Pulmonary endothelial barrier dysfunction is a hallmark of clinical pulmonary edema and contributes to the development of acute lung injury (ALI). Here we reported that ruscogenin (RUS), an effective steroidal sapogenin of Radix Ophiopogon japonicus, attenuated lipopolysaccharides (LPS)-induced pulmonary endothelial barrier disruption through mediating non-muscle myosin heavy chain IIA (NMMHC IIA)‒Toll-like receptor 4 (TLR4) interactions. By in vivo and in vitro experiments, we observed that RUS administration significantly ameliorated LPS-triggered pulmonary endothelial barrier dysfunction and ALI. Moreover, we identified that RUS directly targeted NMMHC IIA on its N-terminal and head domain by serial affinity chromatography, molecular docking, biolayer interferometry, and microscale thermophoresis analyses. Downregulation of endothelial NMMHC IIA expression in vivo and in vitro abolished the protective effect of RUS. It was also observed that NMMHC IIA was dissociated from TLR4 and then activating TLR4 downstream Src/vascular endothelial cadherin (VE-cadherin) signaling in pulmonary vascular endothelial cells after LPS treatment, which could be restored by RUS. Collectively, these findings provide pharmacological evidence showing that RUS attenuates LPS-induced pulmonary endothelial barrier dysfunction by inhibiting TLR4/Src/VE-cadherin pathway through targeting NMMHC IIA and mediating NMMHC IIA‒TLR4 interactions.

肺内皮屏障功能障碍是临床肺水肿的标志，并有助于急性肺损伤 (ALI) 的发展。在这里，我们报道了麦冬的有效甾体皂苷元 ruscogenin (RUS) 通过介导非肌肉肌球蛋白重链 IIA (NMMHC IIA)-Toll 样受体 4 (TLR4) 减弱脂多糖 (LPS) 诱导的肺内皮屏障破坏。 ) 互动。通过体内和体外实验中，我们观察到 RUS 给药显着改善了 LPS 引发的肺内皮屏障功能障碍和 ALI。此外，我们通过串行亲和层析、分子对接、生物层干涉测量和微尺度热泳分析确定了 RUS 直接靶向 NMMHC IIA 的 N 末端和头部结构域。体内和体外内皮 NMMHC IIA 表达的下调取消了 RUS 的保护作用。还观察到 NMMHC IIA 与 TLR4 解离，然后在 LPS 处理后激活肺血管内皮细胞中的 TLR4 下游 Src/血管内皮钙粘蛋白 (VE-cadherin) 信号传导，这可以通过 RUS 恢复。总的来说，这些发现提供了药理学证据，表明 RUS 通过靶向 NMMHC IIA 和介导 NMMHC IIA-TLR4 相互作用来抑制 TLR4/Src/VE-钙粘蛋白通路，从而减轻 LPS 诱导的肺内皮屏障功能障碍。