6-姜酚通过 RUNX1/NF-κB 信号通路改善脂多糖诱导的 ARDS 中的肺泡过度凝血和纤溶抑制。
6-Gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition in LPS-provoked ARDS via RUNX1/NF-κB signaling pathway.
机构信息
Department of Intensive Care Unit, the Affiliated Hospital of Guizhou Medical University, Guiyang, China.
Emergency department, the Affiliated Hospital of Guizhou Medical University, Guiyang, China.
出版信息
Int Immunopharmacol. 2024 Feb 15;128:111459. doi: 10.1016/j.intimp.2023.111459. Epub 2024 Jan 4.
BACKGROUND
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.
METHODS
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).
RESULTS
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.
CONCLUSIONS
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.
背景
肺泡过度凝血和纤维蛋白溶解抑制在急性呼吸窘迫综合征（ARDS）的难治性低氧血症中起核心作用，但缺乏有效的预防和治疗这种病理生理学的药物。我们之前的实验证实，RUNX1 通过 NF-κB 途径促进肺泡过度凝血和纤维蛋白溶解抑制。其他研究表明，6-姜酚通过抑制 NF-κB 信号通路来调节炎症和代谢。我们假设 6-姜酚通过 RUNX1/NF-κB 途径改善 LPS 诱导的 ARDS 中的肺泡过度凝血和纤维蛋白溶解抑制。
方法
通过 LPS 吸入复制大鼠 ARDS 模型。在 LPS 吸入之前，大鼠经腹腔给予不同剂量的 6-姜酚或相同体积的生理盐水（NS）12 小时，然后经气管内吸入 LPS 24 小时。在细胞实验中，用 6-姜酚处理肺泡上皮细胞 II 型（AECII）6 小时，然后再用 LPS 处理 24 小时。下调肺组织和细胞中的 RUNX1 基因。通过 Western-blot（WB）、qPCR 或酶联免疫吸附（ELISA）测定组织因子（TF）、纤溶酶原激活物抑制剂 1（PAI-1）和凝血酶。评估大鼠肺损伤评分、肺水肿和肺胶原 III。还观察了 NF-κB 通路的体内和体外情况。使用药物亲和反应靶稳定性测试（DARTS）证实 6-姜酚与 RUNX1 的直接结合能力。
结果
6-姜酚剂量依赖性地减轻 LPS 诱导的肺损伤和肺水肿。LPS 给药导致肺组织和 AECII 细胞中 TF 和 PAI-1 表达过度，支气管肺泡灌洗液（BALF）中 TF、PAI-1 和凝血酶大量增加，6-姜酚处理均呈剂量依赖性显著降低。LPS 引起的肺组织中胶原 III 水平显著降低。6-姜酚明显抑制 LPS 刺激的 NF-κB 通路激活，表现为 p-p65/总 p65、p-IKKβ/总 IKKβ减少，也抑制 RUNX1 表达。RUNX1 基因敲低或 RUNX1 抑制剂 Ro5-3335 显著增强了 6-姜酚在体内和体外的疗效，但 RUNX1 过表达显著削弱了 6-姜酚对 TF、PAI-1 和 NF-κB 通路的作用。DARTS 结果表明，6-姜酚直接与 RUNX1 分子结合。
结论
我们的实验数据表明，6-姜酚通过 RUNX1/NF-κB 途径改善 LPS 诱导的 ARDS 中的肺泡过度凝血和纤维蛋白溶解抑制。6-姜酚有望成为 ARDS 的有效药物。
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