From the Department of Neurosurgery, Mie University Graduate School of Medicine, Tsu, Japan.
Stroke. 2018 Nov;49(11):2743-2751. doi: 10.1161/STROKEAHA.118.021757.
Background and Purpose- Plasma levels of galectin-3-a matricellular protein-are increased after aneurysmal subarachnoid hemorrhage (SAH), but the functional significance remains undetermined. This study was conducted to evaluate whether modified citrus pectin (MCP; galectin-3 inhibitor) prevents post-SAH early brain injury, focusing on blood-brain barrier disruption. Methods- C57BL/6 male adult mice (n=251) underwent sham or filament perforation SAH modeling, followed by a random intracerebroventricular injection of vehicle or drug at 30 minutes post-modeling. First, vehicle-treated and 0.8, 4, 16, or 32 µg MCP-treated mice were assessed by neuroscore and brain water content at 24 and 48 hours post-modeling. Second, Evans blue extravasation, Western blotting, coimmunoprecipitation and immunostaining were performed in vehicle-treated or 4 µg MCP-treated mice at 24 hours post-modeling. Third, vehicle or R-galectin-3 (recombinant galectin-3) was administered to SAH mice simultaneously with vehicle or MCP, and neuroscore and Evans blue extravasation were evaluated at 24 hours post-modeling. Fourth, vehicle or R-galectin-3 was administered to MCP-treated SAH mice at 24 hours, and neuroscore and IgG immunostaining were evaluated at 48 hours post-SAH. Results- Among tested dosages, 4 µg MCP showed the best neuroprotective effects as to preventing neurological impairments and brain edema at 24 to 48 hours post-SAH. Four micrograms MCP attenuated post-SAH blood-brain barrier disruption and galectin-3 upregulation in brain capillary endothelial cells, associated with inactivation of ERK (extracellular signal-related kinase) 1/2, STAT (signal transducer and activator of transcription)-3, and MMP (matrix metalloproteinase)-9, and the consequent preservation of a tight junction protein ZO-1 (zonula occludens-1). Coimmunoprecipitation assay demonstrated physical interactions between galectin-3 and TLR (Toll-like receptor) 4. R-galectin-3 blocked the neuroprotective effects of MCP. Conclusions- MCP prevents post-SAH blood-brain barrier disruption possibly by inhibiting galectin-3, of which the mechanisms may include binding to TLR4 and activating ERK1/2, STAT-3, and MMP-9. This study suggests galectin-3 to be a novel therapeutic target against post-SAH early brain injury.
背景与目的- 血小板衍生生长因子-3-a（一种基质细胞蛋白）在蛛网膜下腔出血（SAH）后水平升高，但功能意义仍未确定。本研究旨在评估改良柑橘果胶（MCP；半乳糖凝集素-3 抑制剂）是否可预防 SAH 后早期脑损伤，重点关注血脑屏障破坏。方法- C57BL/6 成年雄性小鼠（n=251）行假手术或微栓子法 SAH 模型制作，造模后 30 分钟随机行侧脑室注射载体或药物。首先，神经评分和脑水含量检测行假手术组、0.8、4、16 或 32μg MCP 处理组于造模后 24 小时和 48 小时。其次，于造模后 24 小时行 Evans 蓝渗出、Western blot、免疫共沉淀和免疫组化检测假手术组或 4μg MCP 处理组。再次，于造模同时行重组半乳糖凝集素-3（R-galectin-3）和 MCP 或载体处理 SAH 小鼠，于造模后 24 小时行神经评分和 Evans 蓝渗出检测。最后，MCP 处理组于造模后 24 小时行 R-galectin-3 或载体处理，于 SAH 后 48 小时行神经评分和 IgG 免疫组化检测。结果- 测试剂量中，4μg MCP 在预防 SAH 后 24 至 48 小时神经损伤和脑水肿方面具有最佳的神经保护作用。4μg MCP 减轻了 SAH 后血脑屏障破坏和脑毛细血管内皮细胞中半乳糖凝集素-3 的上调，与 ERK1/2、STAT-3 和 MMP-9 的失活有关，以及紧密连接蛋白 ZO-1（闭锁小带蛋白-1）的保留。免疫共沉淀试验证实了半乳糖凝集素-3 和 TLR（Toll 样受体）4 之间的物理相互作用。R-galectin-3 阻断了 MCP 的神经保护作用。结论- MCP 可能通过抑制半乳糖凝集素-3 来预防 SAH 后血脑屏障破坏，其机制可能包括与 TLR4 结合并激活 ERK1/2、STAT-3 和 MMP-9。本研究表明半乳糖凝集素-3 是治疗 SAH 后早期脑损伤的一种新的治疗靶点。
