酮体代谢对大鼠急性脊髓损伤神经保护作用的实验研究

Effects of ketobodies metabolism on neuroprotection after acute spinal cord injury in rats

目的已有研究证实脊髓损伤后的生酮饮食(ketogenic diet,KD)具有神经保护作用,本文进一步探讨损伤前酮体代谢对于大鼠急性颈脊髓挫伤的氧化应激和细胞凋亡的抑制作用。方法 48只雄性Sprague-Dawley大鼠随机分为KD组和标准饮食组(SD),喂养2周后检测血酮体含量。在电磁伺服材料试验机上行C5脊髓挫伤,其挫伤位移为1.5 mm,挫伤速度达500 mm/s。挫伤后3 h和24 h采集颈脊髓组织。采用免疫荧光双标检测与凋亡和炎症相关的Bax,caspase-3和Bcl-2,CD68等的蛋白表达,ELASA检测挫伤部位反映氧化应激程度的SOD和MDA含量。结果 KD组酮体水平为1.32 mmol/L,明显高于SD组(0.38 mmol/L)(P<0.05)。Bax,Bcl-2,caspase-3以及CD68的蛋白表达在损伤后均呈阳性,其中KD组表达量在两个时间点上均与SD组的差异有显著性(P<0.05)。KD组SOD值在损伤后3h高于SD组,且两组间差异在伤后24 h有显著性(540 pg/ml与281 pg/ml)(P<0.05)。MDA的变化趋势与SOD相反,伤后24 h KD组MDA值为509 pg/ml,明显低于SD组(855 pg/ml)(P<0.05)。结论本研究结果证明SCI前的高酮体水平减少了急性SCI的氧化应激程度、抑制细胞凋亡,为其在SCI神经保护作用提供了实验证据。

Objective Previous studies supported neuroprotective functionof ketogenic diet（KD） after spinal cord injury. The present study was aimed to investigate effects of pre- existing ketobodies metabolism on antioxidant and anti-apoptotic after acute spinal cord in rats. Methods Forty-eight male Sprague-Dawley rats were randomly assigned into 2 groups fed either with KD or standard diet（SD） for up to 2 weeks. Levels of blood ketones was measured for each rat at 2 weeks. All rats were subjected to a 1.5 mm contusion injury at500 mm/s on the C5 spinal cord using an electromagnetic- servo material testing machine. Samples of spinal cord tissues were harvested at 3 h and 24 h after injury. Protein expression of Bax, caspase-3, Bcl-2 and CD68,indicating apoptosis and inflammation, were tested using double immunofluorescence. Contents of superoxide dismutase（SOD） and malondialdehyde（MDA）, reflecting oxidative stress, were measured using ELISA.Results The blood ketone level in the KD group was 1.32 mmol/L, which was significantly higher than that in the SD group（0.38 mmol/L）（P0.05）. Expression of Bax, Bcl-2,caspase-3 and CD68 were positive after injury. A significant difference in expression between groups was seen at 3 h and 24 h after injury. The content of SOD in the KD group was higher than that in the SD group at 3 h after injury, and there was a significant difference between groups at 24 h（540 pg/m L vs 281 pg/ml）（P0.05）. Change of levels of MDA was opposite to the change of SOD. The level of MDA in the KD group was 509 pg/ml, which was significantly lower than the level in the SD group（855 pg/ml）（P0.05）. Conclusion The present results indicated that a pre-existing high level of blood ketones can reduce oxidative stress and apoptosis in the spinal cord after acute contusion injury, suggesting neuroprotective function for acute spinal cord injury under ketobodies metabolism.