代谢型谷氨酸受体负向变构剂JNJ16259685对蛛网膜下腔出血大鼠神经元的保护作用

Protective effect of metabotropic glutamate receptor 1 negative allosteric modulator JNJ16259685 on neuron after subarachnoid hemorrhage in rats

目的探讨代谢型谷氨酸受体1(m GluR1)的负向变构剂JNJ16259685对蛛网膜下腔出血(SAH)后神经功能的保护作用及其机制。方法选取SPF级SD雄性大鼠90只,完全随机分为假手术组(18只)、SAH+安慰剂组(36只)、SAH+JNJ16259685组(36只),采用血管内穿刺法制作SAH模型。术后2、24、48 h,SAH+安慰剂组腹腔注射含5%二甲基亚砜(DMSO)的无菌水,SAH+JNJ组腹腔注射1 mg/kg JNJ16259685(溶解于5%DMSO的无菌水)。SAH后72 h,采用Garcia评分标准评估神经功能缺损,干湿重法检测脑水肿,伊文思蓝法评估血-脑屏障通透性,利用钙测定试剂盒检测线粒体钙离子浓度,免疫荧光观察神经元凋亡。采用Graph Pad 7. 0软件对3组间各指标进行单因素方差分析。结果与假手术组比较,SAH+安慰剂组的Garcia评分[(11. 0±0. 4)分]降低,左右脑半球脑组织水含量[分别为(80. 5±0. 1)%、(80. 3±0. 2)%]、伊文思蓝溢出量(2. 8±0. 2)、基底皮质线粒体钙离子浓度(2. 5±0. 3)、基底皮质和海马CA1区神经元凋亡[活性caspase-3/Neu N阳性细胞数分别为(300±30)个/mm^2、(20±2)个/mm]均增加(均P <0. 05);而SAH+JNJ组Garcia评分[(13. 0±0. 5)分]显著高于SAH+安慰剂组,左右脑半球脑组织水含量[分别为(79. 8±0. 2)%、(79. 3±0. 1)%]、伊文思蓝溢出量(1. 8±0. 2)、基底皮质线粒体钙离子浓度(1. 7±0. 1)、基底皮质和海马CA1区神经元凋亡数目[活性caspase-3/Neu N阳性细胞数分别为(180±10)个/mm^2、(12±2)个/mm]均较SAH+安慰剂组减少(均P <0. 05)。结论 SAH后,JNJ16259685减轻脑水肿及降低血-脑屏障通透性,抑制皮质线粒体钙离子浓度增加,减少神经元凋亡,从而发挥神经保护作用。

Objective To investigate the protective effect and its mechanism of metabotropic glutamate receptor 1（ m GluR1） negative allosteric modulator JNJ16259685 on neuron after subarachnoid hemorrhage（ SAH） in rats. Methods Ninety SPF-grade SD male rats were selected. They were randomly divided into3 groups： sham operation group（ n = 18）,SAH ＋ placebo group（ n = 36）,and SAH ＋ JNJ16259685（ JNJ）group（ n = 36）. A SAH model was induced by intravascular puncture. SAH ＋ placebo group received intraperitoneal injection of aseptic water containing 5 % dimethyl sulfoxide（ DMSO） at 2,24 and 48 h after operation. The SAH ＋ JNJ group was intraperitoneally injected with 1 mg/kg JNJ16259685（ dissolved in sterile water in 5% DMSO）. Garcia scoring criteria were used to assess neurological deficits at 72 h after SAH. Dry and wet weight method was used to detect brain edema. Evans Blue method was used to assess blood-brain barrier permeability. A calcium assay kit was used to detect the mitochondrial calcium ion concentration. Immunofluorescence staining was used to observe neuronal apoptosis. Graph Pad 7. 0 software was used to conduct one-way analysis of variance in all indicators among the 3 groups. Results Compared with the sham operation group,the Garcia score（ 11. 0 ± 0. 4） decreased in the SAH ＋ placebo group. The water content in left and right hemispheres was 80. 5 ± 0. 1% and 80. 3 ± 0. 2% respectively,the Evans blue dye extravasation（ 2. 8 ± 0. 2）,basal cortical mitochondrial calcium ion concentration（ 2. 5 ± 0. 3）,and neuronal apoptosis in basal cortex and hippocampus CA1 region（ the number of active caspase-3/Neu N positive cells was 300 ± 30/mm^2 and 20 ± 2/mm respectively） increased（ all P〈0. 05）; and the Garcia score（ 13. 0 ±0. 5） was significantly higher in the SAH ＋ JNJ group than in the SAH ＋ placebo group. Water content in left and right hemispheres was 79. 8 ± 0. 2% and 79. 3 ± 0. 1% respectively,Evans blue dye extravasation（ 1. 8 ±0. 2）,basal cortex mitochondrial calcium ion concentration（ 1. 7 ±0. 1）,basal cortex and the number of neuronal apoptosis in hippocampal CA1 region（ the number of active caspase-3/Neu N positive cells were 180 ±10/mm^2,12 ±2/mm） reduced compared with the SAH ＋ placebo group（ all P 0. 05）. Conclusion After SAH,JNJ16259685 relieves cerebral edema and reduces blood-brain barrier permeability,inhibits the increase of cortical mitochondrial calcium ion concentration,and reduces neuronal apoptosis,thereby exerting neuroprotective effects.