Changes in the number of CD31⁻CD45⁻Sca-1⁺ cells and Shh signaling pathway involvement in the lungs of mice with emphysema and relevant effects of acute adenovirus infection

Figures & data

Figure 1 RFP expression in frozen lung sections under fluorescence microscope.

Notes: (A) PBS group; (B) CSE group; (C) CSE + adenovirus group.
Abbreviations: RFP, red fluorescence protein; PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Table 1 Lung function $\left(\overline{x}\pm s\right)$

Group	F (bpm)	TV (mL)	Cdyn (mL/cmH2O)	RI (cmH2O·mL^−1·min^−1)
PBS (n=10)	183±23	0.24±0.40	0.074±0.030	0.24±0.14
CSE (n=9)	183±20	0.26±0.31	0.054±0.019*	0.48±0.28*
CSE + adenovirus (n=8)	190±16	0.23±0.33	0.052±0.014*	0.49±0.24*

Notes: Mouse lung function was measured on 28th day after CSE treatment started. F, respiratory frequency; Cdyn, lung dynamic compliance; RI, airway resistance;

* P<0.05 vs PBS.

Abbreviations: TV, tidal volume; CSE, cigarette smoke extract; PBS, phosphate-buffered saline.

Table 2 MLI and DI $\left(\overline{x}\pm s\right)$

Group	MLI (μm)	DI (%)
PBS (n=10)	23.8±0.8	10.7±1.3
CSE (n=9)	42.0±4.2*	38.2±2.1
CSE + adenovirus (n=8)	38.1±2.1*	39.1±2.6*

Note:

* P<0.05 vs PBS.

Abbreviations: MLI, mean linear intercept; DI, destructive index; PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Figure 2 Histological examination of lungs in experimental groups (HE staining).

Notes: (A) PBS group; (B) CSE group; (C) CSE + adenovirus group. Magnification ×100.

Abbreviations: HE, hematoxylin and eosin; PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Figure 3 (A) Peribronchial and (B) perivascular inflammation in lung tissues (HE staining).

Notes: (a) PBS group; (b) CSE group; (c) CSE + adenovirus group. Magnification ×200.

Abbreviations: HE, hematoxylin and eosin; PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Figure 4 Semi-quantitative inflammation scores in (A) peribronchial and (B) perivascular sections in lung tissues from experimental groups.

Notes: PBS group, n=10; CSE group, n=9; CSE + adenovirus group, n=8; *P<0.05 vs PBS; ^#P<0.05 vs CSE.
Abbreviations: PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Figure 5 Selected bivariate scatter plots of lung cell suspension in experimental groups by flow cytometry.

Notes: Anti-CD31 and anti-CD45 antibodies were labeled with fluorescent APC and anti-Sca-1 antibody with fluorescent FITC. The percentage of CD31⁻CD45⁻Sca-1⁺ population in single-cell suspension from the whole lung is indicated by the polygon drawn by the black line. (A) Blank control without fluorescent antibody; (B) PBS group; (C) CSE group; (D) CSE + adenovirus group. Autofluorescence of lung tissues was also confirmed.

Abbreviations: FITC, fluorescein isothiocyanate; FITC-A, antibody with fluorescein isothiocyanate; PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Figure 6 Number of lung CD31⁻CD45⁻Sca-1⁺ cells in experimental groups.

Notes: n=8; *P<0.05 vs PBS; **P<0.05 vs CSE.
Abbreviations: PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Figure 7 mRNA levels of the major components of the Shh signaling pathway in experimental groups: PBS group (n=10), CSE group (n=9), and CSE + adenovirus group (n=8).

Notes: The Shh, Ptch1, and Gli1 mRNA levels were analyzed using real-time RT-PCR. Data are expressed as copy number mean ± SD of the corresponding genes normalized to that of β-actin; **P<0.05 vs CSE.

Abbreviations: mRNA, messenger RNA; PBS, phosphate-buffered saline; CSE, cigarette smoke extract; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation.

Figure 8 Protein levels of the major components in the Shh signaling pathway in experimental groups: (a) PBS group (n=10); (b) CSE group (n=9); (c) CSE + adenovirus group (n=8).

Notes: (A) The Shh, Ptch1, and Gli1 protein levels in lung tissues were analyzed by Western blotting. β-Actin was used as a loading control. (B) Densities of Shh, Ptch1, and Gli1 were normalized against that of β-actin to obtain a fold-change to represent the relative expression of the corresponding molecules. *P<0.05 vs PBS; **P<0.05 vs CSE.
Abbreviations: PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Figure 9 Immunohistochemical detection of Shh signaling pathway molecules.

Notes: (A) Negative controls in which the primary antibody was replaced with PBS; (B) PBS group; (C) CSE group; (D) CSE + adenovirus group. All sections were counterstained with hematoxylin. Magnification ×200.

Abbreviations: PBS, phosphate-buffered saline; CSE, cigarette smoke extract.

Figure 10 Semi-quantitative measurement of Shh, Ptch1, and Gli1 expression in experimental groups by immunohistochemistry.

Notes: PBS group, n=10; CSE group, n=9; CSE + adenovirus group, n=8; *P<0.05 vs PBS; **P<0.05 vs CSE.
Abbreviations: PBS, phosphate-buffered saline; CSE, cigarette smoke extract.