SP600125对蛛网膜下腔出血大鼠海马区神经细胞自噬及神经细胞丢失的影响

Effects of SP600125 on autophagy and neurocyte loss in the hippocampus of rats with subarachnoid hemorrhage

目的适度自噬有利于提高神经细胞的存活率;而自噬过度又会引起细胞的调亡。文中旨在探讨SP600125对蛛网膜下腔出血(SAH)大鼠海马区神经细胞自噬及神经细胞丢失的影响。方法将健康雄性清洁级SD大鼠40只按随机化数字表法分为假手术组、模型组、二甲基亚砜(DMSO组)、SP600125组,每组10只。模型组、DMSO组和SP600125组应用血管内穿刺法制成大鼠SAH模型。采用3-0缝合线引入到右颈外动脉,通过颈内动脉推进刺破大脑前动脉和大脑中动脉分叉处,建SAH模型。假手术组除不刺破血管,其余操作均与模型组一致。SP600125组于造模前30 min,利用脑立体定位仪行侧脑室注射3μg/μL SP600125溶液10μL。假手术组和模型组均注射等体积等渗盐水,DMSO组注射等量的DMSO。24 h处死,HE染色观察各组海马区神经元形态及数量;免疫组化染色及Western blot法对p-JNK蛋白以及自噬标志物Beclin-1和LC3-Ⅱ进行定性定量检测。结果 HE结果显示,与假手术组比较,模型组海马区神经元排列紊乱、细胞多呈多边形、数量明显减少;与模型组比较,SP600125组海马区神经元损伤程度有所减轻、细胞明显增多。与假手术组大鼠海马区Beclin-1、LC3-Ⅱ、p-JNK蛋白平均光密度值(1.56±0.28、1.60±0.30、1.58±0.32)比较,模型组(14.66±4.40、12.62±3.46、12.82±3.68)、DMSO组(13.85±3.85、11.59±4.52、13.03±3.53)和SP600125组(9.86±3.14、6.78±2.56、5.60±2.42)明显升高(P<0.05);SP600125组较模型组明显降低(P<0.05)。与假手术组大鼠海马区Beclin-1、LC3-Ⅱ、p-JNK蛋白表达(0.136±0.014、0.126±0.012、0.102±0.009)比较,模型组(0.474±0.122、0.668±0.130、0.496±0.124)、DMSO组(0.432±0.102、0.628±0.113、0.416±0.094)和SP600125组(0.264±0.106、0.332±0.113、0.219±0.104)明显升高(P<0.05);而SP600125组较模型组明显降低(P<0.05)。结论SP600125对SAH后神经元细胞具有保护作用,其机制可能与SP600125通过抑制JNK信号通路的活性使自噬适度激活有关。

Objective Moderate autophagy helps improve the viability of neurocytes. This study aims to investigate the effect of SP600125 on the autophagy and loss of nerve cells in the hippocampus in rats with subarachnoid hemorrhage（ SHA）. Methods Forty healthy male SD rats were equally randomized into a sham operation,an DMSO group,an SAH model,and an SP600125 group. The SAH model was established by vascular puncture and the rats of the SP600125 group were injected with 10 μL of SP600125（ 3 μg/μL）into the lateral cerebral ventricle at 30 minutes before modeling. Sham group and SAH group were injected with equal volume of normal saline,DMSO group was injected with the same amount of DMSO. The animals were sacrificed at 24 hours after modeling for observation of the changes in the morphology and the number of neurons in the hippocampus by HE staining and qualitative and quantitative determination of the expressions of the p-JNK protein and the autophagy markers beclin-1 and LC3-II by immunohistochemistry and Western blot. Results Compared with the sham operation group,the neurons exhibited a disordered arrangement and the cells were polygonal and decreased in number in the hippocampus of the SAH models,while milder neuronal injury and more cells were observed in the rats of the SP600125 group than in the SAH models. The mean optical density values of Beclin-1,LC3-II and p-JNK in the hippocampus were significantly higher in the SAH models（ 14.66±4.40,12.62±3.46,and 12.82±3.68） and DMSO（ 13.85± 3.85、11.59± 4.52、13.03 ± 3. 53）,and the SP600125 group（ 9. 86 ± 3. 14,6. 78 ±2.56,and 5.60±2.42） than in the sham operation group（ 1.56±0.28,1.60±0.30,and 1.58±0.32）（ P〈0.05）,but markedly lower in the SP600125 than in the SAH model group（ P〈0.05）. The expressions of Beclin-1,LC3-II and p-JNK were remarkably increased in the SAH models（ 0.474±0.122,0.668±0.130,and 0.496± 0.124） and DMSO（ 0.432± 0.102、0.628± 0.113、0.416± 0. 094） and the SP600125 group（ 0.264±0.106,0.332±0.113,and 0.219± 0.104） than in the sham operation group（ 1. 56 ± 0. 28,1. 60 ± 0. 30,and1.58±0. 32）（ P 〈 0. 05）,but significantly decreased in the SP600125 group as compared with the SAH models（ P 〈 0. 05）.Conclusion SP600125 has a protective effect on the neurocytes in the hippocampus of SAH rats,which may be associated with SP600125 moderately activating neuronal autophagy by inhibiting the activity of the JNK signaling pathway.