncel, MY, Erdeve, O, Unverdi, HG, et al.. All-trans-retinoic acid attenuates intestinal injury in a neonatal rat model of necrotizing enterocolitis. Neonatology 2013;104:22–7. https://doi.org/10.1159/000350510.Search in Google Scholar

14. Guven, A, Gundogdu, G, Uysal, B, Cermik, H, Kul, M, Demirbag, S, et al.. Hyperbaric oxygen therapy reduces the severity of necrotizing enterocolitis in a neonatal rat model. J Pediatr Surg 2009;44:534–40. https://doi.org/10.1016/j.jpedsurg.2008.06.008.Search in Google Scholar

15. Ozdemir, R, Yurttutan, S, Sarı, FN, Uysal, B, Unverdi, HG, Canpolat, FE, et al.. Antioxidant effects of N-acetylcysteine in a neonatal rat model of necrotizing enterocolitis. J Pediatr Surg 2012;47:1652–7. https://doi.org/10.1016/j.jpedsurg.2012.02.016.Search in Google Scholar

16. Okur, H, Küçükaydin, M, Köse, K, Kontaş, O, Doğam, P, Kazez, A. Hypoxia-induced necrotizing enterocolitis in the immature rat: the role of lipid peroxidation and management by vitamin E. J Pediatr Surg 1995;30:1416–9. https://doi.org/10.1016/0022-3468(95)90395-x.Search in Google Scholar

17. Nadler, EP, Dickinson, E, Knisely, A, Zhang, XR, Boyle, P, Beer-Stolz, D, et al.. Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis. J Surg Res 2000;92:71–7. https://doi.org/10.1006/jsre.2000.5877.Search in Google Scholar

18. Jilling, T, Lu, J, Jackson, M, Caplan, MS. Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental rat model of neonatal necrotizing enterocolitis. Pediatr Res 2004;55:622–9. https://doi.org/10.1203/01.pdr.0000113463.70435.74.Search in Google Scholar

19. Ohkawa, H, Ohishi, N, Yagi, K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. https://doi.org/10.1016/0003-2697(79)90738-3.Search in Google Scholar

20. Paglia, DE, Valentine, WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–69.Search in Google Scholar

21. Lowry, OH, Rosebrough, NJ, Farr, AL, Randall, RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. https://doi.org/10.1016/s0021-9258(19)52451-6.Search in Google Scholar

22. Yurttutan, S, Ozdemir, R, Canpolat, FE, Oncel, MY, Uysal, B, Unverdi, HG, et al.. Protective effects of colchicine in an experimental model of necrotizing enterocolitis in neonatal rats. J Surg Res 2013;183:156–62. https://doi.org/10.1016/j.jss.2013.01.002.Search in Google Scholar PubMed

23. Ganji, N, Li, B, Lee, C, Filler, R, Pierro, A. Necrotizing enterocolitis: state of the art in translating experimental Research to the bedside. Eur J Pediatr Surg 2019;29:352–60. https://doi.org/10.1055/s-0039-1693994.Search in Google Scholar PubMed

24. Bivalacqua, TJ, Musicki, B, Hsu, LL, Berkowitz, DE, Champion, HC, Burnett, AL. Sildenafil citrate-restored eNOS and PDE5 regulation in sickle cell mouse penis prevents priapism via control of oxidative/nitrosative stress. PLoS One 2013;8:e68028. https://doi.org/10.1371/journal.pone.0068028.Search in Google Scholar PubMed PubMed Central

25. Karabulut, R, Turkyilmaz, Z, Sonmez, K. Comment: oxidative DNA damage and NOX4 levels in children with undescended testes. Eur J Pediatr Surg 2021;31:541.10.1055/s-0040-1713132Search in Google Scholar PubMed

26. Cao, Z, Li, Y. Chemical induction of cellular antioxidants affords marked protection against oxidative injury in vascular smooth muscle cells. Biochem Biophys Res Commun 2002;292:50–7. https://doi.org/10.1006/bbrc.2002.6614.Search in Google Scholar PubMed

27. Bostancı, ME, Hepokur, C, Kisli, E. The effects of sildenafil and N-acetylcysteine the zone of stasis in burns. Ulus Travma Acil Cerrahi Derg 2021;27:9–16.10.14744/tjtes.2020.25679Search in Google Scholar PubMed

28. Margonis, GA, Christoloukas, N, Antoniou, E, Arkadopoulos, N, Theodoropoulos, G, Agrogiannis, G, et al.. Effectiveness of sildenafil and U-74389G in a rat model of colitis. J Surg Res 2015;193:667–74. https://doi.org/10.1016/j.jss.2014.08.064.Search in Google Scholar PubMed

29. Montani, D, Chaumais, MC, Guignabert, C, Günther, S, Girerd, B, Jaïs, X, et al.. Targeted therapies in pulmonary arterial hypertension. Pharmacol Ther 2014;141:172–91. https://doi.org/10.1016/j.pharmthera.2013.10.002.Search in Google Scholar PubMed

30. Yazji, I, Sodhi, CP, Lee, EK, Good, M, Egan, CE, Afrazi, A, et al.. Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling. Proc Natl Acad Sci USA 2013;110:9451–6. https://doi.org/10.1073/pnas.1219997110.Search in Google Scholar PubMed PubMed Central

31. Soydan, G, Sokmensuer, C, Kilinc, K, Tuncer, M. The effects of sildenafil on the functional and structural changes of ileum induced by intestinal ischemia-reperfusion in rats. Eur J Pharmacol 2009;610:87–92. https://doi.org/10.1016/j.ejphar.2009.03.038.Search in Google Scholar PubMed

32. Rodriguez-Miguelez, P, Lee, N, Tucker, MA, Csányi, G, McKie, KT, Forseen, C, et al.. Sildenafil improves vascular endothelial function in patients with cystic fibrosis. Am J Physiol Heart Circ Physiol 2018;315:H1486–h94. https://doi.org/10.1152/ajpheart.00301.2018.Search in Google Scholar PubMed PubMed Central

33. Kosutova, P, Mikolka, P, Balentova, S, Kolomaznik, M, Adamkov, M, Mokry, J, et al.. Effects of phosphodiesterase 5 inhibitor sildenafil on the respiratory parameters, inflammation and apoptosis in a saline lavage-induced model of acute lung injury. J Physiol Pharmacol 2018;69. https://doi.org/10.26402/jpp.2018.5.15.Search in Google Scholar PubMed

34. Cho, SX, Berger, PJ, Nold-Petry, CA, Nold, MF. The immunological landscape in necrotising enterocolitis. Expet Rev Mol Med 2016;18:e12. https://doi.org/10.1017/erm.2016.13.Search in Google Scholar PubMed PubMed Central

35. Yurttutan, S, Ozdemir, R, Canpolat, FE, Oncel, MY, Unverdi, HG, Uysal, B, et al.. Beneficial effects of Etanercept on experimental necrotizing enterocolitis. Pediatr Surg Int 2014;30:71–7. https://doi.org/10.1007/s00383-013-3415-4.Search in Google Scholar PubMed

36. Araújo, S, Duarte-Silva, E, Marinho, CGS, Oliveira, WH, França, MER, Lós, D, et al.. Effect of sildenafil on neuroinflammation and synaptic plasticity pathways in experimental autoimmune encephalomyelitis. Int Immunopharm 2020;85:106581. https://doi.org/10.1016/j.intimp.2020.106581.Search in Google Scholar PubMed

37. Lee, J, Bae, EH, Ma, SK, Kim, SW. Altered nitric oxide system in cardiovascular and renal diseases. Chonnam Med J 2016;52:81–90. https://doi.org/10.4068/cmj.2016.52.2.81.Search in Google Scholar PubMed PubMed Central

38. Jia, H, Guo, Z, Yao, Y. PDE5 inhibitor protects the mitochondrial function of hypoxic myocardial cells. Exp Ther Med 2019;17:199–204. https://doi.org/10.3892/etm.2018.6951.Search in Google Scholar PubMed PubMed Central

39. Hornik, CP, Onufrak, NJ, Smith, PB, Cohen-Wolkowiez, M, Laughon, MM, Clark, RH, et al.. Association between oral sildenafil dosing, predicted exposure, and systemic hypotension in hospitalised infants. Cardiol Young 2018;28:85–92. https://doi.org/10.1017/s1047951117001639.Search in Google Scholar PubMed PubMed Central

40. Cohen, JL, Nees, SN, Valencia, GA, Rosenzweig, EB, Krishnan, US. Sildenafil use in children with pulmonary hypertension. J Pediatr 2019;205:29–34.e1. https://doi.org/10.1016/j.jpeds.2018.09.067.Search in Google Scholar PubMed PubMed Central

Received: 2021-10-04
Accepted: 2022-04-12
Published Online: 2022-05-23

© 2022 Sevcan İpek et al., published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.