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

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。

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.