大鼠脊髓损伤节段线粒体自噬蛋白 及凋亡因子的表达

The expression of mitophagy protein and apoptosis factor in spinal cord injury in rats

中文关键词:  脊髓损伤  线粒体自噬  凋亡

英文关键词:Spinal cord injury  Mitophagy  Apoptosis

中文摘要:

  【摘要】 目的：观察脊髓损伤节段线粒体自噬蛋白与凋亡蛋白的表达情况。方法：将50只清洁级SD雄性大鼠随机分为损伤组（39只）和对照组（11只），损伤组应用Allen′s法建立大鼠脊髓损伤模型，并分为5个不同时间点（损伤后1h、6h、24h、48h、72h），每个时间点7只，在损伤不同时间点处死大鼠，并截取损伤区域约1cm长脊髓节段，通过Western blot检测线粒体自噬蛋白（LC3、NIX）及凋亡蛋白（BNIP3、cleaved caspase-3、cytochrome c）表达水平；另取4只大鼠在损伤后24h截取损伤组织，用透射电镜观察损伤后神经元自噬体结构。对照组不做任何处理，其中7只大鼠用于Western blot分析，4只用于电镜检测。结果：对照组及损伤后1h、6h、24h、48h、72h脊髓损伤组大鼠LC3表达水平分别为0.2893±0.0325、0.3002±0.0474、0.3943±0.1154、0.4818±0.0426、0.5430±0.0865、0.5790±0.0892；NIX表达分别为0.1392±0.0171、0.1431±0.0325、0.1955±0.1379、0.3841±0.1136、0.4043±0.1059、0.4506±0.0174；BNIP3表达水平分别为0.1354±0.0547、0.1896±0.1264、0.2654±0.1341、0.7220±0.1030、0.4713±0.1041、0.3975±0.1505；cleaved caspase-3表达水平分别为0.2806±0.0999、0.4158±0.1137、0.7865±0.4056、0.9354±0.2659、1.0152±0.3441、1.1608±0.2488；细胞色素C表达水平分别为0.1489±0.0300、0.2196±0.0762、0.3162±0.1656、0.4456±0.1180、0.5407±0.1029、0.5812±0.1388。LC3、NIX、cleaved caspase-3和细胞色素C在24h、48h、72h与对照组比较明显增加（P<0.05）；BNIP3在24h、48h与对照组比较明显增加（P<0.05）。进一步通过透射电镜观察损伤后24h细胞超微结构，可观察到双层膜结构自噬体，其内包裹有受损线粒体等细胞器结构。结论：大鼠急性脊髓损伤后可能通过促进NIX蛋白与LC3结合诱导线粒体自噬，减少凋亡水平，从而在脊髓损伤修复中发挥保护作用。

英文摘要:

  【Abstract】 Objectives: To investigate the mitophagy protein and apoptosis after spinal cord injury in animal models by the Allen′s method. Methods: 50 SD male rats were randomly divided into injury group and control group. The injury group was divided into 5 different time points(1h, 6h, 24h, 48h, 72h), each group had 7 rats. At different time points after injury, the rats were sacrificed and selected the injured area about 1cm long. Then the expression levels of mitophagy(LC3, NIX) and apoptosis protein(BNIP3, cleaved caspase-3, cytochrome c) were tested by Western blot. Another 4 rats at 24h after injury were used to observe neuron autophagy by transmission electron microscopy. The control group accepted no treatment, in which 7 rats were used for Western blot analysis, 4 rats were used for electron microscopy. Results: The LC3 expression in control group and at 1h, 6h, 24h, 48h, 72h after injury was 0.2893±0.0325, 0.3002±0.0474, 0.3943±0.1154, 0.4818±0.0426, 0.5430±0.0865, 0.5790±0.0892, respectively. The NIX expression was 0.1392±0.0171, 0.1431±0.0325, 0.1955±0.1379, 0.3841±0.1136, 0.4043±0.1059, 0.4506±0.0174, respectively. The BNIP3 expression was 0.1354±0.0547, 0.1896±0.1264, 0.2654±0.1341, 0.7220±0.1030, 0.4713±0.1041, 0.3975±0.1505, respectively. The cleaved caspase-3 expression was 0.2806±0.0999, 0.4158±0.1137, 0.7865±0.4056, 0.9354±0.2659, 1.0152±0.3441, 1.1608±0.2488, respectively. The cytochrome c expression was 0.1489±0.0300, 0.2196±0.0762, 0.3162±0.1656, 0.4456±0.1180, 0.5407±0.1029, 0.5812±0.1388, respectively. The protein expressions of LC3, NIX, cleaved caspase-3 and cytochrome c in 24h, 48h, 72h model group significantly increased compared with those in control group（P<0.05）. The protein expression of BNIP3 in 24h, 48h model group significantly increased compared with that in control group（P<0.05）. The ultrastructure of cells was observed by transmission electron microscope after 24h. The double membrane structure of autophagy was observed covered with damaged mitochondria and other organelles. Conclusions: After acute spinal cord injury, rats may promote mitophagy and reduce apoptosis by promoting the binding of NIX protein to LC3 and thus play a protective role in the repair of spinal cord injury.