吸入一氧化氮对急性肺损伤大鼠内源性一氧化氮及纤溶酶原激活物抑制剂-1表达的影响及其相关研究

Effects of inhaled nitric oxide on intrapulmonarynitric oxide production and expression of plasminogen activator inhibitor-1 in lipopolysaccharide-induced acute lung injury

目的 探讨早期吸入一氧化氮（NO）对急性肺损伤大鼠纤溶酶原激活物抑制剂-1（PAI-1）mRNA和蛋白表达的影响及其意义;观察吸入NO后急性肺损伤大鼠诱生性NO合酶（iNOS）和内源性NO的变化及其与PAI-1表达的关系.方法 采用内毒素（LPS）二次打击方法建立4～5周SD大鼠急性肺损伤模型.对照组和LPS组分别随机给予吸入空气（A）、20×10-6 NO,干预24 h.应用荧光实时定量PCR方法测定大鼠肺组织PAI-1 mRNA水平,免疫组织化学测定2组大鼠肺组织PAI-1蛋白的表达水平,并测定肺组织iNOS活性和NO水平;同时行肺组织病理评分和纤维素染色.结果 造模后气体干预24 h时肺组织PAI-1 mRNA和蛋白表达在NO干预的LPS-NO组较LPS-A组显著降低（4.94 ±0.52比5.56±0.27;1.31 ±0.40比1.69 ±0.16,P均＜0.05）.同时NO干预后LPS-NO组iNOS活性和肺组织NO水平均显著低于LPS-A组[（0.84±0.36）U/mg prot比（2.30±0.25） U/mg prot;（1.90±0.84） μmol/g prot比（3.38±0.73） μmol/g prot,P均＜0.05）.iNOS活性与PAI-1 mRNA和蛋白表达呈正相关（r=0.481,P=0.005;r =0.667,P=0.000）;肺组织NO水平与PAI-1 mRNA和蛋白表达呈正相关（r=0.532,P=0.002;r =0.784,P=0.000）.肺病理评分在干预24 h时,LPS-NO组较LPS-A组下降,差异有统计学意义（4.28±0.94比6.12±1.51,P＜0.05）.光镜下LPS-NO组大鼠肺纤维素沉积较吸入空气的大鼠有所减少.结论 早期吸入20×10-6 NO可抑制急性肺损伤大鼠PAI-1的高表达,缓解纤溶失衡,减少纤维蛋白沉积,减轻肺损伤;吸入NO可减少急性肺损伤时肺组织iNOS活性和NO产量,此作用与PAI-1表达下调密切相关,因此可将研究内源性NO系统调节PAI-1表达的信号通路作为下一步的研究方向,为设计新疗法提供思路.

Objective To explore the effects of inhaled nitric oxide（NO） on expression of plasminogen activator inhibitor-1 （PAI-1） mRNA and protein in the early-stage of experimental acute lung injury （ALI） in a rat model.And to investigate the relationship between endogenous NO system including inducible nitric oxide synthase （iNOS）and intrapulmonary NO production and expressions of PAI-1 in ALI.Methods In the study,endotoxemia followed by the second attack due to intratracheal injection of lipopolysaccharide （LPS） in rats caused ALI.Male SD rats aged 4-5 weeks （clean conventional rats,180-200 g） were randomly assigned to 2 groups：saline control （C） group,LPS-treated （LPS） group,and the 2 groups were randomly allocated to subgroups exposed to air （A） or 20 × 10-6 NO.They were sacrificed for 24 h.Expressions of PAI-1 mRNA of the lung tissue were evaluated by real-time polymerise chain reaction; PAI-1 proteins were determined by immunohistochemistry.NO production in the lung tissues and pulmonary iNOS activity were measured.Meanwhile,histopathological lung injury scores were evaluated and modified martius acid fuchsin brilliant blue（MSB） stains was performed to evaluate fibrin of the lung tissues.Results At 24 h time point with intervention of iNO,PAI-1 mRNA and protein levels in LPS-NO subgroup were decreased compared with those in LPS-A subgroup （4.94 ± 0.52 vs 5.56 ± 0.27 ; 1.31 ± 0.40 vs 1.69 ± 0.16,all P 〈 0.05）.Meanwhile,iNOS activity and NO productions in LPS-NO subgroup were lower than those of LPS-A subgroup [（0.84 ± 0.36） U/mg prot vs （2.30 ± 0.25） U/mg prot ; （1.90 ± 0.84） μmol/g prot vs （3.38 ± 0.73） μmol/g prot,all P 〈 0.05].iNOS activity had significant correlation with expression of PAI-1 mRNA and protein in lung tissue （r =0.481,P =0.005 ; r =0.667,P =0.000） ; NO production had significant correlation with expression of PAI-1 mRNA and protein in lung tissue（r =0.532,P =0.002; r =0.784,P =0.000）.At 24 h time point,the histopathologic lung injury scores in LPS-NO subgroup were decreased in contrast to LPS-A subgroup （4.28 ±0.94 vs 6.12 ± 1.51,P 〈 0.05）.Fibrin deposition evaluated by modified MSB stains in LPS subgroups was found in alveolar space,lumen of blood vessel and mesenchymal ;LPS subgroup with NO appeared a decreasing trend in contrast to LPS subgroup with air.Conclusions Inhaled nitric oxide of 20 × 10 6 can suppress elevated expression of PAI-1 in ALI induced by endotoxin.This inhaled NO can improve the imba-lance of plasminogen activation system and alleviate lung injury.Meanwhile,inhaled NO down-regulates intrapulmonary iNOS activity as well as endogenous NO productions in rats with 2 hits of LPS induced ALI.These changes also have a close correlation with down-regulation of PAI-1 mRNA and protein.Thus,regulation of endogenous NO system on the expression of PAI-1 will be the future direction of new therapies for ALI.