高氧致慢性肺疾病新生大鼠肺组织尿激酶型纤溶酶原激活因子和纤溶酶原激活物抑制因子1表达的动态改变及意义

Changes of u-PA and PAI-1 expression in the lung tissue of neonatal rats after inhaling high concentration oxygen

目的慢性肺疾病(Chronic lung disease,CLD)的特征性病理改变是肺发育受阻和肺间质纤维化,因此胶原等细胞外基质(extracellular matrix,ECM)重塑在肺纤维化形成中的作用已成为当今的研究热点。纤溶系统也是调节 ECM 降解和基质金属蛋白酶(MMPs)活性的重要因素。这里我们制备了吸入高浓度氧(高氧)致新生大鼠 CLD 的模型,探讨尿激酶型纤溶酶原激活因子(urokinase-plasminogen activator,u-PA)和纤溶酶原激活物抑制因子(plasminogen activator inhibitor-1,PAI-1)在新生大鼠 CLD 中的动态变化和意义。方法足月新生大鼠生后12h 内,分别于0.90~0.95氧和空气中持续暴露,于1、3、7,14、21 d 光镜下观察不同时间点的病理改变,应用 Western Blotting 和RT-PCR 的方法分别检测肺组织 u-PA、PAI-1蛋白及 mRNA 表达的动态改变。结果(1)病理改变:3 d 时高氧组可见炎性反应;7 d 时肺发育障碍,间隔增宽,胶原纤维沉积。(2)u-PA 表达:高氧暴露3d 时,蛋白表达较对照组明显升高(115.52±7.10 vs 96.51±6.33),P<0.01;高氧组在7~21 d(97.66±7.98,99.91±7.60,103.23±6.24)与对照组比较(112.43±6.01,123.25±8.35,103.23±6.24)降低,P 值分别为<0.05,<0.01,<0.01。mRNA 表达在高氧暴露3 d 时较对照组明显升高(1.18±0.07 vs 0.99±0.05),P<0.01,7 d 至21 d(1.01±0.06,1.10±0.12,1.27±0.06)与对照组比较(1.15±0.08,1.51±0.32,1.60±0.24)降低,P 均<0.01。(3)PAI-1表达:高氧暴露后7~21 d,蛋白表达(147.83±12.27,149.07±11.17,161.42±13.08)明显高于同期对照组(116.18±10.67,113.73±15.58,126.60±8.59),P 值分别<0.01,<0.05,<0.01;mRNA 表达在7-21 d(1.49±0.28,1.46±0.31,1.51±0.33)也高于同期对照组(0.94±0.01,0.94±0.03,0.98±0.03),P<0.05,<0.01和<0.05。结论新生大鼠高氧暴露早期,肺组织 u-PA/PAI一1mRNA 及蛋白表达增强,纤溶和降解活性增强;高氧暴露中晚期,u-PA/PAI-1mRNA 及蛋白表达降低,纤溶和降解活性降低,与肺纤维沉积平行,提示 u-PA/PAI-1失衡参与了高氧致 CLD 的纤维化形成过程。

Objective Arrested lung development and lung fibrosis are characteristic pathological changes in chronic lung disease(CID).Therefore,the rote of extracellular matrix(ECM)remodeling in lung fibrosis has been emphasized recently.Plasmin system is also an important factor to modulate ECM degradation and matrix metalloproteinase(MMP)system.In this study,the authors established an animal model of CLD induced by inhaling high concentration oxygen(hyperoxia)to investigate the changes and functions of urokinase-plasminogen activator(u-PA)and plasminogen activator inhibitor-1(PAI-1)in CTD.Methotis Flull.term newborn rats were continuously exposed to oxygen(0.90-0.95 O2)or room air within 12 h after birth.On day 1,3,7,14,21 in hyperoxia groups and air controls,lung pathology in newborn rats were observed.The changes of u-PA and PAl-1 protein and mRNA expression were measured by Western blotting and RT-PCR Results(1)The pathological findings of the lung tissue:on day 3,there was a few inflammatory cells exuded out,bleeding,edema,and interstitial cells increased in hyperoxia group.On day 7 and thereafter,the terminal air space size of the oxygen-exposed rat became large,there was inflammatory response and more interstitial cells,interstitium was thicker,and collagen deposited.(2)u-PA expression:On day 3.the u-PA protein expression increased in hyperoxia group compared with controls(115.52±7.10 vs.96.51±6.33),P<0.01.On day 7 to day 21,u-PA protein expression(97.66±7.98,99.91±7.60.103.23±6.24)was lower than in the control groups(112.43±6.01,123.25±8.35,103.23±6.24),P<0.05,<0.01 and<0.O1,respectively.u-PA mRNA increased on d 3 in hyperoxia group compared with controls(1.18±0.07vs.0.99±0.05),P<0.01.On d 7 to 21,mRNA expression(1.01±0.06,1.10±0.12,1.27±0.06)was lower than that in the controls(1.15±0.08,1.51±0.32,1.60±0.24)too,P<0.01.(3)PAI-l expression:From d 7 to 21 of oxygen exposure.PAI-1 protein expression(147.83±12.27,149.07±11.17,161.42±13.08)increased compared with the controls(116.18±10.67,113.73±15.58,126.60±8.59),P<0.01,<0.05 and<0.01,respectively.mRNA expression(1.49±0.28,1.46±0.3l,1.51±0.33)increased compared with the control group(O.94±0.01,0.94±0.03,0.98±0.03),P<0.05,<0.01 and<0.05,respectively.Conclusions In tlle early stage of hyperoxic exposure.the balance of u.PA/PAI.1 mRNA and protein increased,plasmin and degradation activity increased,which may increase the degradation of ECM in lung base membrane.During the middle and late stage.the expression of u-PA/PAI-1 mRNA and protein decreased,plasmin and degradation activity were lower,in parallel to thicker interstitium,suggesting that the imbalance of u-PA/PAI-1 may also play a role in lung fibrosis in CLD induced by hyperoxia.