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Objective: to establish the model of neonatal rats with hyperoxia-induced lung injury; observed hyperoxia-induced lung injury acute phase of lung tissue P38 mitogen activated protein kinase (P38 mitogen-activated protein kinase, P38 MAPK) phosphorylation matrix metalloproteinase-2 ,9 (matrix metalloproteinases, MMP-2, 9) and its specific tissue inhibitor -1 (tissue inhibitor of metalloproteinases-1, TIMP-1) mRNA expression levels change; explore P38 -MAPK phosphorylation Gao hyperoxic lung injury in the acute phase inflammatory response and MMP-2, MMP-9, TIMP-1 mRNA of the expression. Methods: Animal groups and animal model: 72 3 to 5 days newborn Sprague Dawley (SD) rats were randomly divided into three groups: Ⅰ air group; II hyperoxia saline (hyperoxia group); III high oxygen the SB203580 (intervention group), 24. Then the experimental time points 3,7 talent for two subgroups of 12; each subfractions lavage group and the non-lavage group, six each. Ⅱ, Ⅲ group oxygen tank chamber to keep the oxygen concentration of 90% to 95%, the group Ⅰ placed in the air; daily at the same point in time of group III tail vein injection of SB203580 (p38-MAPK phospho-specific suppression agent, Sigma, USA, 5 mg / kg); group II tail vein injection of normal saline. 2, specimen collection: each group is divided into ① lavage group and (2) non-lavage group two subgroups. Bronchoalveolar lavage with normal saline lavage group at each time point were anesthetized animals, white blood cell count in BALF was measured specimens from bronchoalveolar lavage fluid (bronchoalveolar lavage fluids, BALF); non-lavage group animals were sacrificed at different time points, portion of right lung Country of lung tissue, detecting the phosphorylation of p38 MAPK, MMP-2, mm P-9, TIMP-1 mRNA in the lung homogenates of total protein; remaining right lung tissue is weighed and placed in an oven measuring lung The wet / dry weight ratio. Left lung fixed, embedded, sectioned for routine HE staining, the pathological changes of lung tissue, archive photographs; detection of lung tissue phosphorylation of p38 MAPK (p-p38 MAPK) (Western blot law): modified RIPA buffer fluid routine extraction of the protein in lung tissue, BCA colorimetric method (Bio-Rad protein detection kit) Determination of the concentration of the extracted protein. Each histone Sampling 100μg SDS-PAGE electrophoresis, transferred to a PVDF membrane. Closed blocking buffer at room temperature for 1 h, phospho-P38 MAPK antibody (1:1000, purchased from the United States SANTA CRUZ) overnight at 4 ° C incubation, the secondary antibody (1:2000) was incubated for 1 h, ECL significant color. With Q550CW computer image analysis system analysis and processing of protein bands, the protein content of the target band correction volume (Adj volume) / the GAPDH corrected volume (Adj volume). 4, lung tissue MMP-2, MM P-9, TIMP-1 mRNA detection (RT-PCR method): (1) by Trizol total RNA extraction kit (Invitrogen, USA)'s instructions. Total RNA was extracted from lung tissue RNA extraction solution (Trizol Reagent). (2) reverse transcriptase to synthesize the complementary DNA (cDNA) chain. (3) PCR amplification (primers and amplification conditions see Experimental Procedure). (4) of the product analysis: the amplified product was purified by 2% agarose gel electrophoresis and ethidium bromide staining, and computer image analysis system Q550CW analysis of electrophoretic bands. MRNA levels of the target product to the target band correction volume (ADJ volume) / the GAPDH corrected volume (Adj volume). Results: 1. General condition: Air group were the spirit of good status, activities, flexible, stable weight gain during the experiment; hyperoxia rats 3 days after the spirit of the poor response to 7 days after the weight gain is not obvious, shortness of breath, slow activities; SB203580 intervention rats state of mind is a bit weak, slightly reduced activity, weight gain is not obvious, a little shortness of breath. Lung tissue biopsy: hyperoxia group at 3 days alveolar significant bleeding and inflammatory cell exudation, 7 day injuries were aggravated lung interval widened, structural disorder of the lung tissue. The SB203580 intervention group and the hyperoxia group inflammation and reduced lung interval widened obvious. Lung wet / dry weight ratio (W / D): the Experiment 3 days hyperoxia group higher than the air group (5.5104 ± 0.5001 vs 4.2706 ± 0.3884, p lt; 0.01); the experiment seven days, further increase in the ratio ( 5.7671 ± 0.3599 vs 4.3977 ± 0.2313, p lt; 0.01). Of SB203580 intervention group and hyperoxia group compared to the same point in time the ratio was significantly reduced to (Experiment 3 days, 4.7411 ± 0.3112 vs 5.5104 ± 0.5001, p lt; 0.05; Experiment 7 days, 4.6761 ± 0.2504 vs 5.7671 ± 0.3599, p lt; 0.01 ). Lung homogenates of total protein (Total Protein, TP) content detection: Experiment 3 days, hyperoxia group TP content higher than the air group (1.0463 ± 0.2113 vs 0.7323 ± 0.0297, p lt; 0.05); 7 days, high TP content of oxygen groups continued to increased (1.2226 ± .1751 vs. 0.9416 ± 0.1365, p lt; 0.05); SB203580 intervention group with the high-oxygen group comparison, Experiment 3 days TP content no significant difference (1.0262 ± 0.1281 vs 1.0463 ± 0.2113, p gt; 0.05) at day 7 was significantly decreased (0.9297 ± 0.0642 vs 1.2226 ± 0.1751, p lt; 0.05) BALF in white blood cell count: Experiment 3 days, the white blood cell counts in BALF in hyperoxia group was significantly higher than the air control group, the difference was statistically significant (42.32 ± 4.78 vs 11.73 ± 3.86, p lt; 0.01); Experiment 7 days, white blood cells the count further increase (126.48 ± 18.88 vs 18.75 ± 3.74, P lt; 0.01). SB203580 intervention group with the high-oxygen group, at the same time points leukocyte count was significantly decreased (experiment 3 days, 21.35 ± 3.21 vs 42.32 ± 4.78, p lt; 0.01; experiment 7 days, 33.75 ± 8.63 vs 126.48 ± 18.88, p lt; 0.01 ). Lung tissue phosphorylation of P38 MAPK (p-P38 MAPK) expression: the air lung tissue p-p38 MAPK only a weak expression. Experiment 3 days, the high-oxygen group, p-p38 MAPK expression was significantly enhanced compared with the corresponding time points Air group, the difference was statistically significant (1.506 ± 0.160 vs 0.627 ± 0.024, p lt; 0.01); Experiment 7 days, p-p38 MAPK was further enhanced (1.998 ± 0.176 vs 0.664 ± 0.034, p lt; 0.01). SB203580 intervention group and the hyperoxia group compared to the same point in time p-p38 MAPK expression significantly decreased, the difference was statistically significant (Experiment 3 days, 0.936 ± 0.051 vs 1.506 ± 0.160, p lt; 0.01; Experiment 7 days, 1.319 ± 0.082 vs 1.998 ± 0.176, p lt; 0.01). 7 lung tissue MMP-2, MMP -9, TIMP-1 mRNA of the expression: air group, MMP-2, MMP-9, TIMP-1 mRNA of the expression level is low. The hyperoxia group MMP-2 (Experiment 3 days, 3.428 ± 0.316 vs1.681 ± 0.092, increased 103%, p lt; 0.01; Experiment 7 days, 1.149 ± 0.129 vs 0.490 ± 0.034, increased 134%, p lt; 0.01), MMP-9 (Experiment 3 days, 0.383 ± 0.011 vs 0.133 ± 0.006, increased 190%, P lt; 0.01; Experiment 7 days 0.329 ± 0.008 vs 0.142 ± 0.008, increased 132%, P lt; 0.01) TIMP-1 mRNA (Experiment 3 days, 0.294 ± 0.015 vs 0.112 ± 0.010, increased 54%, p lt; 0.05; Experiment 7 days, 0.449 ± 0.009 vs 0.155 ± 0.006, increased 40%, p lt; 0.05) expression was significantly increased, compared to the same point in time air group, the difference was statistically significant, and MMP-2, MMP-9 mRNA increased in magnitude compared with the TIMP-1 mRNA increased rate; the SB203580 intervention group the Gao oxygen group, the same point-in-time MMP-2 (Experiment 3 days, 2.104, ± 0.094vs3.428 ± 0.316, reduced 39%, p lt; 0.01; experiments seven days 0.620 ± 0.027vs 1.149 ± 0.129, reduced 46%, p lt; 0.01), MMP 9 (Experiment 3 days, of 0.208 ± 0.012vs 0.383 ± 0.011, reduced 46%, p lt; 0.01; the experimental 7 days, 0.198 ± 0.0112 vs 0.329 ± 0.008, reduced by 57%, p lt; 0.01), TIMP-1 ( Experiment 3, 0.197 ± 0.013 vs 0.294 ± 0.015, reduced 18%, p lt; 0.05; seven days of experiment, 0.273 ± 0.018 vs 0.449 ± 0.009, reduced by 14%, p lt; 0.05) mRNA expression was decreased, the difference was statistically meaning, and MMP-2, MMP-9 mRNA decreased compared with the TIMP-1 mRNA reduction. Conclusion: The long-term high concentrations of oxygen can lead to neonatal rats with acute lung injury and pathological changes of lung tissue local congestion oozing and bleeding, inflammation, rupture of pulmonary interval, alveolar cavity to expand and disorders of the lung structure Organization; 2 p -P38 MAPK in neonatal rats with acute hyperoxic lung injury in lung tissue expression levels were significantly increased, and further increased with the severity of lung injury, suggesting that activation of the p38 MAPK signal transduction pathway may be involved in hyperoxia-induced lung injury pathogenesis of regulation; 3. hyperoxia-induced lung injury in lung tissue MMP-2, MMP-9, TIMP-1 mRNA of expression were significantly increased, and MMP-2, MMP-9/TIMP-1 in balance is changed, show that over-expression of matrix metalloproteinases (MMPs) and the imbalance between the expression of specific tissue inhibitor of matrix metalloproteinases (TIMPs) in the development and prognosis of hyperoxia-induced lung injury play an important role; P38 MAPK-specific phosphorylation inhibitor SB203580 by inhibiting P38 MAPK phosphorylation, reduce hyperoxic lung injury in the acute phase of the inflammatory response, and the impact of MMP-2, MMP-9, TIMP-1 mRNA of expression, suggesting that p38 MAPK signal transduction pathway activation may be involved in hyperoxia-induced lung injury in the acute phase inflammatory response and MMP-2, MMP-9, TIMP-1 mRNA of the expression regulation provides a new way for the prevention and treatment of neonatal the Gao oxygen lung injury.
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