Hu Jinhui, Wu Zhixin, Wang Huawei, Geng Haifeng, Huo Jie, Zhu Xueping, Zhu Xiaoli
Department of Neonatology, Children's Hospital of Soochow University, Suzhou, People's Republic of China.
Neonatal Medical Center, Huai'an Maternity and Child Health Care Hospital, Xuzhou Medical University, Huai'an, People's Republic of China.
J Inflamm Res. 2022 Aug 25;15:4891-4906. doi: 10.2147/JIR.S371906. eCollection 2022.
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.
We first performed in vivo studies using neonatal C57BL/6 mice in which we induced BPD by exposing them to a hyperoxic environment (85% O). The mice were divided into room air (RA; 21% O), 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.
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.
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.
支气管肺发育不良（BPD）是早产儿常见且严重的并发症。据报道，维生素D（VitD）可预防BPD；然而，其在线粒体介导的和MEK1/2-ERK1/2信号通路中的作用尚未见报道。
我们首先使用新生C57BL/6小鼠进行体内研究，通过将它们暴露于高氧环境（85%氧气）来诱导BPD。将小鼠分为常氧组（RA；21%氧气）、RA+VitD组、BPD组和BPD+VitD组。采用苏木精-伊红染色和Masson三色染色评估肺损伤。使用酶联免疫吸附测定（ELISA）、逆转录定量聚合酶链反应（RT-qPCR）和TUNEL检测法测量炎症和细胞凋亡。然后，我们分析了分为相同组别的BEAS-2B细胞，另外增加了一个BPD+VitD+抑制剂组。通过透射电子显微镜、线粒体膜电位和蛋白质免疫印迹法评估线粒体凋亡。然后我们使用VDR-shRNA使BEAS-2B细胞中的维生素D受体（VDR）沉默。通过RT-qPCR、流式细胞术和蛋白质免疫印迹法检测细胞中的炎症、凋亡率以及MEK1/2和ERK1/2的磷酸化形式。
与RA组肺相比，BPD组肺的平均线性截距、间隔厚度和异常纤维化增加，而肺泡计数减少。给予VitD能够改善BPD组肺的表型。BPD+VitD组肺中白细胞介素-6（IL-6）、干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）的表达以及凋亡率降低。VitD预处理可恢复BEAS-2B细胞中异常的线粒体形态，减少线粒体膜损伤，并降低裂解的半胱天冬酶-3、Bax和Bcl-2的表达。给予VitD还可降低暴露于高氧环境的细胞中IL-6、IFN-γ和TNF-α的信使核糖核酸（mRNA）水平，以及pMEK1/2和pERK1/2的表达和凋亡率。
我们得出结论，VitD治疗通过靶向线粒体途径并经由MEK1/2-ERK1/2信号通路改善了BPD中的细胞凋亡和炎症，从而支持其在这种病症中的潜在治疗用途。
