自噬对氧糖剥夺再氧合(OGD/R)大鼠大脑皮层神经元凋亡的抑制作用[J].,Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology

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自噬对氧糖剥夺再氧合(OGD/R)大鼠大脑皮层神经元凋亡的抑制作用[J].
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology Pub Date : 2022-07-01
Lei Zhang ₁ , Yang Li ₂ , Yuqi Hu ₂ , Hui Xu ₂ , Chao Zhu ₂ , Xu Wang ₂ , Dong Zhao ₃ , Xin He ₄

Affiliation

目的观察自噬对氧糖剥夺复氧（OGD/R）后大脑皮层神经元凋亡的影响。方法选择24小时内出生的新生SD大鼠18只。通过机械胰蛋白酶消化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。CCK-8法和乳酸脱氢酶(LDH)法检测细胞活力，流式细胞仪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)双标记检测细胞凋亡。微管相关蛋白1轻链3（LC3）的mRNA表达，逆转录PCR检测P62和胱抑素3（caspase-3），Western blot检测LC3、P62和caspase-3的蛋白表达。测定感染红色荧光蛋白-绿色荧光蛋白-LC3（RFP-GFP-LC3）腺病毒的神经元的自噬荧光强度，透射电镜观察自噬体的超微结构变化。结果与对照组相比，OGD/R组神经细胞活性明显降低，LDH释放水平明显升高，细胞凋亡明显增加；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。添加晚期自噬抑制剂BafA1后RFP的荧光强度显着降低。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。

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[Inhibitory effect of autophagy on apoptosis of rat cerebral cortical neurons after oxygen-glucose deprivation and reoxygenation (OGD/R)].

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.

更新日期：2022-07-01

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