Low hyper-oxygen exposure induces p21-dependent p53-independent senescence in alveolar cells

Abstract

Bronchopulmonary dysplasia (BPD) is a chronic pulmonary condition predominantly affecting premature neonates who necessitate oxygen therapy. Currently, BPD is classified into two types-old and new BPD-that differ in histology and pathology. The new BPD is observed in premature infants exposed to gentle ventilation and low oxygen concentrations, emphasizing the disruption of normal development. This study assessed the effects of low-to-high oxygen concentrations on rat alveolar epithelial L2 cells, aiming to mimic clinical scenarios. Exposure to 40 % oxygen induced p53-independent p21 expression in alveolar cells, resulting in G1-cell cycle exit cellular senescence. The inhibition of autophagy induced senolysis in L2 cells exposed to 40 % oxygen. Alveolar epithelial cells exhibit distinct responses to varying oxygen concentrations. Elucidating the interaction between senescence and autophagy is crucial for understanding the pathogenesis of novel bronchopulmonary dysplasia (BPD) in premature infants, thereby identifying potential preventive strategies.

Keywords: alveolar epithelial cells; autophagy; bronchopulmonary dysplasia; cellular senescence; oxygen therapy; premature infants.

Figure 1

Hyperoxia reduced L2 cellular viability: (a) The results of bioactivity at varying oxygen concentrations and durations. Data are shown as mean ± SEM from three independent experiments. Statistical significance is indicated by *P < 0.05, **p < 0.01, ***p < 0.001 and ****P < 0.0001. (b) Live cell counting in various treatment groups. Data are shown as mean ± SEM. P-values was determined by One-way-ANOVA. Statistical significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, where values were compared to the 20% oxygen group by post hoc analysis.

Figure 2

High oxygen concentration induced apoptosis in L2 cells: (a) The proportion of apoptotic L2 cells under varying oxygen concentrations, detected using annexin V and propidium iodide. (b) The percentage of apoptotic L2 cells at various oxygen concentrations and durations. (c) Representative western blot analysis shows the expression of cleaved-caspase-3 protein at various oxygen concentrations and durations. Quantification of cleaved-caspase3 abundance was normalized to the corresponding β-actin level. Data are shown as mean ± SEM from three independent experiments. P-values were determined by one-way ANOVA. Statistical significance is indicated by *P < 0.05, where values are compared to various treatment group at the same duration by post hoc analysis.

Figure 3

Various oxygen concentrations induce different patterns of cell cycle arrest in L2 cell lines: (a) Representative histograms demonstrating the proportion of L2 cells in each phase of the cell cycle under varying oxygen concentrations. (b) Mean percentage of cells in each phase of the cell cycle at various oxygen concentrations and durations. Cells are stained with propidium iodide. Means and standard error bars are based on four repeats. Cell cycle distribution is significantly blocked in the G0/G1 phase when L2 cells are cultivated in 40 % oxygen. At 85 % oxygen, the cell cycle is blocked in the G2/M phase, with an increase in apoptosis (subG1).

Figure 4

Forty percent oxygen induced senescence in L2 cells: (a) Cellular proliferation is measured using 5-ethynyl-2'-deoxyuridine immunochemical detection staining(green). Nuclei were stained with DAPI (blue), and the merged view shows the overlap. The quantification of EdU-positive cells was performed by counting cells from three random fields per group across three independent experiments. (b) Senescent cells of L2 cells are detected by β-galactosidase staining under 40 % oxygen exposure. Quantification of SA-β gal-positive cells was performed by counting cells from three random fields per group across three independent experiments. (c) Cellular senescence was stable and irreversible even return optimal growth conditions (20% oxygen environment). After oxygen exposure, collect the cells at the designated time points and count the total number of viable cells using a cell counter. Data are shown as mean ± SEM from three independent experiments. Statistical significance is indicated by *P < 0.05 and **p < 0.01.

Figure 5

Forty percent oxygen induced senescence through p53-independent p21-dependent signaling pathway in L2 cells: (a) Representative western blot analysis of pH2A.X, p53, p53(s15), p53(s37), p16 and p21 protein expression at various oxygen concentrations and durations. (b) Quantification of the proteins shown in (A) was normalized to β-actin under corresponding oxygen concentrations and durations. (c) p21 small interfering RNA increased the death of L2 cells under 40 % oxygen exposure. Data are shown as mean ± SEM from three independent experiments. Statistical significance is indicated by *P < 0.05 and **p < 0.01.

Figure 5

Forty percent oxygen induced senescence through p53-independent p21-dependent signaling pathway in L2 cells: (a) Representative western blot analysis of pH2A.X, p53, p53(s15), p53(s37), p16 and p21 protein expression at various oxygen concentrations and durations. (b) Quantification of the proteins shown in (A) was normalized to β-actin under corresponding oxygen concentrations and durations. (c) p21 small interfering RNA increased the death of L2 cells under 40 % oxygen exposure. Data are shown as mean ± SEM from three independent experiments. Statistical significance is indicated by *P < 0.05 and **p < 0.01.

Figure 6

Cytokines secretion by senescent L2 cells after exposure to 40 % oxygen are detected using enzyme-linked immunosorbent assay. A significant increase in IL-17A compared to control group. Data are shown as mean ± SEM from three independent experiments. Statistical significance is indicated by *P < 0.05, **p < 0.01, ***p < 0.001 and ****P < 0.0001. Abbreviations: INF-γ, interferon-γ; TNF-α, tumor necrosis factor-α; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Figure 7

Autophagy inhibition induces cell death in senescent L2 cells: (a) Cellular viability is reduced by autophagy inhibitor, bafilomycin. Data are shown as mean ± SEM from three independent experiments. Statistical significance is indicated by *P < 0.05, **p < 0.01, ***p < 0.001 and ****P < 0.0001. (b) Bafilomycin reduces the levels of senescence-associated-β-galactosidase-positive cells in the 40 % oxygen group. Quantification of SA-β gal-positive cells was performed from three fields per group in three independent experiments. (c) Representative western blot analysis indicated protein expression at 40 % oxygen concentration and duration. Protein quantification was normalized to β-actin. Bafilomycin reduced p21 expression and increased p53 expression in L2 cells. Statistical significance is indicated by *P < 0.05, **p < 0.01 and ***p < 0.001.

Figure 8

The mechanism graph of this study. Exposure to 40 % oxygen induced p53-independent p21 expression in alveolar cells, which caused G1 cell cycle exit and cellular senescence. Autophagy inhibition induced senolysis of senescent L2 cells.