Alveolar hypercoagulation and fibrinolytic inhibition play a central role in refractory hypoxemia in acute respiratory distress syndrome (ARDS), but it lacks effective drugs for prevention and treatment of this pathophysiology. Our previous experiment confirmed that RUNX1 promoted alveolar hypercoagulation and fibrinolytic inhibition through NF-κB pathway. Other studies demonstrated that 6-gingerol regulated inflammation and metabolism by inhibiting the NF-κB signaling pathway. We assume that 6-gingerol would ameliorate alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/ NF-κB pathway in LPS-induced ARDS. Rat ARDS model was replicated through LPS inhalation. Before LPS inhalation, the rats were intraperitoneally treated with different doses of 6-gingerol or the same volume of normal saline (NS) for 12 h, and then intratracheal inhalation of LPS for 24 h. In cell experiment, alveolar epithelial cell type II (AECII) was treated with 6-gingerol for 6 h and then with LPS for another 24 h. RUNX1 gene was down-regulated both in pulmonary tissue and in cells. Tissue factor (TF), plasminogen Activator Inhibitor 1(PAI-1) and thrombin were determined by Wester-blot (WB), qPCR or by enzyme-linked immunosorbent (ELISA). Lung injury score, pulmonary edema and pulmonary collagen III in rat were assessed. NF-κB pathway were also observed in vivo and in vitro. The direct binding capability of 6-gingerol to RUNX1 was confirmed by using Drug Affinity Responsive Target Stability test (DARTS). 6-gingerol dose-dependently attenuated LPS-induced lung injury and pulmonary edema. LPS administration caused excessive TF and PAI-1 expression both in pulmonary tissue and in AECII cell and a large amount of TF, PAI-1 and thrombin in bronchial alveolar lavage fluid (BALF), which all were effectively decreased by 6-gingerol treatment in a dose-dependent manner. The high collagen Ⅲ level in lung tissue provoked by LPS was significantly abated by 6-gingerol. 6-gingerol was seen to dramatically inhibit the LPS-stimulated activation of NF-κB pathway, indicated by decreases of p-p65/total p65, p-IKKβ/total IKKβ, and also to suppress the RUNX1 expression. RUNX1 gene knock down or RUNX1 inhibitor Ro5-3335 significantly enhanced the efficacies of 6-gingerol in vivo and in vitro, but RUNX1 over expression remarkably impaired the effects of 6-gingerol on TF, PAI-1 and on NF-κB pathway. DARTS result showed that 6-gingerol directly bond to RUNX1 molecules. Our experimental data demonstrated that 6-gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/NF-κB pathway in LPS-induced ARDS. 6-gingerol is expected to be an effective drug in ARDS.

Alveolar type II (ATII) cells produce pulmonary surfactant (PS) essential for maintaining lung function. The aberration or depletion of PS can cause alveolar collapse, a hallmark of acute respiratory distress syndrome (ARDS). However, the intricacies underlying these changes remain unclear. This study aimed to elucidate the mechanisms underlying PS perturbations in ATII cells using transcriptional RNA-seq, offering insights into the pathogenesis of ARDS. ATII cells were identified using immunofluorescence targeting surface-active protein C. We used 24-h lipopolysaccharide (LPS)-induced ATII cells as an ARDS cell model. The efficacy of the injury model was gauged by detecting the presence of tumour necrosis factor-α and interleukin-6. RNA-seq analysis was performed to investigate the dynamics of PS deviation in unaltered and LPS-exposed ATII cells. Whole-transcriptome sequencing revealed that LPS-stimulated ATII cells showed significantly increased transcription of genes, including Lss, Nsdhl, Hmgcs1, Mvd, Cyp51, Idi1, Acss2, Insig1, and Hsd17b7, which play key roles in regulating cholesterol biosynthesis. We further verified gene levels using real-time quantitative PCR, and the results showed that the mRNA expression of these genes increased, which was consistent with the RNA-seq results. Our study revealed pivotal transcriptional shifts in ATII cells after LPS exposure, particularly in nine key lipid and cholesterol metabolism genes. This altered expression might disrupt the lipid balance, ultimately affecting PS function. This finding deepens our understanding of the aetiology of ARDS and may lead to new therapeutic directions.

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.

Background: Sepsis is a globally recognized health issue that continues to contribute significantly to mortality and morbidity in intensive care units (ICUs). The association between mean arterial pressure (MAP) and prognosis among patients with patients is yet to be demonstrated. Objective: The aim of this study was to explore the association between MAP and 28-day mortality in ICU patients with sepsis using data from a large, multicenter database. Methods: This is a retrospective cohort study. We extracted data of 35,010 patients with sepsis from the MIMIC-IV (Medical Information Mart for Intensive Care) database between 2008 and 2019, according to the Sepsis 3.0 diagnostic criteria. The MAP was calculated as the average of the highest and lowest readings within the first 24 hours of ICU admission, and patients were divided into 4 groups based on the mean MAP, using the quadruple classification approach. Other worst-case indications from the first 24 hours of ICU admission, such as vital signs, severity of illness scores, laboratory indicators, and therapies, were also gathered as baseline data. The independent effects of MAP on 28-day mortality were explored using binary logistic regression and a two-piecewise linear model, with MAP as the exposure and 28-day mortality as the outcome variables, respectively. To address the nonlinearity relationship, curve fitting and a threshold effect analysis were performed. Results: A total of 34,981 patients with sepsis were included in the final analysis, the mean age was 66.67 (SD 16.01) years, and the 28-day mortality rate was 16.27% (5691/34,981). The generalized additive model and smoothed curve fitting found a U-shaped relationship between MAP and 28-day mortality in these patients. The recursive algorithm determined the low and high inflection points as 70 mm and 82 mm Hg, respectively. Our data demonstrated that MAP was negatively associated with 28-day mortality in the range of 34.05 mm Hg-69.34 mm Hg (odds ratio [OR] 0.93, 95% CI 0.92-0.94; P<.001); however, once the MAP exceeded 82 mm Hg, a positive association existed between MAP and 28-day mortality of patients with sepsis (OR 1.01; 95% CI 1.01-1.02, P=.002). Conclusions: There is a U-shaped association between MAP and the probability of 28-day mortality in patients with sepsis. Both the lower and higher MAP were related with a higher risk of mortality in patients with sepsis. These patients have a decreased risk of mortality when their MAP remains between 70 and 82 mm Hg.

Background: Andrographolide (Andro) has been confirmed to ameliorate alveolar hypercoagulation and fibrinolysis inhibition via NF-κB pathway in acute respiratory distress syndrome (ARDS), but the specific target of Andro is unknown.Purpose: Our aim is to explore the specific target of Andro through which the drug exerted its effects on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS.Methods: AECII was treated with different doses of Andro for 1 h, and then stimulated with LPS for 24 h. Expressions of tissue factor (TF), plasminogen activator inhibitor (PAI)-1 and tissue factor pathway inhibitor (TFPI) were detected. Concentrations of thrombin-antithrombin complex (TAT), pro-collagen type III peptide (PIIIP), antithrombin III (ATIII) and activated protein C (APC) in cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). NF-κB signaling pathways activation was simultaneously determined. AECII with p65 down-/over-expression were used as control.Results: Andro effectively inhibited TF and PAI-1 and promoted TFPI expressions on AECII induced by LPS stimulation. Andro also significantly suppressed the productions of TAT and PIIIP but promoted ATIII and APC secretions from the LPS-treated cell. Furthermore, Andro application obviously inhibited NF-κB signaling pathway activation provoked by LPS, as shown by decreased level of phosphorylation (p‑)-IKKβ/IKKβ, p-p65/p65 and p65 DNA binding activity. The effects of Andro on those factors were obviously strengthened by down- but were weakened by up-regulation of p65 gene in AECII cell.Conclusions: Our data demonstrates that targeting AECII is the mechanism by which Andro ameliorates alveolar hypercoagulaiton and fibrinolytic inhibition via NF-κB pathway in ARDS. Andro is worth to be clinically further studied in ARDS treatment.

Alveolar hypercoagulation and fibrinolytic inhibition are critical mechanisms contributing to refractory hypoxemia in acute respiratory distress syndrome (ARDS). The nuclear factor kappa‐B (NF‐κB) pathway is known to play a role in these processes. Lysophosphatidic acid receptor 1 (LPAR1) has been identified as being associated with the NF‐κB pathway. We hypothesize that LPAR1 may regulate alveolar hypercoagulation and fibrinolytic inhibition in ARDS, potentially through modulation of the NF‐κB pathway. The rat model of acute respiratory distress syndrome (ARDS) was induced via inhalation of lipopolysaccharide (LPS). In some rats, pulmonary tissue was subjected to either overexpression or knockdown of the LPAR1 gene using lentiviral‐mediated transfection prior to LPS exposure. The impact of LPAR1 modulation on alveolar hypercoagulation, fibrinolytic inhibition, acute lung injury, and the NF‐κB signaling pathway in the ARDS rat model was investigated. In vitro, the expression level of LPAR1 in LPS‐stimulated type II alveolar epithelial cells (AECII) was monitored over time. Additionally, we assessed the influence of LPAR1 on the expression of tissue factor (TF) and plasminogen activator inhibitor‐1 (PAI‐1), as well as its role in the NF‐κB signaling pathway within these cells. The potential influence of LPAR1 on the hypercoagulation and fibrinolytic inhibition in LPS‐induced ARDS via the NF‐κB signaling pathway was also examined. An increase in LPAR1 expression was observed in the lung tissue of rats with ARDS. Our findings indicated that overexpression of LPAR1 significantly intensified lung injury and the wet‐to‐dry (W/D) ratio, both of which are induced by LPS, while the knockdown of LPAR1 substantially reduced these effects. Inhalation of LPS resulted in elevated expression levels of TF, plasminogen activator inhibitor‐1 (PAI‐1), as well as increased activity of TF, PAI‐1, and thrombin in bronchoalveolar lavage fluid (BALF). These effects were exacerbated by LPAR1 overexpression but were notably diminished by LPAR1 knockdown. Overexpression of LPAR1 further activated the LPS‐induced NF‐κB signaling pathway, whereas reduced LPAR1 expression significantly dampened NF‐κB activation. In cellular studies, LPAR1 levels began to rise 6 h post‐LPS stimulation, peaked at 24 h, and subsequently declined. LPS stimulation also increased TF and PAI‐1 expression and activated the NF‐κB signaling pathway. The enhancements of TF and PAI‐1 expressions observed in LPAR1‐overexpressing conditions were markedly counteracted by LPAR1 knockdown. Rescue experiments validated that the inhibition of the NF‐κB signaling pathway significantly reversed the upregulation of TF and PAI‐1 caused by LPAR1 overexpression in LPS‐stimulated RLE‐6TN cells. The findings of our study provide evidence that LPAR1 exacerbates LPS‐induced alveolar hypercoagulation and the inhibition of fibrinolysis in LPS‐induced ARDS through the NF‐κB signaling pathway. The LPAR1/NF‐κB signaling axis may represent a promising target for further investigation into the molecular mechanisms underlying ARDS.