Role of Carbon Monoxide in Oxidative Stress-Induced Senescence in Human Bronchial Epithelium

Abstract

Prolonged or excessive stimulation from inhaled toxins may cause oxidative stress and DNA damage that can lead to stress-induced senescence in epithelial cells, which can contribute to several airway diseases. Mounting evidence has shown carbon monoxide (CO) confers cytoprotective effects. We investigated the effects of CO on oxidative stress-induced senescence in human airway epithelium and elucidated the underlying molecular mechanisms. Here, CO pretreatment reduced H₂O₂-mediated increases in total reactive oxygen species (ROS) production and mitochondrial superoxide in a human bronchial epithelial cell line (BEAS-2B). H₂O₂ treatment triggered a premature senescence-like phenotype with enlarged and flattened cell morphology accompanied by increased SA-β-gal activity, cell cycle arrest in G0/G1, reduced cell viability, and increased transcription of senescence-associated secretory phenotype (SASP) genes. Additionally, exposure to H₂O₂ increased protein levels of cellular senescence markers (p53 and p21), reduced Sirtuin 3 (SIRT3) and manganese superoxide dismutase (MnSOD) levels, and increased p53 K382 acetylation. These H₂O₂-mediated effects were attenuated by pretreatment with a CO-containing solution. SIRT3 silencing induced mitochondrial superoxide production and triggered a senescence-like phenotype, whereas overexpression decreased mitochondrial superoxide production and alleviated the senescence-like phenotype. Air-liquid interface (ALI) culture of primary human bronchial cells, which becomes a fully differentiated pseudostratified mucociliary epithelium, was used as a model. We found that apical and basolateral exposure to H₂O₂ induced a vacuolated structure that impaired the integrity of ALI cultures, increased goblet cell numbers, decreased SCGB1A1+ club cell numbers, increased p21 protein levels, and increased SASP gene transcription, consistent with our observations in BEAS-2B cells. These effects were attenuated in the apical presence of a CO-containing solution. In summary, we revealed that CO has a pivotal role in epithelial senescence by regulating ROS production via the SIRT3/MnSOD/p53/p21 pathway. This may have important implications in the prevention and treatment of age-associated respiratory pathologies.

Figure 1

CO suppressed H₂O₂-induced increase in intracellular total ROS and mitochondrial superoxide in airway epithelial cells. (a, c, and f) Representative fluorescent images and (b, d, e, g, and h) quantification of intracellular CO and ROS levels in live BEAS-2B cells. (a and b) Cells were stained with CO probe (red) and Hoechst (blue) for 45 min in the presence of different concentrations (0-100%) of CO-containing solution. BEAS-2B cells were treated with (c–e) carboxy-H₂DCFDA (green), CO probe (Red), or (f–h) MitoTracker (green) MitoSOX (red) in control (KH solution) or 50% CO-containing solution, followed by exposing to 100 μM H₂O₂ for 1 h. The fluorescence level in each group was normalized by the cell number indicated by Hoechst. The data were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test, and expressed as the mean ± SEM (n =4). ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 2

CO attenuated H₂O₂-induced cellular senescence. (a) Representative images and (b) quantification data of SA-β-galactosidase positive (blue) BEAS-2B cells pretreated with control or 50% CO-containing solution followed by H₂O₂ exposure for 96 h. (c) Flow cytometric cell cycle distribution assay to detect the proportion of BEAS-2B cells in G1, S, and G2/M phases and (d) CCK-8 assay to detect cell viability for cells pretreated with a control or 50% CO-containing solution followed by H₂O₂ exposure for 48 h. (e) mRNA expression of SASP genes was detected by RT-qPCR with pretreated with control, 50% CO-containing solution followed by H₂O₂ exposure for 12 h. The data were analyzed by one-way ANOVA (b and d) or two-way ANOVA (c and e) followed by Tukey's multiple comparisons test and expressed as the mean ± SEM (n =3). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 3

SIRT3 mediated the inhibitory effects of CO on H₂O₂-induced expression of senescence markers. (a, c, and e) Representative western blots and (b, d, and f) quantification data showing protein expression levels. (a and b) BEAS-2B cells pretreated with control or 50% CO-containing solution followed by H₂O₂ exposure for 48 h. (c–f) BEAS-2B cells were transfected with SIRT3 siRNA or overexpression plasmid for 48 h followed by H₂O₂ exposure for 24 h. β-ACTIN was used as loading control for all experiments. The data were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test, and expressed as the mean ± SEM (n =3). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

Figure 4

SIRT3 regulated mitochondrial superoxide production and airway epithelial cell viability. BEAS-2B cells were transfected with a SIRT3 siRNA or overexpression plasmid for 48 h (a–c), then stained with MitoTracker (green) and MitoSOX (red) to measure mitochondrial superoxide production and (d) CCK-8 assay to measure cell viability. The data were analyzed by Student's t-test and expressed as the mean ± SEM (n =4). ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 5

Characterization of mucociliary phenotype and cell-specific markers of ALI cultures derived from primary HBECs. (a) Hematoxylin & Eosin (H&E) staining and (b) Periodic acid Schiff (PAS) staining demonstrated mucociliary phenotype of the ALI cultures at day 28. These multilayered pseudostratified epithelium models were composed of at least ciliated cells and goblet cells located on the apical side. Immunofluorescence for cell-specific markers of (c) ciliated cells (α-tubulin, green)/goblet cells (MUC5B, red), (d) ciliated cells (α-tubulin, green)/club cells (SCGB1A1, red), and nuclear counterstain (Hoechst, blue). (e) Transepithelial electrical resistance (TEER) and (f) mRNA levels of epithelial cell type-specific markers (TP63, KRT5, MUC5AC, SCGB1A1, FOXJ1) were detected each week after being air-lifted. The data were expressed as the mean ± SEM. Scale bar is equal to 50 μm.

Figure 6

CO attenuated H₂O₂-induced structural impairment and change of cell type-specific markers in ALI cultures. (a) PAS staining showed mucus-producing goblet cells and a vacuolated structure upon H₂O₂ treatment. Immunofluorescence for cell-specific markers of (b) ciliated cells (α-tubulin, green)/goblet cells (MUC5B, red) and (c) ciliated cells (α-tubulin, green)/club cells (SCGB1A1, red) with nuclear counterstain (Hoechst, blue). Scale bar is equal to 100 μm.

Figure 7

CO attenuated H₂O₂-induced increase in SASP and p21 in ALI cultures. (a) mRNA expression of SASP genes was detected by RT-qPCR from ALI cultures exposure to 50% CO-containing solution and H₂O₂ for 12 h. The data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test, and expressed as the mean ± SEM (n =4). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. (b) Immunofluorescence for ciliated cells (α-tubulin, green)/senescence marker (p21, red), with nuclear counterstain (Hoechst, blue) after exposure to in total 4 times of 50% CO-containing solution and H₂O₂. Scale bar is equal to 100 μm.

Figure 8

Schematic summary of the role and underlying mechanisms of CO in oxidative stress-induced senescence in human airway epithelium. The oxidative stress might lead to cellular senescence by causing DNA damage. In turn, the senescent cells could also augment oxidative stress by SASP and form a vicious cycle. The present study verified that H₂O₂-induced airway epithelium senescence, and the effects could be partially reversed by CO. This protective mechanism, at least in part, by inhibiting the mitochondrial ROS production and mediated by the mitochondrial deacetylase SIRT3.