细胞自噬缓解氧-糖剥夺诱导原代培养小鼠脑皮质神经元缺血损伤

Autophagy decreases OGD-induced ischemic injury of mouse primary cultured cortical neurons

目的在离体细胞水平,探讨氧-糖剥夺(OGD)致原代培养小鼠脑皮质神经元缺血损伤过程中,细胞自噬的变化情况和可能作用。方法出生24 h内C57BL/6J乳鼠脑皮质神经元原代培养7 d后,分为常氧(Nor.)、15、30、60、90和120 min OGD后24 h复糖复氧等6组,每组n=6;免疫印迹(Western blot)法检测细胞自噬相关蛋白Beclin-1表达和LC3Ⅱ/Ⅰ比值;借助MTT比色法和乳酸脱氢酶(LDH)漏出率,检测神经元的损伤;用细胞自噬下游抑制剂巴弗洛霉素A1(Baf A1100 nmol/L)抑制自噬后,观察神经元的损伤。结果与对照组相比,随OGD时间的延长,自噬相关蛋白Beclin-1表达水平和LC3Ⅱ/Ⅰ比值显著升高,30 min达到峰值,之后略有回降(P<0.05);在OGD处理30和60 min致使神经元显著损伤的基础上,细胞自噬抑制剂Baf A1可明显加重60 min OGD处理神经元的损伤和提高LC3Ⅱ/Ⅰ的比值(P<0.05)。结论 OGD可诱发原代培养小鼠脑皮质神经元自噬发生,且细胞自噬可缓解OGD处理皮质神经元的缺血损伤。

Objective To explore the dynamic changes of autophagy and its role in oxygen-glucose deprivation （OGD） -induced ischemie injury in primary cultured cortical neurons of mice in vitro. Methods The primary cortical neurons that obtained from postnatal 24 h C57BL/6J mice were cultured for 7 days, and then divided into six groups including normoxia （ Nor. ） , 15, 30, 60, 90 and 120 min OGD/24 h reoxygenation. The effect of OGD treatment on autophagy-related protein level of Beclin-1 and LC3 Ⅱ/Ⅰ ration was determined by Western blot. The colorimetric method of thiazolyl blue tetrazolium bromide （MTT） and the examination of lactate dehydrogenase （LDH） leakage were applied to assess OGD-induced ischemic injury. The autophagy inhibitor Bafilomycin A1 （ BafA1 , 100 nmol/L） was used to explore the effect of autophagy on OGD-induced ischemic injury of cortical neurons. Results Compared with normoxic group, the levels of autophagy-related protein Beclin-1 and LC3 Ⅱ/Ⅰratio significantly increased with the exposure time of OGD treatment, and the increase of Beclin-1 and LC3 Ⅱ/Ⅰ ratio reached their peaks at 30 min of OGD treatment （P 〈 0. 05 ）. Both of 30 and 60 min OGD treatment significantly affected the viability and mortality of cortical neurons （ P 〈 0. 05 ）. In addition, the autophagy inhibitor BafA1 could aggravate 60 min OGD-induced ischemic injury and enhance the LC3 Ⅱ/Ⅰ ratio （P 〈 0.05 ）. Conclusions These results demonstrated that OGD treatment may induce autophagy in primary cultured cortical neurons, and autophagy may protect cortical neurons against OGD-induced ischemic injury.