Effects Of Plasminogen Activator Inhibitor Type-1 On Toll Like Receptor 4 Signaling Pathway In Acute Lung Injury Mice

Part I Character of acute lung injury model with plasminogen activator inhibitor-1 deficient miceBackgroundAlveolar and intravascular fibrin deposition is one of the characteristic pathological changes of ALI/ARDS. Many factors contribute to the changes including thrombin expression induced by tissue factor and increased plasminogen activator inhibitor (PAI-1) synthesis and release. Experimental and clinical studies have demonstrated that PAI-1 in bronchoalveolar lavage fluid (BALF) and plasma increased in ALI/ARDS. The increased level of PAI-1 in BALF was correlated with poor prognosis. But, clinical trial showed that anticoagulant didn't change the process and outcome. It suggests that PAI-1 play an important role in ALI/ARDS besides regulating coagulation and fibrinolysis. So, it is important that explore the mechanism of PAI-1 participating in ALI/ARDS.ObjectiveTo investigate the effect of PAI-1 deficiency in ALI mice induced by LPS.MethodsPAI-1 deficient mice (KO)and wild type C56BL/6J mice (WT) were randomly assigned to Oh group (WT Oh group, KO Oh group),2h group(WT 2h group, KO 2h group),,4h group (WT 4h group, KO 4h group), and 8h group (WT 8h group, KO 80h group), according to LPS administration time and were given LPS (5 mg/kg intratracheal injection) or nothing. ALI was evaluated by determining the lung wet/dry ratio, malondialdehyde (MDA), pulmonary microvascular permeability, coagulation/fibrinolysis alteration and histopathological examination.ResultsMortality in KO ALI mice markedly increased than that in WT ALI mice (40% vs.10% respectively, P<0.05). The ALI score increased significantly for the KO 8h group compared with WT 8h group (8.5±0.5 vs. 11.1±0.6 respectively, P<0.05). Microvascular permeability of lung tissue in ALI mice significantly increased; Compared with WT mice at the same time point, the microvascular permeability of KO 4h and KO 8h significantly increased (62.28±8.62 vs.78.63±7.82,81.02±11.35 vs.98.02±10.21 respectively), P<0.05. Lung tissue wet/dry ratio began to increase at 2 hours after LPS treatment and the ratio in KO 8h group mice was markedly higher than WT 8h group mice(6.36±0.23 vs.5.48±0.45), P<0.05. There were no differences at lung tissue MDA between WT ALI mice and KO ALI mice. Abnormal coagulation and fibrinolysis were observed in all ALI mice, whereas no difference was appreciable.ConclusionCompared with WT ALI mice, PAI-1 deficient ALI mice had higher mortality with aggravated inflammatory response. However, alteration in coagulation and fibrinolysis in both genotypes mice was in similar extent.Part II Change of TLR4 signaling pathway in acute lung injury mice with plasminogen activator inhibitor-1 deficiencyBackgroundStudies suggested that PAI-1 participate in inflammatory and immune response. But, the role of PAI-1 in lung injury has been controversial. Renckens et al found that deletion of PAI-1 was not protective in lung infection induced by pneumococcal pneumonia. However, Rijneveld et al found that PAI-1 knockout increased mortality and worsened lung injury when mice were challenged with gram negative bacteria kelebessila. It suggests that PAI-1 plays different roles in Gram negative and Gram positive bacterial infections. The reason may be related to the characteristics of two types of pathogenic bacteria. Gram-negative bacilli easily led to acute lung injury via LPS.Cascade of inflammatory and immune response initiated through Toll like receptors (TLRs) pathway is the pathophysiological basis of acute lung injury. LPS is specific recognized by TLR4. LPS recognition and signal transduction is the key of host defense to Gram-negative bacteria. What role does PAI-1 play in ALI induced by LPS? We supposed that PAI-1 may contribute to ALI/ARDS process by regulating TLR4 signal pathway. It is worthy of further study.ObjectivesTo investigated the effect of PAI-1 deficiency in TLR4 pathway and cytokine in ALI mice induced by LPS. MethodsMice model of ALI was established by intratracheal lipopolysaccharide (LPS) instillation. PAI-1 deficient mice and wild type mice were randomized into Oh group (WT Oh group, KO Oh group) and 8h group (WT 8h group, KO 8h group) according to LPS administration time. IL-6, IL-10 and IFN-y protein level in BALF, plasma and lung tissue were measured by ELISA. IL-6, TLR4, SOCS1 and IRAK-M mRNA level were determined by real-time PCR. Immunohistochemistry were performed for IRAK-M protein in lung tissue. SOCS1, IRAK-M, phosphorylated ERK (P-ERK) and phosphorylated JNK (P-JNK) in each group were determined by Western blot.ResultsELISA examinations showed that IL6, IL-10 and IFN-y protein level in BALF increased in all ALI mice, compared with Oh mice (P<0.05). Furthermore, cytokines levels in KO 8h group were higher than WT 8h group. The ELISA results of cytokines in lung tissue gave the same pattern. And, the levels of IL-6 and IFN-y in plasma of KO 8h mice were significantly increased than WT 8h. However, IL-10 level in plasma of all ALI mice didn't change between before and after LPS exposure. Compared with Oh mice, IL-6 mRNA in KO 8h and WT 8h mice increased 3.2 times and 1.84 times respectively. Compared with WT 8h mice, IL-6mRNA in KO 8h mice increase 2.4 times. After LPS treatment, IRAK-M mRNA and SOCS1 mRNA in lung tissue of WT mice were significantly increased, but these trends weren't observed in KO mice. TLR4 mRNA level increased in WT and KO mice after LPS exposure, and compared with WT 8h mice, TLR4 mRNA level were significantly increased (6.5 times, P<0.05). By immunohistochemistry analysis, WT 8h mice were shown to have more IRKA-M protein than KO 8h mice (IOD 3290±358.9 vs.1113.4±229.2 respectively), P<0.05, P＜0.05. Western blot analysis of IRAK-M and SOCS1 showed that there were sparely expression in both WT Oh mice and KO Oh mice. ALI mice had more IRAK-M expression, especially WT 8h. And, SOCS1 protein in WT 8h mice increased significantly compared with WT Oh mice. However, SOCS1 protein expressions were no difference between KO 8h and WT Oh. Compared with WT 8h mice, p-JNK and p-ERK were more obviously in KO 8h mice.ConclusionsPAI-1 deficiency increased TLR4 expression and inhibited IRAK-M and SOCS1 in ALI mice. PAI-1 deficiency increased cytokines release in BALF, plasma and lung tissue, especially IL-6 and IFN-y. Furthermore, PAI-1 deficiency increased JNK and ERK phosphorylation. PartⅢEffects of heparin or urokinase treatment on Toll like receptor 4 and coagulation in acute lung injury mice with PAI-1 deficiencyBackgroundStudies showed that the level of PAI-1 in BALF increased in ARDS patients. It suggests that lung is prone to coagulation in ARDS. The increased level of PAI-1 in BALF related to the poor outcome of ALI/ARDS. It indicated that the alteration of PAI-1 was the important factor of ALI/ARDS. And, anticoagulation therapy maybe improves the prognosis of ALI/ARDS. Heparin and urokinase are generally used to treat thromboembolic disease, initial stage of DIC and anticoagulation in vitro. A limited number of related studies used heparin or urokinase to treat lung injury, and conclusions were inclusive with poor overall outcome. The mechanism of heparin or urokinase acting on lung injury remains to be unraveled. The first two parts of the test results showed that compared with WT mice, no significant change in coagulation/fibrinolysis and mortality increased in PAI-1 knockout ALI mice. TLR4 signaling pathway enhanced, cytokines release augmented. Whether anticoagulant drug is the same as the impact of PAI-1 on TLR4 signaling pathway? It is worth to study.ObjectiveTo investigate the potential effects of heparin or urokinase on TLR4 signaling pathway and coagulation in ALI mice. To investigate the relationship between heparin or urokinase effects and PAI-1 gene.MethodsPAI-1 deficient mice (KO) and wild type mice (WT) were randomly divided into control groups (WT control group, KO control group), heparin treatment group (WT heparin treatment group, KO treatment group) and urokinase treatment groups (WT urokinase treatment group, KO urokinase group). Control group were administered with LPS (5mg/kg) intratracheal injection. Heparin treatment groups and urokinase treatment group were administered with heparin 400IU/kg or urokinase 50000 u/kg respectively via caudal vein plus LPS intratracheal treatment. ELISA was used to measure the level of IL-6, IL-10 and IFN-γin serum and lung tissue; Real-time PCR was used to measure the expression of IL-6, TLR4, SOCS1 and IRAK-M mRNA in lung tissue. Western blots were used to measure the expression of IRAK-M and SOCS1 protein;ResultsMortality was 10%, survival time 22±1.3 h in WT control group; Mortality was 40%, survival time 16.2±2.1 h in KO control group. Compared with WT control group or KO control group, mortality in WT heparin treatment group or KO heparin group was no difference. Furthermore, compared with WT control group or KO control group, mortality in WT urokinase treatment group or KO urokinase group was no difference. ALI score was 8.5±0.5 or 11.1±0.6, pulmonary microvascular permeability 81.02±11.35 or 98.02±10.21, W/D ratio 5.48±0.51 or 6.36±0.23 in WT control group and KO control group respectively. Compared with control ALI mice of same genotypes, ALI score (6.4±0.4,7.3±0.5), pulmonary microvasucar permeability (60.32±14.3,72.56±10.12)and W/D ratio (4.32±0.60,5.0±0.50) significantly decreased in WT heparin treatment group and KO heparin treatment group. Urokinase treated with similar result with heparin therapy. Compared with controlled ALI mice, heparin or urokinase treatment can improve the coagulation/fibrinolytic abnormalities. But, there was no difference in all indicators between KO mice and WT mice after drug treatment. The level of IL-6 was significantly decreased after heparin or urokinase treatment. Furthermore, the rate of descent of IL-6 was similar in two genotypes mice up after heparin or urokinase treatment. WT and KO ALI mice had no significantly change in the level of IL-10 and IFN-y in plasma and lung tissue before and after heparin or urokinase treatment. In accordance with those finding in ELISA, heparin or urokinase treatment decrease IL-6 mRNA expression. The results of real-time PCR and western-blot analysis showed that TLR4, IRAK-M and SOCS1 expression were not influenced by heparin or urokinase treatment.ConclusionAlthough heparin or urokinase therapy improved coagulation and fibrinolysis of ALI mice, no difference was observed between WT and KO mice. Heparin or urokinase treatment decreased ALI score, pulmonary microvascular permeability and W/D ratio, but these didn't alter mortality of ALI. Drug showed their beneficial effects on mice ALI/ARDS model, merely decreasing IL-6 release of plasma and lung tissue. The expression of TLR4 and its negative regulators including IRAK-M, SOCS1 were not influence by heparin or urokinase treatment. There were no correlation between heparin or urokinase effects and PAI-1.