To investigate whether therapeutic hypothermia augments the restorative impact of protein kinase C-β (PKC-β) and Nox2 inhibition on an in vitro model of human blood–brain barrier (BBB). Cells cultured in normoglycaemic (5.5 mM) or hyperglycaemic (25 mM, 6 to 120 h) conditions were treated with therapeutic hypothermia (35 °C) in the absence or presence of a PKC-β inhibitor (LY333531, 0.05 μM) or a Nox2 inhibitor (gp91ds-tat, 50 μM). BBB was established by co-culture of human brain microvascular endothelial cells (HBMECs) with astrocytes (HAs) and pericytes. BBB integrity and function were assessed via transendothelial electrical resistance (TEER) and paracellular flux of sodium fluorescein (NaF, 376 Da). Nox activity (lucigenin assay), superoxide anion production (cytochrome-C reduction assay), cellular proliferative capacity (wound scratch assay) and actin cytoskeletal formation (rhodamine-phalloidin staining) were assessed both in HBMECs and HAs using the specific methodologies indicated in brackets. Therapeutic hypothermia augmented the protective effects of PKC-β or Nox2 inhibition on BBB integrity and function in experimental setting of hyperglycaemia, as evidenced by increases in TEER and concomitant decreases in paracellular flux of NaF. The combinatory approaches were more effective in repairing physical damage exerted on HBMEC and HA monolayers by wound scratch and in decreasing Nox activity and superoxide anion production compared to sole treatment regimen with either agent. Similarly, the combinatory approaches were more effective in suppressing actin stress fibre formation and maintaining normal cytoskeletal structure. Therapeutic hypothermia augments the cerebral barrier-restorative capacity of agents specifically targeting PKC-β or Nox2 pathways.

目的 研究低温治疗是否增强蛋白激酶 C-β (PKC-β) 和 Nox2 抑制对人血脑屏障 (BBB) 体外模型的恢复影响。在正常血糖（5.5 mM）或高血糖（25 mM，6至120小时）条件下培养的细胞在不存在或存在PKC-β抑制剂（LY333531，0.05 μM）或Nox2的情况下用治疗性低温（35°C）进行处理抑制剂（gp91ds-tat，50 μM）。 BBB是通过人脑微血管内皮细胞（HBMEC）与星形胶质细胞（HA）和周细胞共培养建立的。通过跨内皮电阻 (TEER) 和荧光素钠 (NaF, 376 Da) 的细胞旁通量评估 BBB 完整性和功能。使用括号中所示的具体方法对 HBMEC 和 HA 中的 Nox 活性（光泽精测定）、超氧阴离子产生（细胞色素C还原测定）、细胞增殖能力（伤口划痕测定）和肌动蛋白细胞骨架形成（罗丹明-鬼笔环肽染色）进行评估。在高血糖实验环境中，治疗性低温增强了 PKC-β 或 Nox2 抑制对 BBB 完整性和功能的保护作用，TEER 增加和伴随的 NaF 细胞旁通量减少证明了这一点。与单独使用任一药物的治疗方案相比，组合方法在修复伤口划伤对 HBMEC 和 HA 单层造成的物理损伤以及降低 Nox 活性和超氧阴离子产生方面更有效。同样，组合方法在抑制肌动蛋白应力纤维形成和维持正常细胞骨架结构方面更有效。 低温治疗可增强专门针对 PKC-β 或 Nox2 通路的药物的脑屏障恢复能力。