Vitamin D Ameliorates Apoptosis and Inflammation by Targeting the Mitochondrial and MEK1/2-ERK1/2 Pathways in Hyperoxia-Induced Bronchopulmonary Dysplasia

Jinhui Hu,1,2,* Zhixin Wu,1,* Huawei Wang,1 Haifeng Geng,1 Jie Huo,1,3 Xueping Zhu,1 Xiaoli Zhu4 1Department of Neonatology, Children’s Hospital of Soochow University, Suzhou, People’s Republic of China; 2Neonatal Medical Center, Huai’an Maternity and Child Health Care Hospital, Xuzhou Medical University, Huai’an, People’s Republic of China; 3Department of Neonatology, Yangzhou Maternity and Child Health Care Hospital, Yangzhou, People’s Republic of China; 4Department of Intervention, The First Affiliated Hospital of Soochow University, Suzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xueping Zhu, Department of Neonatology, Children’s Hospital of Soochow University, No. 92 Zhongnan Street, Industrial Park, Suzhou, Jiangsu, 215025, People’s Republic of China, Tel +86-13073304816, Fax +86-512-80693599, Email zhuxueping4637@hotmail.com Xiaoli Zhu, Department of Intervention, The First Affiliated Hospital of Soochow University, 899 Pinghai Road, Gusu District, Suzhou, Jiangsu, 215006, People’s Republic of China, Tel +86-13013805898, Fax +86-512-65233905, Email zhuxiaoli90@hotmail.comPurpose: Bronchopulmonary dysplasia (BPD) is a common and severe complication in preterm infants. Vitamin D (VitD) has been reported to protect against BPD; however, its role in the mitochondria-mediated and MEK1/2-ERK1/2 pathways has not yet been reported.Methods: We first performed in vivo studies using neonatal C57BL/6 mice in which we induced BPD by exposing them to a hyperoxic environment (85% O2). The mice were divided into room air (RA; 21% O2), RA+VitD, BPD, and BPD+VitD groups. Hematoxylin and eosin and Masson’s trichrome staining were used to evaluate lung injury. Inflammation and apoptosis were measured using ELISA, RT-qPCR, and TUNEL assays. We then analyzed BEAS-2B cells divided into the same groups along with an additional BPD+VitD+inhibitor group. Mitochondrial apoptosis was evaluated by transmission electron microscopy, mitochondrial membrane potential, and Western blotting. We then used VDR-shRNA to silence the Vitamin D Receptor (VDR) in the BEAS-2B cells. The inflammation, apoptotic rate, and the phosphorylated forms of MEK1/2 and ERK1/2 in cells were detected by RT-qPCR, flow cytometry, and Western blotting.Results: The mean linear intercept, septal thickness, and abnormal fibrosis increased, while radial alveolar count decreased in BPD lungs compared to RA lungs. VitD administration was able to ameliorate the phenotype in BPD lungs. IL-6, IFN-γ, and TNF-α expression and the apoptotic rate decreased in the BPD+VitD lung group. VitD pretreatment restored abnormal mitochondrial morphology, reduced mitochondrial membrane loss, and reduced the expression of cleaved caspase-3, Bax, and Bcl-2 in BEAS-2B cells. VitD administration also reduced IL-6, IFN-γ, and TNF-α mRNA, as well as pMEK1/2 and pERK1/2 expression and apoptosis rate in cells exposed to hyperoxia.Conclusion: We concluded that VitD treatment ameliorated apoptosis and inflammation by targeting the mitochondrial pathway and via the MEK1/2-ERK1/2 signaling pathway in BPD, thus supporting its potential therapeutic use in this condition.Keywords: bronchopulmonary dysplasia, vitamin D, hyperoxia, apoptosis, inflammation, mitochondria, MEK1/2-ERK1/2