Establishment and evaluation of an in vitro blast lung injury model using alveolar epithelial cells

Abstract

Background: Gas explosion is a fatal disaster commonly occurred in coal mining and often causes systematic physical injuries, of which blast lung injury is the primary one and has not yet been fully investigated due to the absence of disease models. To facilitate studies of this field, we constructed an in vitro blast lung injury model using alveolar epithelial cells.

Methods: We randomly divided the alveolar epithelial cells into the control group and blast wave group, cells in the blast wave group were stimulated with different strengths of blast wave, and cells in the control group received sham intervention. Based on the standards we set up for a successful blast injury model, the optimal modeling conditions were studied on different frequencies of blast wave, modeling volume, cell incubation duration, and cell density. The changes of cell viability, apoptosis, intracellular oxidative stress, and inflammation were measured.

Results: We found that cell viability decreased by approximately 50% at 6 h after exposing to 8 bar energy of blast wave, then increased with the extension of culture time and reached to (74.33 ± 9.44) % at 12 h. By applying 1000 ~ 2500 times of shock wave to 1 ~ 5 × 105 cells /ml, the changes of cell viability could well meet the modeling criteria. In parallel, the content of reactive oxide species (ROS), malonaldehyde (MDA), interleukin 18 (IL-18), tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β) increased in the blast wave group, while superoxide dismutase (SOD) and Glutathione -S- transferase (GST) decreased, which were highly consistent with that of human beings with gas explosion-induced pulmonary injury.

Conclusion: An in vitro blast lung injury model is set up using a blast wave physiotherapy under 8 bar, 10 Hz blast wave on (1 ~ 5) ×105 alveolar epithelial cells for 1 000 times. This model is flexible, safe, and stable, and can be used for studies of lung injury caused by gas explosion and blast-associated other external forces.

Keywords: alveolar epithelial cells; blast injury; gas explosion; lung injury model; oxidative stress response.

Figure 1

Model construction process using a blast wave therapy instrument type HM08CJ based on the alveolar epithelial cells.

Figure 2

Effects of different blast wave energy and post exposure culture time on L2 cell viability. (A–F) show the changes of cell viability after 4, 6, 8, 10, 12, and 24 h post exposure culture time. Impulse energy gradient is set at each time point for comparison. Data represent mean ± standard deviation; n = 5. *P < 0.05.

Figure 3

Changes of cell viability under different blast wave frequencies, modeling volumes, modeling concentrations and cell plating number. (A) Different impulse frequencies are set for comparison. (B) Different modeling volumes are set for comparison. (C) Different modeling concentrations are set for comparison. (D) Different cell plating number is set for comparison. Group 0 represents the control group and data represent mean ± SD; n = 5. *P < 0.05. NS, not significant, P > 0.05.

Figure 4

Effects of blast wave stimulation on viability and apoptosis of L2 and A549 cells. (A) Images of Calcein-AM/PI staining showing the living and membrane-ruptured L2 and A549 cells. Green, living cells; red, membrane-ruptured cells. (B) Dot plot showing the apoptosis of L2 and A549 cells. (C, D) Quantitative analysis and comparison of cell viability between the control and blast wave groups of L2 and A549 cells. (E) Comparison of L2 cell apoptosis between groups. (F) Comparison of A549 cell apoptosis between groups. (G) Comparison of A549 cell viability between groups. Data represent mean ± SD; n = 10. *P < 0.05. FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 5

Oxidative stress and inflammatory response of blast wave stimulated alveolar epithelial cells. (A, B) Representative images of immunofluorescence staining showing the content of ROS in L2 and A549 cells. (C, D) Quantitative analysis and comparison of intracellular ROS between groups. (E–G) Representative images of immunofluorescence staining showing the expression of IL-18, TNF-α, and TGF-β in L2 cells. (H–J) Representative images of immunofluorescence staining showing the expression of IL-18, TNF-α, and TGF-β in A549 cells. (K–M) Quantitative analysis and comparison the intracellular MDA, GST, and SOD of L2 cells. Data represent mean ± SD; n = 10. *P < 0.05. IL-18, interleukin 18; TNF-α, tumor necrosis factor alpha; TGF-β, transforming growth factor beta; ROS, reactive oxide species; MDA, malonaldehyde; GST, Glutathione -S- transferase; SOD, superoxide dismutase.