1Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China. 2Department of Physiology and Pharmacology, Loma Linda University, Loma Linda, CA.
Crit Care Med. 2013 Dec;41(12):e466-74. doi: 10.1097/CCM.0b013e31829a8246.
Brilliant blue G, a selective P2X7 receptor antagonist, exhibits neuroprotective properties. This study examined whether brilliant blue G treatment ameliorates early brain injury after experimental subarachnoid hemorrhage, specifically via inhibiting p38 mitogen-activated protein kinase-related proapoptotic pathways.
Controlled in vivo laboratory study.
Animal research laboratory.
One hundred fifty-four adult male Sprague-Dawley rats weighing 280-320 g.
Subarachnoid hemorrhage was induced in rats by endovascular perforation. Experiment 1 implemented sham-operated rats (sham) and subarachnoid hemorrhage animals, which received vehicle (subarachnoid hemorrhage + vehicle), brilliant blue G (subarachnoid hemorrhage + brilliant blue G), or brilliant blue G plus 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) (subarachnoid hemorrhage + brilliant blue G + BzATP). The animals were intraperitoneally treated with brilliant blue G (30 mg/kg) at 30 minutes after subarachnoid hemorrhage. BzATP (50 μg/rat), a P2X7 receptor agonist, was intracerebroventricularly administered. Experiment 2 implemented sham-operated rats (sham) and subarachnoid hemorrhage animals, which received vehicle (subarachnoid hemorrhage + vehicle), scramble small interfering RNA (subarachnoid hemorrhage + scramble small interfering RNA), or P2X7 receptor small interfering RNA (subarachnoid hemorrhage + P2X7 receptor small interfering RNA). Subarachnoid hemorrhage grading, neurobehavioral score, and brain edema were evaluated at 24 and 72 hours after surgery. The expression of phosphorylated p38 mitogen-activated protein kinase, phosphorylated extracellular signal-regulated kinases, phosphorylated c-Jun N-terminal kinases, P2X7 receptor, Bcl-2, and cleaved caspase-3 in the left cerebral hemisphere were determined by Western blot. Neuronal apoptosis was examined by double immunofluorescence staining using P2X7 receptor, terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling, and neuronal nuclei.
Brilliant blue G significantly improved neurobehavioral function and ameliorated brain water content at 24 and 72 hours after subarachnoid hemorrhage. BzATP reversed these treatment effects. Brilliant blue G attenuated neuronal apoptosis in the subcortex, which was associated with decreased expression of phosphorylated p38 mitogen-activated protein kinase and cleaved caspase-3 and an increased expression of Bcl-2 in the left cerebral hemisphere. The beneficial effects of P2X7 receptor small interfering RNA were also mediated by a p38 mitogen-activated protein kinase pathway.
Inhibition of P2X7 receptor by brilliant blue G or P2X7 receptor small interfering RNA can prevent early brain injury via p38 mitogen-activated protein kinase after subarachnoid hemorrhage.
亮蓝 G 是一种选择性 P2X7 受体拮抗剂，具有神经保护作用。本研究探讨了亮蓝 G 是否通过抑制 p38 丝裂原活化蛋白激酶相关促凋亡途径来改善实验性蛛网膜下腔出血后的早期脑损伤。
体内对照实验研究。
动物研究实验室。
154 只成年雄性 Sprague-Dawley 大鼠，体重 280-320g。
通过血管内穿孔诱导蛛网膜下腔出血。实验 1 实施假手术大鼠（假手术）和蛛网膜下腔出血动物，接受载体（蛛网膜下腔出血+载体）、亮蓝 G（蛛网膜下腔出血+亮蓝 G）或亮蓝 G+2'（3'）-O-（4-苯甲酰苯甲酰）腺苷 5'-三磷酸（BzATP）（蛛网膜下腔出血+亮蓝 G+BzATP）。蛛网膜下腔出血后 30 分钟，动物腹腔内给予亮蓝 G（30mg/kg）。BzATP（50μg/大鼠），一种 P2X7 受体激动剂，经侧脑室给药。实验 2 实施假手术大鼠（假手术）和蛛网膜下腔出血动物，接受载体（蛛网膜下腔出血+载体）、 scramble 小干扰 RNA（蛛网膜下腔出血+ scramble 小干扰 RNA）或 P2X7 受体小干扰 RNA（蛛网膜下腔出血+ P2X7 受体小干扰 RNA）。术后 24 小时和 72 小时评估蛛网膜下腔出血分级、神经行为评分和脑水肿。通过 Western blot 测定左大脑半球中磷酸化 p38 丝裂原活化蛋白激酶、磷酸化细胞外信号调节激酶、磷酸化 c-Jun N 末端激酶、P2X7 受体、Bcl-2 和 cleaved caspase-3 的表达。通过 P2X7 受体、末端脱氧核苷酸转移酶介导的尿嘧啶 5'-三磷酸生物素 nick 末端标记和神经元核的双重免疫荧光染色检测神经元凋亡。
亮蓝 G 可显著改善蛛网膜下腔出血后 24 小时和 72 小时的神经功能，并减轻脑水含量。BzATP 逆转了这些治疗效果。亮蓝 G 减轻了皮质下的神经元凋亡，这与左大脑半球中磷酸化 p38 丝裂原活化蛋白激酶和 cleaved caspase-3 的表达减少以及 Bcl-2 的表达增加有关。P2X7 受体小干扰 RNA 的有益作用也通过 p38 丝裂原活化蛋白激酶途径介导。
亮蓝 G 或 P2X7 受体小干扰 RNA 抑制 P2X7 受体可通过蛛网膜下腔出血后的 p38 丝裂原活化蛋白激酶预防早期脑损伤。
