[The effect of melatonin on the regulation of collagen accumulation and matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 mRNA and protein in a murine model of chronic asthma].
OBJECTIVE
To investigate the effect of melatonin on the regulation of collagen accumulation and the underlying mechanisms.


METHODS
Ninety-six BALB/c mice were randomly divided into five groups. The mice were injected i.p. with normal saline in the control group (n = 21) and the asthma group (n = 22), melatonin in the melatonin group (n = 23), dexamethasone in the dexamethasone group (n = 24), and melatonin antagonist luzindole in the luzindole group (n = 7) respectively. The mice of the control group, asthma group, melatonin group, dexamethasone group were sensitized and repeatedly challenged with ovalbumin for 2, 4, 8 weeks(the number of control group were 7, 7, 7 respectively at 2, 4, 8 weeks; the number of asthma group were 7, 7, 8 respectively at 2, 4, 8 weeks; the number of melatonin group were 7, 8, 8 respectively at 2, 4, 8 weeks; the number of dexamethasone group were 7, 8, 8 respectively at 2, 4, 8 weeks). The mice of the luzindole group (n = 7) were sensitized and repeatedly challenged with ovalbumin for 2 weeks, to establish the murine model of chronic asthma. Masson staining was used to stain collagen fibers and immunohistochemistry to stain protein expression. MetaMorph image analysis software was used to measure the positive area per micrometer perimeter of basement membrane (Wcol/Pbm), to evaluate collagen accumulation in lung tissue. The immuno-positive area per unit area (PA/UA) was measured to evaluate the protein level of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA levels.


RESULTS
The collagen area per unit perimeter of basement membrane (Wcol/Pbm) in the melatonin group was (11.8 +/- 1.3), (12.3 +/- 1.1), (12.7 +/- 1.4) microm(2)/microm respectively at 2, 4, and 8 weeks, which were decreased as compared with (14.5 +/- 1.5), (15.8 +/- 1.8), (16.2 +/- 1.4) microm(2)/microm in the asthma group (t(d) = 3.89, 5.96, 5.50 respectively, all P < 0.01). The PA/UA of MMP-9 was (9.7 +/- 4.9) pixel, (14.8 +/- 4.9) pixel, (11.0 +/- 6.8) pixel respectively at 2, 4, 8 weeks in the melatonin group, (15.65 +/- 6.11) pixel, (26.2 +/- 6.9) pixel, (24.6 +/- 6.0) pixel respectively at 2, 4, 8 weeks in the asthma group, the difference being statistically significant between the two groups (t(d) = 3.00, 4.83, 5.50 respectively at 2, 4, 8 weeks, all P < 0.01). The MMP-9 mRNA level was 0.80 +/- 0.40, 0.68 +/- 0.15, 0.67 +/- 0.24 in the melatonin group at 2, 4, 8 weeks respectively, and was 1.48 +/- 0.29, 1.40 +/- 0.50, 1.20 +/- 0.40 in the asthma group at 2, 4, 8 weeks respectively (t(d) = 3.92, 4.50, 3.29 respectively at 2, 4, 8 weeks, all P < 0.01). The Wcol/Pbm [(11.6 +/- 1.3), (12.3 +/- 1.0), (13.0 +/- 1.7) microm(2)/microm], protein [(12.5 +/- 5.6), (14.0 +/- 4.7), (13.6 +/- 4.8) pixel] and mRNA (0.69 +/- 0.11, 0.61 +/- 0.16, 1.10 +/- 0.40) level were reduced in the dexamethasone group as compared with the asthma group at 2, 4, 8 weeks respectively. The above mentioned parameters in the luzindole group were not statistically different as compared with the asthma group at 2 weeks.


CONCLUSIONS
The results suggest that early treatment with melatonin inhibits airway collagen accumulation, probably mediated by the inhibition of MMP-9.
Zhou
Li
L
Department of Respiratory Research institute. First Affiliated Hospital, Anhui Medical University, Hefei 230032, China.
Qian
Zhao-xia
ZX
Li
Feng
F
Liu
Rong-yu
RY
D000818:Animals; D001249:Asthma / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004195:Disease Models, Animal; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D008550:Melatonin / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*; D014363:Tryptamines / Q000494:pharmacology
D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D014363:Tryptamines; C057154:luzindole; D003907:Dexamethasone; D003094:Collagen; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse; D008550:Melatonin