Bronchopulmonary dysplasia (BPD) is a chronic pulmonary disease seen in premature newborns who required mechanical ventilation and oxygen therapy due to acute respiratory distress.1,2 Although its etiopathogenesis is not completely understood, it is believed to be multifactorial, influenced by prematurity, oxidative lung lesion and inflammation, like chorioamnionitis, hyperoxia, infection, respirator-induced injury and many other adverse stimuli. The lesion alters the normal growth and maturation of the lungs caused by deteriorated alveolar and vascular growth, leading to the abnormal lung structure of BPD.2–4 It was reported for the first time more than 40 years ago, but the nature of BPD has been modified with the increasing use of prenatal corticosteroids, surfactant therapy, new ventilatory strategies and other treatments.5,6 Nonetheless, BPD continues to be an important cause of neonatal morbidity and mortality, as improved perinatal care has increased the survival rate of extremely premature newborns (<28 weeks of gestation).2,4,7 These children are at high risk for having long-term lung injury due to the significant deterioration in lung function produced by the prolonged use of respirators during neonatal intensive care unit stays. After hospital discharge, these children may also have frequent hospitalizations, recurring respiratory exacerbations, intolerance to exercise and other signs of late cardiorespiratory disease, in addition to adverse neurodevelopmental evolution.4 Clinical and laboratory studies suggest that early intervention strategies that directly preserve survival and lung cell function should prevent the appearance of BPD.3,7 However, we still do not have treatments that can effectively attenuate lung lesions and promote lung growth in order to reduce the incidence and severity of BPD. As a consequence, it is necessary to identify new treatment options for the prevention of severe pulmonary injury in the course of BPD.

The Nigella sativa (NS) plant, commonly known as “black seed”, is used as a medicinal plant for the treatment of many diseases.8 In clinical and experimental studies, several therapeutic effects of NS and its extracts have been reported in respiratory diseases.9–12 In NS extracts, many enriched bioactive molecules have been identified, both in fixed oil as well as essential oil. It has been demonstrated that much of the biological activity of NS is due to thymoquinone, which is the main component of the essential oil and fixed oil.8 It has been observed that the volatile oil contains 18.4%–24% thymoquinone and also 46% monoterpenes (e.g. p-cymene, a-pinene).13 Several papers have identified that the extracts of NS have anti-inflammatory and antimicrobial properties (against several germs) as well as antioxidant properties due to its activity of free-radical elimination.14 In addition, its inhibitory action of human neutrophil elastase has been determined15 along with its immunomodulation and cytoprotection activity in many systems of the body with a low grade of toxicity in clinical and experimental studies.8,16 However, the effects of NS oil (NSO) on lungs that have undergone hyperoxic injury have not yet been researched. The objective of this study was therefore to investigate the favorable effects of NSO on the lungs of rat pups with BPD induced by hyperoxia.

For the statistical analysis, the SPSS (v15.0, IL, USA) program was used. Normal data distributions were examined graphically with the Shapiro–Wilks test. Comparisons of numerical variables were done with an ANOVA and the Bonferroni correction. Discrete variables were compared with a χ2 test. Differences with a P value <.05 were considered statistically significant. Data were expressed as mean±standard deviation (SD). The statistical power was analyzed. Taking a magnitude effect value of 0.60 and an alpha error of 0.05 and a beta error of 0.20 to obtain a statistical power of 80%, 10 animals were observed to be sufficient in each group.

At the end of the experiment, 3 rat pups from the hyperoxia group and one from the hyperoxia+NSO group had died. However, there were no significant differences between the 2 groups regarding survival rate (P>.05). In addition, mean birth weight of the pups in the study groups was 4.9±0.4g in the control group, 5.0±0.3g in the hyperoxia group, and 5.1±0.2g in the hyperoxia+NSO group. No significant differences were observed in birth weight among the three groups (P>.05). At the end of the study, the mean bodyweight of the pups in the control group was 16.9±1.5g, but the mean weights in the hyperoxia+NSO and hyperoxia groups were 13.7±1.3g and 11.1±0.8g, respectively, and the mean weight of the pups from the hyperoxia+NSO group was significantly higher than that of the pups from the hyperoxia group (P<.05).

The severity of the lung lesions found was from grade 1 to grade 4 in the histopathologic evaluation (Figs. 1A–C and 2). No alveolar wall thickening or cellular infiltrates were observed in the groups with O2 at 95%+NSO and O2 at 21%. We identified significant changes in the histologic grade and an improvement in the 95% O2+NSO group compared with the group with 95% O2 alone (P<.05). Thus, there was less severe lung lesion in the pups that had been treated with NSO (Fig. 1A–C). The radial alveolar count also showed a recuperation after treatment with NSO (Fig. 2), reflected in a greater number of intact alveoli in this group, with significantly higher radial alveolar counts than those observed in the untreated 95% O2 group (P<.05). In addition, we also observed a better density of lamellar body membrane protein in the NSO group (P<.05). The subepithelial area coefficient demonstrated fibrosis and edema in the 95% O2 group, and this thickening was recuperated with the NSO treatment (P<.05). The epithelium of the intact bronchioles of the 3 groups did not present histological differences, and the epithelial area coefficient showed no significant changes (P>.05).

Fig. 1.

Hematoxylin-eosin stain (A–C, 100×), Masson's trichrome stain (D–I, 400×) and immunohistochemistry for lamellar body membrane protein (J–L, 400×). There is partial destruction of lung tissue (panel B, black arrows), cell infiltration (panel E, white arrows), edema (black arrow) and fibrosis (white arrow) (panel H) in the 95% O2 group, which is not observed in the 21% O2 or the 95% O2+NSO group. Panels J–L show the immunohistochemistry for the lamellar body membrane protein in each group.

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Fig. 2.

Histogram of the histopathologic evaluation, radial alveolar count, subepithelial/epithelial area coefficients and lamellar body membrane protein counts (cells positive for lamellar bodies/mm2) in each group. The asterisk (*) indicates significant changes (P<.05) compared with the groups with 21% O2 and 95% O2+NSO.

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The biochemical analysis was done in samples of lung tissue obtained from the study groups. The levels of GSH-Px and SOD in the tissue were significantly higher in the hyperoxia+NSO group than in the hyperoxia group, and it determined a reduction in oxidative stress (P<.05) (Table 1). However, the levels of MDA and MPO in the tissues of the hyperoxia+NSO group were significantly lower than those of the hyperoxia group, which indicated a reduction of lipid peroxidation due to reactive oxygen species (ROS) and a reduction in the infiltration of neutrophils/inflammation with NSO treatment (Table 1). Furthermore, the levels of MDA and MPO of the control group continued to be lower than those of the hyperoxia+NSO group, in addition to the higher levels of GSH-Px and SOD (P<.05) (Table 1).

In the present study, we have evaluated the favorable effects of NSO on rat pups with hyperoxia-induced BPD. Our results show that the administration of NSO reduced the pulmonary histopathologic lesion and preserved lung cells. The biochemical analysis identified an increase in antioxidant activities, reduced oxidant levels and a reduction in lipid peroxidation and the infiltration of neutrophils-inflammation in the lung tissues damaged by hyperoxia. As far as we know, this is the first study that examines the effects of NSO in an experimental model of BPD in rats.

BPD has a complex pathogenesis that is not sufficiently understood. In addition to the immaturity of the lungs, it has been suggested that the effects of the oxidative lesion that causes the generation of ROS molecular species play a crucial role in the pathogenesis of BPD.1–4,25–27 ROS are produced by the cellular metabolism of molecular oxygen and the activation of neutrophils and macrophages in the lungs. ROS cause oxidative stress by means of the oxidation of lipids, proteins and DNA.28 Consequently, ROS can produce a tissue lesion that leads to the pathologic alterations that are observed in newborns with BPD.25 In addition, premature newborns show an exclusive vulnerability to oxidative stress, caused by high concentrations of additional oxygen during respiratory support as a consequence of the inadequate and immature antioxidant system at birth as well as the deterioration of upregulation as a response to oxidative stress. Consequently, premature babies present increased risk for developing lung damage induced by ROS.26,29 Antioxidant enzymes like SOD and GSH-Px are important for the elimination of ROS.26 It has been suggested that tissue levels of SOD and GSH-Px may reflect ROS levels.30,31 In addition, MDA, which is a reliable indicator of lipid peroxidation and oxidative stress in tissues, is the product of degradation originated by the oxidation of polyunsaturated fatty acids by ROS activity.32 Thus, ROS can be evaluated indirectly with the determination of MDA and the levels of some antioxidant enzymes like SOD or GSH-Px in tissue.30–32

N. sativa and the oil obtained from it are reservoirs of different bioactive compositions, and they have been demonstrated to reduce tissue injury thanks to their antioxidant activities, especially by reducing oxidative stress through their activity of eliminating radicals.8,33 Moreover, it has been observed that NSO promotes the activity of antioxidant enzymes like GSH-PX and SOD, and it reduces the lipid peroxidation of the biological membranes thanks to their antioxidant properties and elimination of ROS.33 In the present study, we have demonstrated that the increased levels of MDA, as well as the reduction of GSH-PX and SOD levels, determined and supported the lung lesion and lipid peroxidation induced by ROS in the lungs of rats subjected to hyperoxia. However, treatment with NSO produced a decrease in MDA levels as well as an increase in GSH-PX and SOD levels in the rat pups subjected to hyperoxia. These data establish that treatment with NSO reduces oxidative stress and lipid peroxidation, and it promotes endogenous antioxidant enzymes during the hyperoxia process in the lungs.

It is believed that inflammation plays a central role in the pathogenesis of BPD. Premature newborns have a high prevalence of inflammatory disorders such as chorioamnionitis in the neonatal period.1–4,25 Furthermore, hyperoxia can also affect morphogenesis due to its chemotactic effects on inflammatory cells like neutrophils and macrophages. Neutrophils and macrophages produce increased inflammatory response against oxygen exposure, as well as a release of ROS at the place of the inflammation, with the consequent lung lesion. In addition, the existing evidence suggests that the lung lesion of BPD is associated with an increase in the content of neutrophils and chemokines in the lungs.25 Consequently, the number of neutrophils grows persistently both in the interstitium as well as in the airspaces of the lungs of newborns with BPD, and the inhibition of the entrance of neutrophils in the hyperoxic lung promotes postnatal lung growth.34,35 However, many studies have identified that NSO inhibits inflammatory lung responses, peribronchial, alveolar and alveolar wall inflammatory cell infiltration with neutrophils and macrophages, and it has immunomodulatory effects on these cells, in addition to antimicrobial properties against different pathogens.8,10–12,16,17,33 Another important factor that has been suggested to contribute to producing lung damage is human neutrophil elastase (HNE). It has been described that HNE plays an important role in the extracellular proteolytic process and is able to fragment many important proteins like elastin, a crucial protein of the extracellular matrix in the lungs. Nevertheless, NS has been demonstrated to have inhibitory effects on the activity of HNE, and it is believed to be a natural anti-elastase agent that acts against HNE.15 It has also been established that the MPO enzyme is specific for the lysosomes of the neutrophils.36 Thus, the evaluation of the migration and neutrophilic infiltration in the lungs can be done indirectly by determining the activity of MPO in lung tissue.34–37 In the present study, the increased MPO activity indicated that the entrance of neutrophils in the lungs could play a central role in lung injury induced by hyperoxia. Nevertheless, our data showed that the activity of MPO in the lungs was reduced with the NSO treatment during the hyperoxic lesion. Consequently, the results obtained suggest that NSO possesses anti-inflammatory properties and prevented the activation of neutrophils in addition to producing antioxidant effects in rat pups subjected to hyperoxia.

In the present study, pulmonary lesion similar to that observed in newborns with BPD was induced by prolonged hyperoxia and showed pathological alterations in the developing lung tissue, which included the following: (a) decreased alveolarization, demonstrated by a decrease in radial alveolar count (RAC) and thickening of the alveolar walls; (b) reduced expression pattern of lamellar bodies, which indicates severe lung lesion through the reduction of the type II cell count containing lamellar bodies; and (c) a significant increase in fibrosis, which is one of the main histopathologic characteristics of lung injury in BPD.4,25,37–40 However, our results indicated the increase in RAC and reduction in the thickness of the alveolar walls, reduced fibrosis as well as improved lamellar body protein staining pattern density in the group of animals with hyperoxia treated with NSO. All of this is evidence that indicates the prevention of lung injury, with better alveolarization and decreased fibrosis. Consequently, given these results, it would suggest that treatment with NSO protects the lungs against the severe damage caused by lung injury induced by hyperoxia, which is an important contributing factor in the development of BPD. These results could be due to the anti-inflammatory, immunomodulator, cytoprotector and antioxidant properties of NSO, in addition to its activity inhibiting HNE.

In conclusion, the therapeutic objectives in BPD are centered around reducing the persisting lesion to a minimum, reducing inflammation, maintaining adequate oxygenation and promoting pulmonary growth in the developing lungs. Treatment with NSO significantly attenuated the pulmonary lesion by maintaining better alveolarization and reducing fibrosis, inflammation and oxidative stress. Thus, NSO seems to be a promising agent for the protection of lung tissues from severe lesion in BPD. However, new studies will be necessary to determine whether treatment with NSO has favorable effects for protecting the lungs from the severe damage produced by the development of BPD.

This study was funded by a grant from the GATA Military School of Medicine and the Fatih University Faculty of Medicine Scientific Research Committee.

The authors declare having no conflict of interests.