高氧、维甲酸对早产鼠肺组织基质金属蛋白酶-2、-9及特异性组织抑制物-1、-2表达的影响

Effect of hyperoxia and retinoic acid on the expression of lung tissue matrix metalloproteinase-2,-9 and tissue inhibitor of metalloproteinase-1,-2 in premature rats

目的探讨基质金属原蛋白-2(MMP-2)、MMP-9、基质金属蛋白酶特异性组织抑制物-1(TIMP-1)和TIMP-2在高氧肺损伤中的作用及维甲酸(RA)的保护作用机制。方法建立高氧(FiO285%)暴露早产SD大鼠肺损伤模型,应用RT-PCR法检测MMP-2、MMP-9、TIMP-1和TIMP-2mRNA表达,采用明胶酶谱检测MMP-2和MMP-9酶原及活酶表达,采用Western blot技术检测TIMP-1和TIMP-2蛋白表达。结果与空气组比较,高氧暴露4、7、14d,MMP-2、MMP-9和TIMP-1 mRNA的表达均显著升高(P均<0.01),MMP-2活酶、MMP-9酶原及活酶和TIMP-1蛋白的表达明显上调(P<0.05);RA对空气暴露下它们的表达均无明显影响(P均>0.05),但不同程度下调高氧暴露后MMP-2、MMP-9、TIMP-1mRNA的表达和MMP-2活酶、MMP-9酶原及活酶的表达,进一步提高TIMP-1蛋白表达;高氧、RA对TIMP-2 mRNA和蛋白的表达均无明显影响(P均>0.05)。结论高氧暴露明显改变MMPs/TIMPs的表达,在肺泡形成关键时期,MMPs/TIMPs之间平衡关系的破坏是造成肺发育受阻和纤维化的重要因素;通过协调MMPs/TIMPs之间的表达,改善肺泡结构,降低肺纤维化程度,从而逆转高氧所致肺损伤,是RA发挥保护作用的重要机制之一。

Objectives To explore the role of matrix metalloproteinase-2, -9 （MMP-2, MMP-9） and tissue inhibitor of metalloproteinase-1, -2 （TIMP-1, TIMP-2） in hyperoxia lung injury and the protective effect of retinoic acid （RA） on hyperoxia lung injury. Methods Model of hyperoxia lung injury was established with exposure the premature Spragne Dawkey （SD） rats to hyper-concentration oxygen of FiO2 85%. The levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA were detected with semi-quantitative reverse transcription polymerase chain reaction （RT-PCR） . The expression of active MMP-2 and pro/active MMP-9 was measured with zymography, the protein abundance of TIMP-1 and TIMP-2 was determined with Western blot. Results Levels of MMP-2, MMP-9 and TIMP-1 mRNA were significantly higher in the model on day 4, day 7, and day 14 of hyperoxia-exposure in comparison with the expression from lungs of normoxic pups （P 〈 0.01 ）. Levels of active MMP-2, pro/active MMP-9 and TIMP-1 were significantly higher after exposure to oxygen than that of room air exposed groups on each experimental day （P 〈 0.05）. RA had no effect on the expression of MMP-2, MMP-9 and TIMP-1 in the pups exposed to room air （P 〉 0.05）. Though both were placed in the hyperoxic environment, the expression levels of mRNA for MMP-2, MMP-9 and TIMP-1 in premature pups treated with RA were significantly lower than non-RA treated pups on each experimental day. Levels of active MMP-2 and pro/active MMP- 9 were decreased markedly after RA treatment in hyperoxia exposed pups. In addition, RA treatment led to a further elevation of TIMP-1 levels. But hyperoxia, RA had no effect on the expression of mRNA TIMP-2 （P 〉 0.05） . Conclusions The expression of MMPs/TIMPs was markedly changed by exposure to hyperoxia. The balance of MMPs/TIMPs was diturbed by hyperoxia injury during alveolarization of lung in premature rat leding to lung development inhibition and lung fibrosis. RA had a protective effect on hyperoxia-induced lung injury by regulating MMPs/TIMPs expression, improving alveolar structure, decreasing fibrosis and reversing the hyperoxia-induced lung injury.