5-Aza-2′-deoxycytidine protects against emphysema in mice via suppressing p16^Ink4a expression in lung tissue

Figures & data

Table 1 Experiment schedule

Day	0	11	15	17	19	22	28
Group
Control	PBS	PBS	PBS	PBS	PBS	PBS	Disposed
CSE	CSE	CSE	PBS	PBS	PBS	CSE	Disposed
CSE + 5-Aza-CdR	CSE	CSE	Aza	Aza	Aza	CSE	Disposed

Abbreviations: CSE, cigarette-smoke extract; 5-Aza-CdR, 5-Aza-2′-deoxycytidine.

Figure 1 Morphological changes in endothelial progenitor cells (EPCs) sourced from bone marrow of C57BL/6J mice during culture.

Notes: (A) Representative microscopy of EPCs cultured with endothelial growth medium 2 in the presence of 5% fetal bovine serum on day 1. EPCs formed were spherical, cell sizes were almost the same, and cells were suspended in the culture medium. (B) On day 4 of the culture, the cells were attached to one other, getting larger, and became oval, spindly, or polygonal. (C) On day 7 of the culture, the cells became fusiform or polygonal in pattern. EPCs contacted one another to attempt to form capillary structures (arrows). Magnification ×100.

Figure 2 Double-positive cells stained with Dil-acLDL and FITC-UEA1 were identified as endothelial progenitor cells.

Notes: Laser-scanning confocal microscopy illustrated that cells on day 7 of the culture displayed red cytoplasm when stained with DiI-acLDL (A), green cytomembrane when combined with FITC-UEA1 (B), orange when double-stained with Dil-acLDL and FITC-UEA1 (C). Magnification ×400.
Abbreviations: Dil-acLDL, DiI-labeled acetylated low-density lipoprotein; FITC-UEA1, fluorescein isothiocyanate-labeled Ulex europaeus agglutinin 1.

Figure 3 Maximal expiratory flow-volume curves.

Notes: In each image, the peaks (upper) represent the expiratory phase and the troughs (lower) the inspiratory phase. Compared with controls (A), the CSE group (B) and CSE + 5-Aza-CdR group (C) showed abrupt ascents, and descending limbs showed a prolonged expiratory phase.

Abbreviations: CSE, cigarette-smoke extract; 5-Aza-CdR, 5-Aza-2′-deoxycytidine; sec, second; div, division.

Figure 4 Lung function.

Notes: C_dyn (mL/cm H₂O) was significantly lower in the CSE group and CSE + 5-Aza-CdR group than controls (P<0.01/P<0.05). R_aw (cm H₂O/mL/min) was significantly higher in the CSE group and CSE + 5-Aza-CdR group than the controls (P<0.01). PEF (mL/second) was significantly lower in the CSE group and CSE + 5-Aza-Cd R group than the controls (P<0.01/P<0.05). T_i/T_e was significantly lower in the CSE group than the controls (P<0.05). There was no statistical difference in T_i/T_e between the CSE + 5-Aza-CdR group and controls (P>0.05). ^☆P<0.05 compared with controls; ^★P<0.01 compared with controls.
Abbreviations: C_dyn, lung dynamic compliance; CSE, cigarette-smoke extract; 5-Aza-CdR, 5-Aza-2′-deoxycytidine; R_aw, airway resistance; PEF, peak expiratory flow; sec, second; T_i, inspiratory time; T_e, expiratory time.

Figure 5 Histomorphological changes in lung tissue.

Notes: Lung tissue in the CSE group (B) exhibited enlarged alveolar space, thinner alveolar septum, and destroyed alveolar wall when compared with controls (A). Lung tissue in the CSE + 5-Aza-CdR group (C) also exhibited enlarged alveolar space, but smaller than that of the CSE group. Magnification ×100.

Abbreviations: CSE, cigarette-smoke extract; 5-Aza-CdR, 5-Aza-2′-deoxycytidine.

Figure 6 Histomorphological changes in lung tissue.

Notes: The mean linear intercept (MLI) was significantly increased in the CSE group compared to controls (P<0.01). Interestingly, the MLI was significantly lower in the CSE + 5-Aza-CdR group than the CSE group (P<0.01), although significantly higher than controls (P<0.01). The destructive index (DI) was significantly increased in the CSE group compared with controls (P<0.01). Interestingly, the DI was significantly lower in the CSE + 5-Aza-CdR group than the CSE group (P<0.05), although significantly higher than controls (P<0.01). The apoptotic index (AI) was significantly increased in the CSE group compared with the controls (P<0.01). Interestingly, the AI was significantly lower in the CSE + 5-Aza-CdR group than the CSE group (P<0.05), although significantly higher than controls (P<0.01). ^★P<0.01 compared with controls; ^△P<0.05 compared with CSE group; ^▲P<0.01 compared with CSE group.
Abbreviations: CSE, cigarette-smoke extract; 5-Aza-CdR, 5-Aza-2′-deoxycytidine.

Figure 7 Apoptosis of lung tissue.

Notes: Numbers of apoptotic cells (arrows) in the alveolar septa in the CSE group (B) and CSE + 5-Aza-CdR group (C) were increased compared to controls (A). The number of apoptotic cells in the CSE + 5-Aza-CdR group (C) was lower than that of the CSE group. Magnification ×400.
Abbreviations: CSE, cigarette-smoke extract; 5-Aza-CdR, 5-Aza-2′-deoxycytidine.

Figure 8 Expression of p16^Ink4a protein in lung tissue (A) and EPCs (B). (C) p16^Ink4a protein-expression comparison.

Notes: p16^Ink4a/β-actin in lung tissue was significantly increased in the CSE group and CSE + 5-Aza-CdR group compared with controls (P<0.01). Interestingly, p16^Ink4a/β-actin in lung tissue was significantly lower in the CSE + 5-Aza-CdR group than the CSE group (P<0.01). p16^Ink4a/β-actin in EPCs was significantly increased in the CSE group and CSE + 5-Aza-CdR group compared with controls (P<0.01 or P<0.05). p16^Ink4a/β-actin in EPCs was significantly lower in the CSE + 5-Aza-CdR group than the CSE group (P<0.05). ^☆P<0.05 compared with controls; ^★P<0.01 compared with controls; ^△P<0.05 compared with CSE group; ^▲P<0.01 compared with CSE group.
Abbreviations: EPCs, endothelial progenitor cells; CSE, cigarette-smoke extract; 5-Aza-CdR, 5-Aza-2′-deoxycytidine; C + A, CSE + 5-Aza-CdR; N, normal (controls).

Figure 9 Expression of p16^Ink4a mRNA.

Notes: p16^Ink4a mRNA in lung tissue was significantly increased in the CSE group and CSE + 5-Aza-CdR group compared with controls (P<0.01). Interestingly, p16^Ink4a mRNA in lung tissue was significantly lower in the CSE + 5-Aza-CdR group than the CSE group (P<0.05). p16^Ink4a mRNA in EPCs was significantly increased in the CSE group and CSE + 5-Aza-CdR group compared with controls (P<0.01/P<0.05). p16^Ink4a mRNA in EPCs was significantly lower in the CSE + 5-Aza-CdR group than the CSE group (P<0.01). ^☆P<0.05 compared with controls; ^★P<0.01 compared with controls; ^△P<0.05 compared with CSE group; ▲P<0.01 compared with CSE group.
Abbreviations: CSE, cigarette-smoke extract; 5-Aza-CdR, 5-Aza-2′-deoxycytidine; EPCs, endothelial progenitor cells.