Study on the Correlation between MDM2/p53 Interaction and Apoptosis of AEC II in Hyperoxic Acute Lung Injury

Abstract

Background: Hyperoxic acute lung injury (HALI) is a pathological condition characterized by lung damage resulting from the inhalation of high concentrations of oxygen. The incidence of this disease has been increasing annually in clinical practice. Therefore, in-depth research into the pathogenesis of HALI is of great significance. This study aims to reveal the underlying regulating mechanism of mouse double minute 2 (MDM2)/p53 on Type II Alveolar Epithelial Cells (AEC II) apoptosis in HALI through in vivo and in vitro experiments. Methods: AEC II cells were isolated, purified, and cultured from healthy male Sprague Dawley (SD) rats. Subsequently, these cells were infected with an adenovirus expressing MDM2/p53 to establish a model of HALI. The proliferation activity of AEC II cells was assessed using the cell counting kit (CCK)-8 assay, and the expressions of MDM2, p53, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, and Caspase-9 mRNA were measured through real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Additionally, cell apoptosis was evaluated through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry (FCM). Arterial blood gas analysis was performed to measure the oxygenation index (OI) and respiratory index (RI). Early apoptosis was detected by measuring mitochondrial membrane potential (MMP), and expressions of MDM2, p53, Bcl-2, Bax, Caspase-3, and Caspase-9 proteins were examined through Western blot analysis. Finally, lung tissue pathology was assessed using the hematoxylin-eosin (HE) staining method. Results: In H₂O₂-induced AEC II cells, an increase in apoptosis and a significant decrease in cell viability were observed, accompanied by a notable decrease in MDM2 expression and an evident increase in p53 expression (p < 0.05). Either over-expression of MDM2 or interference with p53 significantly reduced H₂O₂-induced AEC II apoptosis (p < 0.05), while interfering with MDM2 or over-expressing p53 significantly promoted H₂O₂-induced AEC II apoptosis (p < 0.05). Over-expression of MDM2 led to a significant reduction in the expressions of p53, Bax, Caspase-3, and Caspase-9, as well as a decrease in the RI and wet-to-dry weight ratio in the HALI model (p < 0.05). It also resulted in an increase in the OI and Bcl-2 expression in the HALI model (p < 0.05), alleviated the pathological state of lung tissue, and restored mitochondrial transmembrane potential in the HALI model (p < 0.05). The p53 molecular inhibitor, Pifithrin-α, significantly promoted the reduction of RI and wet-to-dry weight ratio and increased OI in the HALI model (p < 0.05). Moreover, the Pifithrin-α inhibitor was found to improve lung tissue structure in the HALI model. Conclusion: MDM2 plays a dual role in preventing the apoptosis of AEC II and protecting lung tissue by inhibiting p53 expression.