Vitamin D by targeting hyperoxia-induced bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is a common and serious complication of premature infants. The incidence of BPD has increased over the past few years due to improved survival in very preterm births. 1 The poor prognosis of patients with BPD usually persists into adulthood. Despite the detrimental long-term effects of BPD on respiratory function, there are currently no effective treatment options due to its mechanism of interaction with multifactorial etiologies, including hyperoxia and inflammation. Therefore, there is an urgent need to study the pathogenesis of BPD and identify potential therapeutic opportunities through new pharmacological approaches.

Vitamin D (VitD) has multiple biological effects on calcium homeostasis and metabolism. 2 More recently, VitD has been implicated in other processes such as cell proliferation and anti-inflammatory activity. 3,4 Another study showed that some chronic lung diseases are associated with VitD deficiency. 5 In addition, preterm infants are more likely to develop VitD deficiency and BPD than term infants. 6 VitD has been shown to attenuate lung injury, inflammation and apoptosis in hyperoxia-induced mouse models. 7,8 Finally, preclinical studies of BPD have shown that VitD has potential effects on lung development as it regulates cell growth and apoptosis. 9 , 10

The mitogen-activated protein kinase (MAPK) pathway involves MEK1/2 and ERK1/2 kinases, which regulate cell proliferation, inflammation, and apoptosis, and is known to play a key role in cancer by activating this pathway through phosphorylation of key targets. 11,12 Many growth factors trigger activation of the MEK1/2-ERK1/2 signaling pathway, and upregulation of phosphorylated forms has been detected in many solid tumors and lung diseases. 13 A recent study demonstrated increased expression of phosphorylated MEK1/2 and ERK1/2 in the lung of an experimental acute lung injury model. 14

It is increasingly recognized that inflammation severely affects the progression and severity of BPD, and chorioamnionitis and sepsis are important risk factors for the development of BPD. 15,16 Apoptosis is a common form of programmed cell death. A previous study showed that apoptosis induced by BCL2-binding component 3 (BBC3) is closely related to the mechanism of BPD. 17 Mitochondrial apoptosis is the most commonly dysregulated form of apoptosis and has been shown to play an important role in cancer therapy. 18 However, to our knowledge, there are few reports on the effects of mitochondria and the MEK1/2-ERK1/2 pathway on VitD administration in BPD inflammation and apoptosis. We hypothesized that VitD treatment reduces inflammation and apoptosis through mitochondrial-mediated and MEK1/2-ERK1/2 signaling pathways involved in BPD. To test this hypothesis, we first investigated the effects of VitD administration on lung structure, inflammation, and apoptosis in the BPD C57BL/6 mouse model. We found that VitD treatment restored alveolarization and reduced fibrosis and improved inflammation and apoptosis in BPD mouse neonates. We then examined the effect of VitD pretreatment on mitochondrial apoptosis in BEAS-2B cells exposed to hyperoxia. We found that VitD pretreatment improved mitochondrial-mediated apoptosis. Finally, we detected reduced inflammation and apoptosis by chemically inhibiting or genetically silencing the vitamin D receptor (VDR) in the MEK1/2-ERK1/2 signaling pathway in BEAS-2B cells. These results suggest that early intervention in VitD may provide a new strategy for the treatment of BPD in preterm infants.

Materials and methods

Animals and Treatment
One-day-old C57BL/6 mice were purchased from the JOUNN laboratory (Suzhou, China). Postnatal day 2 (PN2) neonatal mice were randomly divided into four groups: (1) RA control group, intraperitoneal injection of saline in room air (RA), exposed to 21% O2; (2) RA+VitD group, RA was intraperitoneally injected with calcitriol; (3) BPD mice were maintained under hyperoxia (85% O 2 ); (4) BPD+VitD group was intraperitoneally injected with calcitriol under hyperoxia. As previously described, PN2 mouse neonates were exposed to 85% O 2 (hyperoxia, BPD) or 21% O 2 (normoxia, RA) until 14 days of age (PN14). 19 All mice received daily intraperitoneal injections of 5 µg/Kg calcitriol (AbMole, USA) or saline control from PN2 to PN14 as previously described. 20 PN14 mice were anesthetized to remove their lungs, which were frozen in liquid nitrogen for later use (Supplementary Figure 1A). The animal protocol was approved by the Ethics Committee of Soochow University (protocol number 2013LW003), and the experiments were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.

Lung histology and morphology
Lungs were excised and fixed in 10% formalin for 24 hours, embedded in paraffin, and stained with hematoxylin and eosin (HE). Lung morphometric measures, including mean linear intercept (MLI), radial alveolar count (RAC), and septal thickness, were calculated as previously described. 21,22 The MLI was calculated using Image J software by measuring the average distance between the alveolar septal tissue intersection and the overlay counting grid. RAC was calculated as the number of septa intersected by each line from the center of the respiratory bronchioles to the distal acinus. Alveolar septal wall thickness was estimated using ImageJ software. Masson's trichrome staining was used to assess peribronchial and alveolar fibrosis. Masson's trichrome staining was performed to assess collagen deposition in the alveoli by determining the percentage of staining using Image J software. 9 Lung injury scoring parameters, including clearing, edema, alveolar wall thickening, and neutrophil infiltration, were scored in five randomly selected regions per section. The severity of pathological features was scored on a scale of 0, 1, 2, 3, or 4, indicating no/very minor, minor, moderate, severe, or very severe injury, respectively. The total score is the average score calculated for each group. At least three sections per sample and at least six fields per section were evaluated with blinded random selection. Three technical replicates were assessed with 10 mice per group.

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