自噬对氧化应激引起的脊髓神经元细胞损伤的作用及其机制

The Effect of Autophagy on the Injury of Primary Spinal Cord Neurons Induced by the Oxidative Stress

氧化应激是脊髓损伤(spinal cord injury,SCI)后脊髓神经元细胞继发性损伤的重要机制,但是如何缓解氧化应激目前仍然不明确。该文培养SD(Sprague Dawley)大鼠原代脊髓神经元细胞,使用不同浓度的H-2O_2作用于神经元细胞12 h后,Western blot检测LC3-II、Beclin-1和P62蛋白质水平变化,分析自噬水平,电镜和绿色荧光蛋白标的记微管相关蛋白轻链3(green fluorescent proteinmicrotubule-associated protein 1 light chain 3,GFP-LC3)转染观察自噬的数量。ATG5 si RNA转染抑制自噬和雷帕霉素(rapamycin)促进细胞自噬,并使用CCK-8和TUNEL(terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling)染色分析自噬对氧化应激下神经元细胞的作用。结果显示,随着H-2O_2浓度增加,LC3-II和Beclin-1蛋白质水平显著升高,而P62蛋白质水平显著下降(P<0.05)。透射电镜和共聚焦观察发现,10、50μmol/L H-2O_2可以增加神经元细胞中自噬体的数量。与对照组比较,H-2O_2明显抑制神经元细胞的活力(P<0.05),ATG5 si RNA抑制自噬水平后,H-2O_2作用下的细胞活力进一步下降,而雷帕霉素促进自噬后却可以提高细胞的活力(P<0.05)。TUNEL和膜联蛋白V/PI(annexin V/PI)染色结果发现,雷帕霉素可以抑制H-2O_2引起的神经元细胞凋亡(P<0.05)。该研究结果提示,脊髓神经元细胞处于氧化应激状态下时,自噬代偿性激活并保护神经元细胞。

Oxidative stress is the important secondary damage to spinal cord neurons after spinal cord injury （SCI）. However, how to alleviate the damage remains unclear. In the present study, primary spinal cord neurons of Sprague Dawley （SD） rats were cultured and treated with different concentrations of H202 for 12 h. The protein levels of LC3-II, Beclin-1 and P62 determined by Western blot were used to reflect the level of autophagy in the neurons. Transmission electron microscopy （TEM） and GFP-LC3 transfection were used to analyze the number of autophagosomes. The effect of autophagy on cellular viability and apoptosis was detected by CCK-8 assay and TUNEL, after the inhibition and activation of autophagy by ATG5 siRNA and rapamycin treatment. An dose- dependent increase in the levels of LC3-II, Beclin-1 and P62 was observed in the H202-treated neurons （P〈0.05）.Autophagosome number was increased by 10 and 50 μmol/L H2O2 detected by TEM and confocal microscopy. Compared with the control group, H2O2 inhibited cellular viability significantly （P〈0.05）, however the inhibition was exacerbated by the ATG5 siRNA and attenuated by the rapamycin （P〈0.05）. In addition, rapamycin could inhibited the apoptosis induced by the H202 （P〈0.05）. These results indicated that autophagy was activated in spinal cord neurons adaptively, and autophagy protected neurons from apoptosis induced by oxidative stress.