Hydrogen coadministration slows the development of COPD-like lung disease in a cigarette smoke-induced rat model

Figures & data

Table 1 Primers of reverse transcription PCR analysis for genes

Primer	Primer sequence (5′–3′)	Product length (bp)
GADPH-F	TGAACGGGAAGCTCACTG	119
GADPH-R	GCTTCACCACCTTCTTGATG
TNF-α-F	CCTCTTCTCATTCCTGCTC	124
TNF-α-R	CTTCTCCTCCTTGTTGGG
IL-6-F	ATCTGCTCTGGTCTTCTGG	78
IL-6-R	TCTGGCTTTGTCTTTCTTGT
IL-17-F	TCATCCCTCAAAGCTCAGCG	167
IL-17-R	TTCATTGCGGTGGAGAGTCC
IL-23-F	AAAGGAGGTTGATAGAGGGT	241
IL-23-R	TCTTAGTAGATCCATTTGTCCC
MMP-12-F	GTGCCCCAGAGGTCAAGATG	245
MMP-12-R	GCATGGGCTAGTGTACCACC
TIMP-1-F	CCTCTGGCATCCTCTTGTTG	161
TIMP-1-R	ATAACGCTGGTATAAGGTGGTC
Caspase-3-F	TGGCATTGAGACAGACAG	150
Caspase-3-R	CAAAGGGACTGGATGAAC
Caspase-8-F	CTGACTGGAGTGAACTATG	267
Caspase-8-R	GTGTGAAGATGGGCTGTG

Figure 1 Effect of hydrogen on body weight and lung function in a cigarette smoke-induced rat model.

Notes: (A) Rats’ body weights before and after smoking. (B) Lung function in rats. (n=10 for each group; ^##P<0.01 compared with COPD group; *P<0.05, **P<0.01).
Abbreviations: COPD, chronic obstructive pulmonary disease; FEV0.2, forced expiratory volume in 0.2 second; FEV0.2/FVC, forced expiratory volume in 0.2 second/forced vital capacity; FVC, forced vital capacity.

Figure 2 Impact of hydrogen on inflammatory cells in rats with COPD-like lung disease.

Notes: The inflammatory cells in BALF were measured to compare the effect of hydrogen in rats with COPD-like lung disease. (n=10 for each group; ^#P<0.05, ^##P<0.01 compared with COPD group; **P<0.01).
Abbreviations: BALF, bronchoalveolar lavage fluid; COPD, chronic obstructive pulmonary disease.

Figure 3 Effect of hydrogen on the pathologic lung changes in rats with COPD-like lung disease.

Notes: (A) Rat lung pathologic observations (HE ×400). (B) MLI, MAN, and BWT/D were measured to compare the effect of hydrogen in rats with COPD-like lung disease. (n=10 for each group. ^##P<0.01 compared with the COPD group, *P<0.05, **P<0.01).
Abbreviations: BWT/D, bronchial wall thickness/diameter; COPD, chronic obstructive pulmonary disease; HE, hematoxylin and eosin; MAN, mean alveolar number; MLI, mean linear intercept.

Figure 4 Effect of hydrogen on changes in the ultrastructure of COPD-like lung disease rats.

Notes: (A) Lung tissue observation of five groups under SEM. (B) Lung tissue observation of five groups under TEM. (C) Cardiac tissue observation of five groups under TEM.
Abbreviations: COPD, chronic obstructive pulmonary disease; SEM, scanning electron microscope; TEM, transmission electron microscope.

Figure 5 Changes in cardiovascular indices and RVHI after hydrogen coadministration to COPD-like lung disease rats.

Notes: Comparisons of mPAP, LVEF, RVAWd, LVAWd, LVPWd, and RVHI. (n=10 for each group; ^##P<0.01 compared with COPD group, *P<0.05, **P<0.01).
Abbreviations: COPD, chronic obstructive pulmonary disease; LVAWd, end-diastolic left ventricular anterior wall thickness; LVEF, left ventricular ejection fraction; LVPWd, end-diastolic left ventricular posterior wall thickness; mPAP, mean pulmonary arterial pressure; RVAWd, end-diastolic right ventricular anterior wall thickness; RVHI, right ventricular hypertrophy index.

Figure 6 Effect of hydrogen on the expression of TNF-α, IL-6, IL-17, and IL-23 in the lung obtained from COPD-like lung disease rats, found by immunohistochemical staining.

Notes: (A) Immunohistochemical staining of the lung sections for TNF-α, IL-6, IL-17, and IL-23. (B) Quantitative analysis of the expression of TNF-α, IL-6, IL-17, and IL-23 in the lung of COPD-like lung disease rats. (n=10 for each group. ^#P<0.05; ^##P<0.01 compared with COPD group. *P<0.05; **P<0.01). Each experiment was repeated three times and similar results were obtained.

Abbreviations: COPD, chronic obstructive pulmonary disease; IL, interleukin; MOD, mean optical density; TNF-α, tumor necrosis factor alpha.

Figure 7 Effect of hydrogen on the expression of MMP-12, TIMP-1, caspase-3, and caspase-8 in the lung obtained from COPD-like lung disease rats, found by immunohistochemical staining.

Notes: (A) Immunohistochemical staining of the lung sections for MMP-12, TIMP-1, caspase-3, and caspase-8. (B) Quantitative analysis of the expression of MMP-12, TIMP-1, caspase-3, and caspase-8 in the lung of COPD-like lung disease rats. (n=10 for each group; #P<0.05, ##P<0.01 compared with COPD group; *P<0.05, **P<0.01). Each experiment was repeated three times and similar results were obtained.

Abbreviations: COPD, chronic obstructive pulmonary disease; MMP, matrix metalloproteinase; MOD, mean optical density; TIMP, tissue inhibitor of metalloproteinase.

Figure 8 Effect of hydrogen on the expression of TNF-α, IL-6, IL-17, IL-23, MMP-12, TIMP-1, caspase-3, and caspase-8 in the lung obtained from COPD-like lung disease rats, determined by Western blotting.

Notes: (A) Western blotting of the lung sections for TNF-α, IL-6, IL-17, IL-23, MMP-12, TIMP-1, caspase-3, and caspase-8. (B) The target protein bands were densitometrically analyzed normalized to β-actin. (^#P<0.05, ^##P<0.01 compared with COPD group; *P<0.05, **P<0.01). Each experiment was repeated three times and similar results were obtained.
Abbreviations: COPD, chronic obstructive pulmonary disease; IL, interleukin; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; TNF-α, tumor necrosis factor alpha.

Figure 9 Effect of hydrogen on the mRNA expression of TNF-α, IL-6, IL-17, IL-23, MMP-12, TIMP-1, caspase-3, and caspase-8 in the lung obtained from COPD-like lung disease rats, analyzed by RT-PCR.

Notes: The mRNA levels of TNF-α, IL-6, IL-17, IL-23, MMP-12, TIMP-1, caspase-3, and caspase-8 were analyzed with RT-PCR. (n=10 for each group; ^#P<0.05, ^##P<0.01 compared with COPD group; *P<0.05, **P<0.01).
Abbreviations: COPD, chronic obstructive pulmonary disease; IL, interleukin; MMP, matrix metalloproteinase; mRNA, messenger RNA; RT-PCR, real-time polymerase chain reaction; TIMP, tissue inhibitor of metalloproteinase; TNF-α, tumor necrosis factor alpha.