Zhang Lei, Li Yang, Hu Yuqi, Xu Hui, Zhu Chao, Wang Xu, Zhao Dong, He Xin
Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832000; Department of Neurosurgery Xinjiang Production and Construction Corps Tenth Division Beitun Hospital, Beitun 836000, China.
Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832000, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2022 Jul;38(7):590-597.
Objective To observe the effect of autophagy on apoptosis of cerebral cortical neurons after oxygen-glucose deprivation and reoxygenation (OGD/R). Methods Eighteen newborn SD rats born within 24 hours were selected. The cortical neurons were extracted by mechanical trypsin digestion and cultured for 7 days. The OGD/R model was established. The rats were randomly divided into 3 groups: control group, OGD/R group, and OGD/R combined with Bafilomycin A1(BafA1) group. Cell viability was detected by CCK-8 assay and lactate dehydrogenase (LDH) assay, and apoptosis was detected by flow cytometry combined with fluorescein isothiocyanate labeled annexin V/propidium iodide (annexin V-FITC/PI) double labeling. The mRNA expressions of microtubule associated protein 1 light chain 3 (LC3), P62 and cystatin 3 (caspase-3) were detected by reverse transcription PCR, and the protein expressions of LC3, P62 and caspase-3 were detected by Western blot analysis. The autophagy fluorescence intensity of neurons infected with red fluorescent protein-green fluorescent protein-LC3 (RFP-GFP-LC3) adenovirus was measured, and the ultrastructural changes of autophagosomes were observed by transmission electron microscopy. Results Compared with the control group, the activity of nerve cells in OGD/R group decreased significantly while the release level of LDH increased significantly and the apoptosis increased significantly; It was also detected the increased mRNA and protein expressions of LC3, P62 and caspase-3, increased fluorescence intensity of RFP, increased autophagosomes and more gathered autophagy lysosomes. The fluorescence intensity of RFP decreased significantly after adding late autophagy inhibitor BafA1. Conclusion OGD/R induces autophagy and apoptosis of neurons, and aggravates neuronal injury and apoptosis after inhibition of autophagy.
目的 观察自噬对氧糖剥夺复氧（OGD/R）后大脑皮质神经元凋亡的影响。方法 选取出生24小时内的新生SD大鼠18只。采用机械胰酶消化法提取皮质神经元并培养7天。建立OGD/R模型。将大鼠随机分为3组：对照组、OGD/R组、OGD/R联合巴弗洛霉素A1（BafA1）组。采用CCK-8法和乳酸脱氢酶（LDH）法检测细胞活力，采用流式细胞术结合异硫氰酸荧光素标记的膜联蛋白V/碘化丙啶（annexin V-FITC/PI）双染法检测细胞凋亡。采用逆转录PCR检测微管相关蛋白1轻链3（LC3）、P62和胱天蛋白酶3（caspase-3）的mRNA表达，采用蛋白质免疫印迹法检测LC3、P62和caspase-3的蛋白表达。测量感染红色荧光蛋白-绿色荧光蛋白-LC3（RFP-GFP-LC3）腺病毒的神经元的自噬荧光强度，采用透射电子显微镜观察自噬体的超微结构变化。结果 与对照组相比，OGD/R组神经细胞活性显著降低，LDH释放水平显著升高，细胞凋亡显著增加；还检测到LC3、P62和caspase-3的mRNA和蛋白表达增加，RFP荧光强度增加，自噬体增多，自噬溶酶体聚集增多。加入晚期自噬抑制剂BafA1后，RFP荧光强度显著降低。结论 OGD/R诱导神经元自噬和凋亡，抑制自噬后加重神经元损伤和凋亡。
