| Partâ… Expression and Role of PAI-1 and Correlation Factors in a Rat Model of Acute Lung injuryBackgroud:Acute lung injury(ALI) and acute respiratory distress syndrome(ARDS) are common,life-threatening causes of acute hypoxemic respiratory failure that is associated with both intrapulmonary and extrapulmonary disorders and with no left atrial hypertension.It is well established that acute lung injury and acute respiratory distress syndrome are characterized by local and intense inflammatory responses. Fibroproliferation,with deposition of collagen is an early response to lung injury and an important therapeutic target as well as anti-inflammation.Plasminogen activator inhibitor(PAI)-1 is the key inhibitor of the plasminogen activation system(PAS). The level of PAI-1 gene expression correlates closely with the amount of collagen deposition in lung tissues.PAI-1 blocking MMPs may prevent the extracellular matrix degradation.Enhanced plasminogen activation leads to increased levels of active hepatocyte growth factor(HGF) within the alveolar spaces and contributes to reducing the extent of subsequent fibrosis.Inhibition of the plasminogen system by increased expression of PAI-1 in lung injury leads to abnormal accumulation of fibrin and play an essential role in lung remodeling.Objectives:The objectives of the experimental study are 1) to observe changes of PAI-1 expression in a rat model of acute lung injury so as to search suitable therapeutic opportunity;2) to explore expression and role of the factors related to PAI-1 in acute lung injury.Methods:Male SD rats aged 4～5weeks(clean conventional rats,180～200g) were primed by lipopolysaccharide(LPS)(0.1mg/kg) via intraperitoneal challege and then followed by a second dose of LPS(1mg/kg) that were given intratracheally 16h later,wheras control groups of rats were given normal saline.LPS-treated rats were sacrificed at 2h,4h,8h,16h,24h,48h and saline-treated rats at 2～4h(marked as baseline)(n=8～10).Measuring of blood gas analysis,histopathological lung injury score,total proteins(TP) and white blood cell(WBC) count in bronchoalveolar lavage fluid(BALF),pulmonary myeloperoxidase(MPO), pulmonary malondialdehyde(MDA) and wet-to-dry lung weight(another six rats sacrificed at baseline,at 4h and 24h respectively) were measured.PAI-1 antigen levels in both plasma and BALF were also measured.Meanwhile,expression of PAI-1, uPA,MMP-9 and HGF mRNA of the lung tissue were evaluated by real-time polymerise chain reaction;PAI-1,uPA,MMP-9 and HGF proteins were determined by immunohistochemistry.Modified MSB stains were performed to evaluate fibrin of the lung tissue.Results:1.Establishing acute lung injury in a rat model:At 2～48h time points,arterial partial pressure of oxygen(PaO2)were decreased when compared to baseline(P＜0.05),and were lower than 60mmHg during 4～8h.In LPS-treated rats,the histopathological lung injury score values at 2h were significantly higherthan that of baseline and the score values in time points after 2h were higher than that of LPS group at 2h(P＜0.05).They were manifested by widerspread neutrophil infiltration, edema,hemorrhage and alveolar atelectasis in the lungs.During 2h～48h after second hit of LPS,TP concentration and WBC counts in BALF,pulmonary MPO were all higher than baseline(P＜0.05) with a trend of progressing.At 4h～48h time points, pulmonary MDA were increased compared with baseline(P＜0.05) with a trend of progressing.At 4h,24h time points,wet-to-dry lung weight showed a trend of increasing.2.Expression of PAI-1 in the lung tissue:After 2h with two LPS attacks,PAI-1 antigen levels in plasma showed a trend of increasing and PAI-1 levels were increased significantly at 8～16h time points(P＜0.05).PAI-1 antigen levels in BALF and PAI-1 proteins were increased compared with baseline during 2～48h(P＜0.05)with a trend of progressing.Expression of PAI-1 mRNA in lung were increased gradually in LPS groups during 2h～24h(P＜0.05) and decreased at 48h.PAI-1 antigen levels in plasma and BALF,expression of PAI-1 mRNA and proteins had a significant correlation with each other;PAI-1 antigen levels in plasma and BALF,PAI-1 proteins had significant correlations with histopathological lung injury scores(P＜0.05).3.Expression of the factors related to PAI-1 in the lung tissue: â‘ Expression of uPA mRNA was increased rapidly after second LPS hit,and at 2h was higher than baseline(P＜0.05).But then uPA mRNA had no significant change during 4～48 time points compared with baseline(P＞0.05).uPA proteins showed a increasing trend at 2h compared with baseline and were decreased at 4h. After 8h with two LPS attacks,uPA proteins were lower than that at 2h and at 24h lower than baseline(P＜0.05).â‘¡PAI-1/uPAmRNA was increased at 2～48h time points in contrast to baseline and PAI-1/uPAmRNA during 8～16h was higher than that at 2h(P＜0.05). PAI-1/uPA ratio was increased at 4h compared with baseline and was significant higher after 8h than that at 4h(P＜0.05).Fibrin deposition in the lung tissure showed the semblable trend with PAI-1 protein and PAI-1/uPA.PAI-1 mRNA/uPA mRNA and PAI-1/uPA had significant correlations with histopathological lung injury scores(P＜0.01 ).â‘¢Expression of MMP-9 mRNA tended to be increased compared with baseline at 2～24h time points and tended to be decresed at 48h in contrast to at 24h(P＞0.05).At 4～24h time points,MMP-9 proteins were higher than baseline(P＜0.05).â‘£Expression of HGF mRNA tended to be increased at 2～4h time points compared with baseline(P＞0.05) and was higher than baseline at 8～24h(P＜0.05).At 4～24h time points,HGF proteins were higher than baseline(P＜0.05).Conclusions:1.Expression of PAI-1 is early elevated in rats with two hits of LPS induced acute lung injury.The prolonged time course of the increase in PAI-1 levels causes imbalance between PAI-1 and uPA and results in depression of the plasminogen activation system in the lung tissue.PAI-1 plays a pivotal role in lung injury and repair and it is important in the progression of ALI.The elevated levels of PAI-1 antigen in BALF corresponding to PAI-1 proteins in the lung tissue predicts severity of ALI.2.Expression of MMP-9 and HGF are increased at first and then decreased.They are decreased earlier than PAI-1 and it may contribute to abnormal deposition of extracellular matrix and lung remodeling. Partâ…¡Effects of Inhaled Nitric Oxide and Hyperoxia on Expression of PAI-1 in a Rat Model of Acute Lung InjuryBackgroud:Fibrin deposition in the alveolar and lung microvasculature likely results from disordered coagulation and fibrinolysis,triggered by inflammation in ALI.The degree of alterations in coagulation and fibrinolysis may be an important pathogenetic and prognostic determinant of mortality in ALI/ARDS.Modulation of fibrin deposition in the lung through targeting modulation of fibrinolysis may be an important therapeutic target in ALI/ARDS.Locally increased amplification of plasminogrn activator inhiitor-1(PAI-1) is largely responsible for the fibrinolytic defect in the acutely injuried lung.A variety of strategies are being explored to develop inhibitors of PAI-1. Inhibition of PAI-1 has been reported with nitric oxide.Inhaled nitric oxide(iNO) has been demonstrated to improve oxygen by selective pulmonary vasolation to alter ventilation-perfusion mismatching in refractory ARDS.It was reported that iNO prevents pulmonary vascular endothelial cell dysfunction in early-stage of acute lung injury.It's unknown that effects of early intervention with inhaled nitric oxide on expression of PAI-1 in ALI.iNO is usually used in combination with high oxygen supply in critical conditions.An animal study showed that lungs of mice exposed to hyperoxia overproduced plasminogen activator inhibitor-1(PAI-1).Whether iNO together with high oxygen supply would have effects on expression of PAI-1 in ALI remains unknown.NO is synthesized from the amino acid L-arginine by NO synthase (NOS) and released by various cells after chemical and mechanical activation. Exogenous NO may influence endogenous NO system.The relation between exogenous NO and endogenous NO system and the effect on expression of PAI-1 in ALI are still unclear.Objectives:The objectives of the experimental study are 1) to explore the effects of inhaled nitric oxide and/or oxygen of high concentration on expression of PAI-1 in early-stage of experimental acute lung injury in a rat model;2) to explore expression and role of correlation factors of PAI-1 with above-mentioned intervention;3) to explore the effects of inhaled nitric oxide and/or oxygen of high concentration on endogenous NO system in experimental acute lung injury.Methods: Male SD rats aged 4～5weeks(clean conventional rats,180～200g) received lipopolysaccharide(LPS)(0.1mg/kg ) with intraperitoneal challege followed by a second LPS(1mg/kg) attack via intratracheal injection 16h later,wheras control rats received normal saline.LPS-treated(LPS) rats(n=8～10 per subgroup) and control(C) rats(n=6～8 per subgroup) were randomly allocated to subgroups exposed to:air(A),95%oxygen(O),20ppm iNO(NO),95%oxygen and 20ppm iNO(ONO).They were sacrificed at 4h,24h,48h(NO and/or 95%oxygen exposure for 24h).Expression of PAI-1,uPA,MMP-9 and HGF mRNA of the lung tissue were evaluated by real-time polymerise chain reaction;PAI-1,uPA,MMP-9 and HGF proteins were determined by immunohistochemistry.NO production in the lung tissues and pulmonary NOS activity including total NOS(tNOS),inducible NOS(iNOS) and constitutive NOS(cNOS) were measured.Meanwhile, histopathological lung injury scores were evaluated and modified MSB stains were performed to evaluate fibrin of the lung tissues.Results:1.Altered expression of PAI-1 and related factors:â‘ PAI-1 in lung tissure:At 4 and 24h time points,expression of PAI-1 mRNA in LPS-A subgroup and LPS-O subgroup were elevated in contrast to control;with intervention of iNO,PAI-1 mRNA levels in LPS-NO subgroup and LPS-ONO subgroup were decreased compared with other LPS subgroups.At 48h,only in LPS-O subgroup PAI-1 mRNA levels were higher than control and with iNO that in LPS-ONO subgroup was lower than LPS-O subgroup(P＜0.05).At 4h,expression of PAI-1 proteins in LPS-NO subgroup were lower than other LPS subgroups and there were no statistical difference between LPS-NO subgroup and control.At 24h,All LPS subgroups had higher PAI-1 protein levels than corresponding control subgroups(P＜0.05).It tended to be decreased in LPS-NO and LPS-ONO subgroups in contrast to other LPS subgroups(P＞0.05).At 48h,with iNO,PAI-1 protein levels in LPS-NO and LPS-ONO were decreased compared with corresponding LPS subgroups(P＜0.05).â‘¡uPA in lung tissure:At 4h,24h and 48 time points,there were no significant difference in uPA mRNA levels between LPS subgroups and corresponding control.At 24h,uPA protein levels in LPS-A subgroup and LPS-O subgroup were lower than correspongding control and that in LPS subgroups with iNO were increased in contrast to LPS subgroups without iNO(P＜0.05). â‘¢PAI-1/uPA in lung tissue:At 4h,PAI-1 mRNA/uPA mRNA in all LPS subgroups was higher than correponding controls.At 24h and 48h,only in LPS-A and LPS-O subgroups the ratios were higher than correponding controls.At 48h,the ratios in LPS subgroups with iNO were decreased in contrast to LPS subgroups without iNO (P＜0.05).At 4h,PAI-1/uPA in LPS-NO and LPS-ONO subgroups tended to be decreased compared with LPS-A and LPS-O subgroups respectively.At 24h,the ratios in all LPS subgroups were higher than corresponding controls.At 48h the radios in LPS-NO subgroup were decreased and there were no difference between LPS-NO subgroup and control.At 24h and 48h,the ratios in LPS subgroups with iNO were decreased in contrast to LPS subgroups without iNO(P＜0.05).â‘£MMP-9 in lung tissue:At 24h,MMP-9 mRNA levels in all LPS subgroups were elevated in contrast to corresponding controls(P＜0.05).There were no difference among all LPS subgroups(P＞0.05).At 4h and 24h,MMP-9 protein levels in all LPS subgroups tended to be elevated in contrast to corresponding controls(P＞0.05).At 48h,only LPS-O subgroup was higher than control(P＜0.05).⑤HGF in lung tissue:At 4h and 24h,HGF mRNA levels in all LPS subgroups appeared increasing trend compared with corresponding controls(P＞0.05),and LPS-NO subgroup were marked higher than control(P＜0.05).At 48h,there were no difference in HGF mRNA levels between all LPS subgroups and corresponding controls(P＞0.05).At 4h and 24h,HGF protein levels in all LPS subgroups were higher than corresponding controls(P＞0.05),and at 48h,there were no difference between all LPS subgroups and corresponding controls(P＞0.05).2.Altered endogenous NO system consistent with expression of PAI-1:â‘ iNOS activity in lung tissue:At 4h,24h and 48h,iNOS activity in all LPS subgroups showed a trend of increasing in contrast to corresponding controls,while LPS-A subgroups at all time points and LPS-O subgroup at 4h,24h iNOS activity was higher than corresponding controls,iNOS activity in LPS subgroups with iNO tended to decrease in contrast to LPS subgroups without iNO,and at 24h,LPS-NO and LPS-ONO subgroups were lower than LPS-A subgroup(P＜0.05).â‘¡cNOS activity in lung tissue:At 24h,cNOS activity in LPS-A subgroup was significantly lower than control and other LPS subgroups.And at 48h,cNOS activity in LPS-NO subgroup was increased in contrast to LPS-A subgroup(P＜0.05).â‘¢tNOS activity in lung tissue:At 24h and 48h,tNOS activity in all LPS subgroups showed a trend of increasing compared with corresponding controls(P＞0.05).â‘£NO productions in lung tissue:At 4h and 48h,NO productions in all LPS subgroups were increased compared with corresponding controls.At 24h,NO productions in LPS-NO subgroup were decreased in contrast to LPS-A subgroup(P＜0.05);There was no difference between LPS-NO subgroup and control(P＞0.05).⑤Correlations between NO system and PAI-1:At 4h,24h and 48h,iNOS activity had significant correlation with expression of PAI-1 mRNA and protein in lung tissue;cNOS activity had inverse correlation with expression of PAI-1 mRNA and protein in lung tissue;NO production had significant correlation with expression of PAI-1 mRNA and protein in lung tissue(P＜0.05).3.Histopathologic changes in lung tissue:At 4h,24h and 48h,the histopathologic lung injury scores in all LPS subgroups were increased in contrast to corresponding controls.LPS subgroups with iNO appeared a decreasing trend in contrast to LPS subgroups without iNO and at 24h,the scores in LPS-NO subgroup were lower than LPS-A subgroup(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 subgroups with iNO appeared a decreasing trend in contrast to LPS subgroups without iNO.Conclusions:1.Inhaled nitric oxide of 20ppm can suppress elevated expression of PAI-1 in rats with two hits of LPS induced acute lung injury.Exposure to high concentration oxygen prolongs the time course of the increase in PAI-1 mRNA and protein in ALI, while iNO can reduce it.iNO can improve imbalance of plasminogen activation system and alleviate lung injury.2.Inhaled nitric oxide down-regulates intrapulmonary iNOS activity as well as endogenous nitric oxide productions in rats with two hits of LPS induced acute lung injury.These changes also have a closed correlation with down-regulation of PAI-1 mRNA and protein. |
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