线粒体分裂抑制剂-1在大鼠急性脊髓损伤中的保护作用及其机制

发布时间：2018-08-22 19:02

【摘要】：目的旨在检测选择性线粒体分裂抑制剂-1 (Mdivi-1)对大鼠急性脊髓损伤(acute spinal cord injury, ASCI)后线粒体损伤和细胞凋亡的影响,为ASCl的继发性损伤机制以及临床靶点药物治疗的研究提供重要线索。方法SD大鼠随机分为四组：假手术组(Sham组),Sham+Mdivi-1预处理组(1.20mg/kg, Sham+ Mdivi-1组),单纯脊髓损伤组(SCI组)和SCI+Mdivi-1预处理组(1.20 mg/kg, SCI+Mdivi-1组)。SCI组和SCl+Mdivi-1组的大鼠采用Allen's方法制备ASCl模型,Sham+Mdivi-1组和SCI+Mdivi-1组大鼠在脊髓打击之前15 min经尾静脉给予Mdivi-1。 SCI组大鼠分别于术后2、4,8,16和24小时(h)五个时间点处死。Sham组,Sham+Mdivi-1组和SCl+Mdivi-1组的大鼠于脊髓暴露或者损伤16h后处死。首先,采用western blotting检测脊髓损伤后大鼠各时间点线粒体内细胞色素C (Cytochrome C, Cyt C)的释放情况,然后选择ASCl或者暴露后16 h作为检测Mdivi-1作用效果的时间点,采用透射电镜技术检测各组大鼠线粒体形态变化,采用JC-I标记法检测各组大鼠脊髓组织中线粒体膜电位(mitochondrial membrane potentials, MMP)变化,采用可见分光光度计检测各组大鼠脊髓组织中线粒体内三磷酸腺苷(Adenosine triphosphate, ATP),丙二醛(Malondialdehyde, MDA)和还原型谷胱甘肽(Reduced Glutathione, GSH)的变化,采用Western Blot检测各组大鼠脊髓组织线粒体上动力蛋白1(Dynamin-related protein 1, Drp1), Cyt C, Bcl-2相关X蛋白(Bcl-2-associatedX protein, Bax)和B细胞淋巴瘤基因-2(B-cell lymphoma 2, Bcl-2)以及细胞内Caspase-3表达的影响,采用免疫荧光标记技术检测大鼠脊髓组织内caspase-3的表达水平,采用荧光TUNEL法检测各组大鼠脊髓组织中细胞凋亡情况。结果1、Western Blot检测结果显示,与Sham组比较,SCI组中线粒体内Cyt C从4 h开始明显降低,16 h达到最低值,然后逐渐上升至24 h(P0.01)；而细胞浆中的Cyt C从4h开始升高,16 h达高峰,然后逐渐下降(P0.01)。ASCI后16 h线粒体外膜上Drp1, Bax和Bcl-2较Sham组明显升高(P0.01),细胞内Caspase-3表达较Sham组明显增多(P0.01)。与SCI组16 h相比,SCI+Mdivi-1组线粒体内Cyt C表达增高(P0.01),而细胞质内Cyt C表达降低(P0.01)；线粒体外膜上Drp1和Bax表达明显减少(P0.01),细胞内Caspase-3表达明显减少(P0.01),而线粒体外膜上Bcl-2无明显变化；Sham组和Sham+Mdivi-1组之间以上指标的表达无明显变化。2、透射电镜检测结果显示,与Sham组比较,ASCI后16 h线粒体数目明显增多,而截面积明显减小(P0.01)；与SCI组16 h比较,SCI+Mdivi-1组线粒体数目明显减少,而截面积明显增大(P0.01)；但是,Sham组和Sham+Mdivi-1组之间以上指标的表达无明显变化。3、JC-1标记法检测结果显示,ASCl后16 h MMP较Sham组明显降低(P0.01)；与SCI组16 h相比,SCl+Mdivi-1组MMP明显增高(P0.01)；但是,Sham组和Sham+Mdivi-1组之间MMP无明显变化。4、可见分光光度计检测结果显示,ASCl后16 h线粒体内ATP和GSH水平较Sham组明显降低(P＜0.01), MAD水平明显升高(P0.01)；与SCI组16 h相比,SCl+Mdivi-1组线粒体内ATP和GSH水平明显升高(P＜0.01), MAD水平明显降低(P0.01)；但是,Sham组和Sham+Mdivi-1组之间线粒体内ATP, GSH和MAD的水平无明显变化。5、免疫荧光技术结果显示：ASCl后16 h Caspase-3阳性细胞百分数明显增多(P0.01)；与SCI组16 h相比,SCl+Mdivi-1组Caspase-3阳性细胞百分数明显减少(P0.01)。但是,Sham组和Sham+Mdivi-1组之间细胞内Caspase-3阳性细胞百分数无明显变化。6、荧光TUNEL法检测结果显示,ASCI后16 h细胞凋亡百分数较Sham组明显增加(P0.01)；与SCI组16 h相比,SCl+Mdivi-1组细胞凋亡百分数明显降低(P0.01)。但是,Sham组和Sham+Mdivi-1组之间细胞凋亡百分比无明显变化。结论Mdivi-1明显保护了ASCI后MMP和ATP水平,抑制了线粒体氧化损伤和细胞凋亡线粒体途径的激活。
[Abstract]:Objective To investigate the effects of selective mitochondrial splitting inhibitor-1 (Mdivi-1) on mitochondrial damage and apoptosis in rats with acute spinal cord injury (ASCI), and to provide important clues for the study of secondary injury mechanism and clinical target drug therapy of ASCl. Methods SD rats were randomly divided into four groups: sham operation. Group A (Sham group), Sham+Mdivi-1 preconditioning group (1.20 mg/kg, Sham+Mdivi-1 group), SCI+Mdivi-1 preconditioning group (SCI group) and SCI+Mdivi-1 preconditioning group (1.20 mg/kg, SCI+Mdivi-1 group). Rats in SCI and SCl+Mdivi-1 groups were given ASCl model by Allen's method, and rats in Sham+Mdivi-1 and SCI+Mdivi-1 groups were given via caudal vein 15 minutes before spinal cord injury. Rats in the Mdivi-1 SCI group were sacrificed at five time points, 2,4,8,16 and 24 hours (h), respectively. Rats in the Sham group, Sham+Mdivi-1 group and SCl+Mdivi-1 group were sacrificed 16 hours after spinal cord exposure or injury. First, the release of cytochrome C (Cytochrome C, Cyt C) in mitochondria was detected by Western blotting at each time point after spinal cord injury. Then, ASCl or 16 hours after exposure were selected as the time point to detect the effect of Mdivi-1. The morphological changes of mitochondria were detected by transmission electron microscopy (TEM), the changes of mitochondrial membrane potentials (MMP) in spinal cord tissue were detected by JC-I labeling method, and the changes were detected by visible spectrophotometer. Mitochondrial adenosine triphosphate (ATP), malondialdehyde (MDA) and reduced glutathione (GSH) levels in the spinal cord of rats in each group were measured by Western Blot. Dynamin-related protein 1 (Drp1), Cyt C and Bcl-2 phases in the spinal cord of rats in each group were detected by Western Blot. To investigate the effects of Bcl-2-associated X protein (Bax) and B-cell lymphoma gene-2 (Bcl-2) and intracellular Caspase-3 expression, immunofluorescence labeling technique was used to detect the expression of Caspase-3 in rat spinal cord tissues. Fluorescence TUNEL method was used to detect the apoptosis of spinal cord tissues. The results of stern Blot assay showed that compared with Sham group, Cyt C in mitochondria of SCI group decreased significantly from 4 h to 16 h, reached the lowest value, then gradually increased to 24 h (P 0.01); Cyt C in cytoplasm increased from 4 h to 16 h, reached the peak, and then decreased gradually (P 0.01). Drp 1, Bax and Bcl-2 in mitochondria extracellular membrane increased significantly 16 h after ASCI compared with Sham group. Compared with the SCI group, the expression of Cyt C in mitochondria of SCI+Mdivi-1 group was higher (P 0.01), but the expression of Cyt C in cytoplasm was lower (P 0.01), the expression of Drp 1 and Bax in mitochondria outer membrane was significantly lower (P 0.01), and the expression of Caspase-3 in cells was significantly lower (P 0.01). The results of transmission electron microscopy showed that the number of mitochondria and the cross-sectional area of mitochondria increased significantly at 16 h after ASCI compared with Sham group (P 0.01); the number of mitochondria decreased significantly and the cross-sectional area increased significantly at 16 h after ASCI compared with SCI group (P 0.01). However, there was no significant change in the expression of the above indexes between Sham group and Sham+Mdivi-1 group. The results showed that the levels of ATP and GSH in mitochondria were significantly lower at 16 h after ASCl than those in Sham group (P There was no significant change in the levels of GSH and MAD. Immunofluorescence assay showed that the percentage of Caspase-3 positive cells increased significantly at 16 h after ASCl (P 0.01); the percentage of Caspase-3 positive cells in SCl+Mdivi-1 group was significantly lower than that in SCI group at 16 h (P 0.01). The percentage of apoptosis in SCl+Mdivi-1 group was significantly lower than that in SCI group (P 0.01). However, there was no significant change in the percentage of apoptosis between Sham group and Sham+Mdivi-1 group. The level of MP and ATP inhibited mitochondrial oxidative damage and activation of mitochondrial pathway.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

【相似文献】