miR-9 targeting RUNX1 improves LPS-induced alveolar hypercoagulation and fibrinolysis inhibition through NF-κB inactivation in ARDS.

Acute respiratory distress syndrome (ARDS) is a clinical and pathophysiological complex syndrome with high mortality. Alveolar hypercoagulation and fibrinolytic inhibition constitute the core part of the pathophysiology of ARDS. miR-9 (microRNA-9a-5p) plays an important role in the pathogenesis of ARDS, but whether it regulates alveolar pro-coagulation and fibrinolysis inhibition in ARDS remains to be elucidated. We aimed to determine the contributing role of miR-9 on alveolar hypercoagulation and fibrinolysis inhibition in ARDS. In the ARDS animal model, we first observed the miR-9 and runt-related transcription factor 1 (RUNX1) expression in lung tissue, the effects of miR-9 on alveolar hypercoagulation and fibrinolytic inhibition in ARDS rats, and the efficacy of miR-9 on acute lung injury. In the cell, alveolar epithelial cells type II (AECII) were treated with LPS, and the levels of miR-9 and RUNX1 were detected. Then we observed the effects of miR-9 on procoagulant and fibrinolysis inhibitor factors in cells. Finally, we explored whether the efficacies of miR-9 were associated with RUNX1; we also preliminarily examined the miR-9 and RUNX1 levels in plasma in patients with ARDS. In ARDS rats, miR-9 expression decreased, but RUNX1 expression increased in the pulmonary tissue of ARDS rats. miR-9 displayed to attenuate lung injury and pulmonary wet/dry ratio. Study results in vivo demonstrated that miR-9 ameliorated alveolar hypercoagulation and fibrinolysis inhibition and attenuated the collagen III expressions in tissue. miR-9 also inhibited NF-κB signaling pathway activation in ARDS. In LPS-induced AECII, the expression changes of both miR-9 and RUNX1 were similar to those in pulmonary tissue in the animal ARDS model. miR-9 effectively inhabited tissue factor (TF), plasma activator inhibitor (PAI-1) expressions, and NF-κB activation in LPS-treated ACEII cells. Besides, miR-9 directly targeted RUNX1, inhibiting TF and PAI-1 expression and attenuating NF-κB activation in LPS-treated AECII cells. Clinically, we preliminarily found that the expression of miR-9 was significantly reduced in ARDS patients compared to non-ARDS patients. Our experimental data indicate that by directly targeting RUNX1, miR-9 improves alveolar hypercoagulation and fibrinolysis inhibition via suppressing NF-κB pathway activation in LPS-induced rat ARDS, implying that miR-9/RUNX1 is expected to be a new therapeutic target for ARDS treatment.

Xiao C ，Li Q ，Xiao J ，Chen X ，Yuan J ，Li S ，Li W ，Gao D ，Li L ，Liu Y ，Shen F ... -  《-》

6-Gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition in LPS-provoked ARDS via RUNX1/NF-κB signaling pathway.

Li Q ，Xiao C ，Gu J ，Chen X ，Yuan J ，Li S ，Li W ，Gao D ，Li L ，Liu Y ，Shen F ... -  《-》

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

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-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB 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-κB inactivation. 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. 50 μl 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 μg/kg, 10 μg/kg, 50 μg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 μg/kg, 10 μg/kg, 50 μg/kg) were intraperitoneally injected or 10 μg/50 μl 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-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. 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-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/β and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice. TP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.

Yang H ，Qian H ，Liu B ，Wu Y ，Cheng Y ，Zheng X ，Li X ，Yang G ，He T ，Li S ，Shen F ... -  《-》

RUNX1 targeting AKT3 promotes alveolar hypercoagulation and fibrinolytic inhibition in LPS induced ARDS.

Alveolar hypercoagulation and fibrinolytic inhibition are mainly responsible for massive alveolar fibrin deposition, which are closely related with refractory hypoxemia in acute respiratory distress syndrome (ARDS). Our previous study testified runt-related transcription factor (RUNX1) participated in the regulation of this pathophysiology in this syndrome, but the mechanism is unknown. We speculate that screening the downstream genes associated with RUNX1 will presumably help uncover the mechanism of RUNX1. Genes associated with RUNX1 were screened by CHIP-seq, among which the target gene was verified by Dual Luciferase experiment. Then the efficacy of the target gene on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS was explored in vivo as well as in vitro. Finally, whether the regulatory effects of RUNX1 on alveolar hypercoagulation and fibrinolytic in ARDS would be related with the screened target gene was also sufficiently explored. Among these screened genes, AKT3 was verified to be the direct target gene of RUNX1. Results showed that AKT3 was highly expressed either in lung tissues of LPS-induced rat ARDS or in LPS-treated alveolar epithelia cell type II (AECII). Tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) were increasingly expressed both in lung tissues of ARDS and in LPS-induced AECII, which were all significantly attenuated by down-regulation of AKT3. Inhibition of AKT3 gene obviously ameliorated the LPS-induced lung injury as well as the collagen I expression in ARDS. RUNX1 overexpression not only promoted the expressions of TF, PAI-1, but also boosted AKT3 expression in vitro. More importantly, the efficacy of RUNX1 on TF, PAI-1 were all effectively reversed by down-regulation of AKT3 gene. AKT3 is an important target gene of RUNX1, through which RUNX1 exerted its regulatory role on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS. RUNX1/ATK3 signaling axis is expected to be a new target for the exploration of ARDS genesis and treatment.

Xiao C ，Liu J ，Cheng Y ，Wu Y ，Li Q ，Chen X ，Yuan J ，Dong Q ，Li L ，Liu Y ，Shen F ... -  《-》

Andrographolide sulfonate attenuates alveolar hypercoagulation and fibrinolytic inhibition partly via NF-κB pathway in LPS-induced acute respiratory distress syndrome in mice.

Alveolar hypercoagulation and fibrinolytic inhibition are important characteristics during acute respiratory distress syndrome (ARDS), and NF-κB p65 signaling pathway is involved to regulate these pathophysiologies. We hypothesize that targeting NF-κB signal pathway could ameliorate alveolar hypercoagulation and fibrinolyitc inhibition, thus attenuating lung injury in ARDS. We explore the efficacy and the potential mechanism of andrographolide sulfonate (Andro-S) on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS in mice. ARDS was made by lipopolysaccharide (LPS) inhalation in C57BLmice. Andrographolide sulfonate (2.5, 5 and 10 mg/kg) was intraperitoneally given to the mice (once a day for three consecutive days) before LPS administration. NEMO binding domain peptide (NBD), an inhibitor of NF-κB, was used as the positive control and it replaced Andro-S in mice of NBD group. Mice in normal control received saline instead of LPS. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis of alveolar coagulation, fibrinolytic inhibition as well as of pulmonary inflammatory response after 8 h of LPS inhalation. NF-κB signal pathway in lung tissue was simultaneously determined. Andro-S dose-dependently inhibited tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 expressions either in mRNA or in protein in lung tissue of ARDS mice, and it also decreased the concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) while promoting the production of activated protein C (APC) in BALF. Meanwhile, Andro-S effectively inhibited inflammatory response (interleukin 1β and myeloperoxidase) induced by LPS. LPS stimulation dramatically activated NF-κB signal pathway, indicated by increased expressions of phosphorylation of p65 (p-p65), p-IKKα/β and p-IκBα and the higher p65-DNA binding activity, which were all dose-dependently reversed by Andro-S. Andro-S and NBD presented similar efficacies. Andro-S treatment improves alveolar hypercoagulation and fibrinolytic inhibition and attenuates pulmonary inflammation in LPS-induced ARDS in mice partly through NF-κB pathway inactivation. The drug is expected to be an effective choice for ARDS.

Qian H ，Yang H ，Li X ，Yang G ，Zheng X ，He T ，Li S ，Liu B ，Wu Y ，Cheng Y ，Shen F ... -  《-》