Objective To observe the changes of thymus gland differentiation antigen-1(Thy-1) expression and its DNA methylation in human embryonic lung fibroblasts(MRC-5) fibrosis induced by coal dust through human monocytic cell derived macrophages.
Methods We stimulated human monocytes differentiation to macrophages by using 500 nmol/L phorbol 12-myristate 13-acetate(PMA) in 1640 medium for 24 h; collected supernatant of 100 μg/mL coal dust stimulated macrophages after 24 h to stimulate human embryonic lung fibroblasts for 24, 48, and 72 h. The control groups were normal macrophages and normal MRC-5. The protein levels of IL-6 and TGF-β1 of macrophages and TGF-β1 of MRC-5 in the supernatant were determined by enzyme-linked immunoassay; collagen-Ⅰ, collagen-Ⅲ, α-SMA, Thy-1, and DNA methylation transferase(DNMTs)(1, 3a, 3b) mRNA expression levels were detected by real-time fluorescent quantitative PCR; DNA methylation of Thy-1 was detected by nested touchdown specific PCR.
Results The protein expression levels of IL-6 and TGF-β1 in macrophage supernatant of the coal dust groups were significantly increased compared with the control group (5.542±0.953)ng/mL vs(2.498±0.456)ng/mL, (462.435±56.620) ng/L vs (329.971±17.911) ng/L, respectively(P < 0.05). After MRC-5 were stimulated with coal dust treated macrophages for 24, 48, and 72h, the TGF-β1 protein expression levels (223.813±5.723), (263.757±17.254), (326.720±11.263) ng/L as well as collagen-Ⅰ(3.38±0.37, 3.87±0.28, 4.40±0.10), collagen-Ⅲ(1.65±0.12, 2.37±0.19, 2.66±0.28), and α-SMA(2.41± 0.47, 4.76±0.10, 4.23±0.63) mRNA expression levels were significantly higher than those of the control group, and in a timeeffect relationship with r=0.965, 0.876, 0.899, and 0.667, respectively(P < 0.05). The mRNA expression levels of Thy-1 in MRC-5 of the coal dust groups were all lower than those of the control group, and there was a negative correlation(r=-0.945) with the increasing stimulating time of supernatant fluid(0.634±0.014, 0.448±0.055, 0.352±0.044)(P < 0.05). The mRNA expression levels of DNMTs were significantly higher(DNMT1: 1.57±0.12, 2.51±0.33, 7.00±0.71; DNMT3a: 0.74±0.09, 2.89±0.52, 3.31±0.53; DNMT3b: 1.46±0.18, 2.25±0.44, 5.33±0.16), demonstrating a time-effect relationship with r=0.924, 0.890, and 0.937(P < 0.05). Thy-1 DNA methylation levels were increased in the coal dust groups(0.506±0.014, 0.536±0.017, 0.570±0.032), and also with a time-effect relationship(r=0.682, P < 0.05).
Conclusion Human embryonic lung fibroblasts fibrosis is caused by human monocyte-derived macrophages exposed to coal dust. Thy-1 DNA methylation and decreased Thy-1 mRNA expression are identified in this process, which might be one of the important mechanism of epigenetics in lung fibrosis induced by coal dust.
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