Triptolide dose-dependently improves LPS-induced alveolar hypercoagulation and fibrinolysis inhibition through NF-B inactivation in ARDS mice

Background: Alveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-kappa B pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-kappa B pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-kappa B inactivation. Purpose: The aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice. Methods: 50 mu l of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 mu g/kg, 10 mu g/kg, 50 mu g/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-kappa B. TP (1 mu g/kg, 10 mu g/kg, 50 mu g/kg) were intraperitoneally injected or 10 mu g/50 mu l of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type III (PIIIP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-kappa B activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. Results: LPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PIIIP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-kappa B pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKK alpha/beta and pI kappa B alpha, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-kappa B signalling pathway in ARDS mice. Conclusions: TP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-kappa B signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.