BACKGROUND Out of the myriad of complications associated with septic shock, septic-associated encephalopathy (SAE) carries a significant risk of morbidity and mortality. Blood-brain-barrier (BBB) impairment, which subsequently leads to increased vascular permeability, has been associated with neuronal injury in sepsis. Thus, preventing BBB damage is an attractive therapeutic target. Mitochondrial dysfunction is an important contributor of sepsis-induced multi-organ system failure. More recently, mitochondrial dysfunction in endothelial cells has been implicated in mediating BBB failure in stroke, multiple sclerosis and in other neuroinflammatory disorders. Here, we focused on Drp1-mediated mitochondrial dysfunction in endothelial cells as a potential target to prevent BBB failure in sepsis. METHODS We used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in a cell culture as well as in murine model of sepsis. BBB disruption was assessed by measuring levels of key tight-junction proteins. Brain cytokines levels, oxidative stress markers, and activity of mitochondrial complexes were measured using biochemical assays. Astrocyte and microglial activation were measured using immunoblotting and qPCR. Transwell cultures of brain microvascular endothelial cells co-cultured with astrocytes were used to assess the effect of LPS on expression of tight-junction proteins, mitochondrial function, and permeability to fluorescein isothiocyanate (FITC) dextran. Finally, primary neuronal cultures exposed to LPS were assessed for mitochondrial dysfunction. RESULTS LPS induced a strong brain inflammatory response and oxidative stress in mice which was associated with increased Drp1 activation and mitochondrial localization. Particularly, Drp1-(Fission 1) Fis1-mediated oxidative stress also led to an increase in expression of vascular permeability regulators in the septic mice. Similarly, mitochondrial defects mediated via Drp1-Fis1 interaction in primary microvascular endothelial cells were associated with increased BBB permeability and loss of tight-junctions after acute LPS injury. P110, an inhibitor of Drp1-Fis1 interaction, abrogated these defects, thus indicating a critical role for this interaction in mediating sepsis-induced brain dysfunction. Finally, LPS mediated a direct toxic effect on primary cortical neurons, which was abolished by P110 treatment. CONCLUSIONS LPS-induced impairment of BBB appears to be dependent on Drp1-Fis1-mediated mitochondrial dysfunction. Inhibition of mitochondrial dysfunction with P110 may have potential therapeutic significance in septic encephalopathy.

中文翻译：

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通过动力蛋白相关蛋白 1 (Drp1) 介导的线粒体功能障碍在脓毒性脑病中传播血脑屏障损伤。

背景在与感染性休克相关的无数并发症中，感染性脑病（SAE）具有显着的发病率和死亡率风险。随后导致血管通透性增加的血脑屏障 (BBB) 损伤与脓毒症中的神经元损伤有关。因此，预防 BBB 损伤是一个有吸引力的治疗目标。线粒体功能障碍是脓毒症引起的多器官系统衰竭的重要原因。最近，内皮细胞中的线粒体功能障碍与介导中风、多发性硬化症和其他神经炎症性疾病中的 BBB 衰竭有关。在这里，我们专注于 Drp1 介导的内皮细胞线粒体功能障碍作为预防败血症中 BBB 衰竭的潜在靶点。方法 我们使用脂多糖 (LPS) 在细胞培养和败血症小鼠模型中诱导炎症和 BBB 破坏。通过测量关键紧密连接蛋白的水平来评估 BBB 破坏。使用生化测定法测量脑细胞因子水平、氧化应激标记和线粒体复合物的活性。使用免疫印迹和 qPCR 测量星形胶质细胞和小胶质细胞的活化。使用与星形胶质细胞共培养的脑微血管内皮细胞的 Transwell 培养物来评估 LPS 对紧密连接蛋白表达、线粒体功能和异硫氰酸荧光素 (FITC) 葡聚糖通透性的影响。最后，评估暴露于 LPS 的原代神经元培养物的线粒体功能障碍。结果 LPS 在小鼠中诱导强烈的脑炎症反应和氧化应激，这与增加的 Drp1 激活和线粒体定位有关。特别是，Drp1-(Fission 1) Fis1 介导的氧化应激也导致脓毒症小鼠血管通透性调节剂的表达增加。同样，在原代微血管内皮细胞中通过 Drp1-Fis1 相互作用介导的线粒体缺陷与急性 LPS 损伤后 BBB 通透性增加和紧密连接丧失有关。Drp1-Fis1 相互作用的抑制剂 P110 消除了这些缺陷，因此表明这种相互作用在介导脓毒症诱导的脑功能障碍中起关键作用。最后，LPS 介导了对初级皮层神经元的直接毒性作用，而 P110 治疗消除了这种作用。结论 LPS 诱导的 BBB 损伤似乎依赖于 Drp1-Fis1 介导的线粒体功能障碍。P110 抑制线粒体功能障碍可能对脓毒性脑病具有潜在的治疗意义。