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目的研究吸入重组骨桥蛋白(r-OPN)对高氧急性肺损伤的作用及其机制。方法将96只小鼠按随机数字表法分为PBS处理组和r-OPN处理组,每组中12只小鼠置于室内空气中作为对照,其余小鼠置于浓度大于95%的氧气室中,各组分别于24、48和72 h取出12只小鼠评价肺损伤严重程度;逆转录-聚合酶链反应(RT-PCR)法检测肺组织核因子κB(NF-κB)和基质金属蛋白酶2(MMP-2)、MMP-9及其抑制剂TIMP-1、TIMP-2的mRNA表达;免疫组化染色检测肺组织NF-κB蛋白的表达和组织分布。结果长时间暴露于高氧能够引起急性肺损伤。暴露于高氧环境72 h后,PBS处理组小鼠较r-OPN处理组小鼠发生更严重的肺损伤。RT-PCR结果显示PBS处理组小鼠肺组织NF-κB mRNA在暴露于高氧48 h和72 h后表达量较同时段r-OPN处理组明显增加(0.54±0.03比0.21±0.08,0.57±0.07比0.34±0.01,均P<0.05)。r-OPN处理组小鼠在暴露于高氧72 h后TIMP-1 mRNA表达较PBS处理组明显增加(0.89±0.09比0.62±0.12,P<0.05),在暴露于高氧48 h和72 h后TIMP-2 mRNA表达较PBS处理组明显增加(0.85±0.11比0.59±0.23,P<0.01;0.88±0.16比0.56±0.13,P<0.01)。免疫组化结果显示PBS处理组小鼠在暴露于高氧72 h后气道上皮NF-κB蛋白表达明显高于r-OPN处理组(53.26±4.70比32.53±7.28,P<0.05)。结论 r-OPN可通过抑制NF-κB表达及促进TIMPs的表达来抑制MMPs的释放和激活,从而减轻高氧所致的急性肺损伤。
Objective To study the effect and mechanism of inhaled recombinant osteopontin (r-OPN) on acute lung injury induced by hyperoxia. Methods 96 mice were randomly divided into PBS group and r-OPN group according to random number table. 12 mice in each group were placed in indoor air as control, and the other mice were placed in oxygen chamber with concentration greater than 95% , And 12 mice in each group were sacrificed at 24, 48 and 72 h respectively to evaluate the severity of lung injury. The expressions of nuclear factor κB (NF-κB) and matrix metalloproteinases (MMPs) in lung tissue were detected by reverse transcription-polymerase chain reaction (RT- The expression of MMP-2, MMP-9 and TIMP-1 and TIMP-2 mRNA were detected by immunohistochemistry. The expression and distribution of NF-κB protein in lung tissue were detected by immunohistochemical staining. The results of prolonged exposure to hyperoxia can cause acute lung injury. After exposure to hyperoxia for 72 h, PBS-treated mice developed more severe lung injury than r-OPN-treated mice. The results of RT-PCR showed that the expression of NF-κB mRNA in the lungs of PBS-treated mice increased significantly (P <0.05) after 48 h and 72 h of exposure to hyperoxia (0.54 ± 0.03 vs. 0.21 ± 0.08 and 0.57 ± 0.07 vs 0.34 ± 0.01, all P <0.05). Compared with PBS group, the expression of TIMP-1 mRNA in r-OPN-treated mice exposed to hyperoxia for 72 h increased significantly (0.89 ± 0.09 vs 0.62 ± 0.12, P <0.05) TIMP-2 mRNA expression was significantly increased (P <0.01; 0.88 ± 0.16 vs 0.56 ± 0.13, P <0.01) compared with PBS treatment group (0.85 ± 0.11 vs 0.59 ± 0.23, P <0.01). The results of immunohistochemistry showed that the expression of NF-κB in airway epithelium of PBS treated group was significantly higher than that of r-OPN treated group (53.26 ± 4.70 vs. 32.53 ± 7.28, P <0.05) after 72 h exposure to hyperoxia. Conclusion r-OPN can inhibit the release and activation of MMPs by inhibiting the expression of NF-κB and promoting the expression of TIMPs, so as to reduce the acute lung injury induced by hyperoxia.