Effects of permanent ischemia and hypoxia on PC12 cells autophagy

LI Yong-qiang WANG Meng WANG Lai LIU Bin SHI Zhen-yu* DENG Jin-bo*

doi:10.16098/j.issn.0529-1356.2017.03.001

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Acta Anatomica Sinica ›› 2017, Vol. 48 ›› Issue (3) : 242-253. DOI: 10.16098/j.issn.0529-1356.2017.03.001

Neurobiology

Effects of permanent ischemia and hypoxia on PC12 cells autophagy

LI Yong-qiang^{1, 2} WANG Meng³WANG Lai¹LIU Bin³SHI Zhen-yu^{1, 3*} DENG Jin-bo ^1*

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Abstract

Objective To investigate the mechanism of permanent injuries induced by oxygen glucose deprivation treatment and the effect of these injuries on autophagy using the cell model of ischemic and hypoxic injuries. Methods In the present study, PC12 cells were used. To establish the oxygen-glucose deprivation (OGD) model, PC12 cells were cultured with glucose free DMEM medium under the condition of 95% N₂ and 5% CO₂， to imitate the permanent ischemia hypoxia (IH) injuries in vivo. The cells were divided into（1）control group: the cell culture in normal medium with normoxia;（2）OGD groups: the cells were cultured in OGD medium for different times from 0.5 hour to 24 hours, such as OGD 0.5 hour (OGD+0.5 hour), 2 hours（OGD+2 hours), 6 hours（OGD+6 hours), 12 hours（OGD+12 hours), 24 hours（OGD+24 hours) groups. The cell viability and cell apoptosis rate were analyzed with MTT assay and flow cytometry. Lactate dehydrogenase (LDH) release level was tested with the colorimetric method. The expressions of hypoxia inducible factor 1α (HIF-1α, a key regulator in hypoxia), cyclooxygenase 2 (COX2, inflammatory indicator), microtubule-associated protein 1 light chain 3（LC3）and Beclin-1 were examined and analyzed with immunocytochemistry and Western blotting. The ultrastructure of autophagosomes were examined by transmission electron microscopy. The effect of different OGD times on ischemic and hypoxic injuries and autophagy was explored. Results The cell viability decreased in OGD groups with time prolongation, compared with control group, the decrease was significant and difference appeared a statistical significance after 6 hours OGD (P<0.05). Cell apoptosis and necrosis rates showed positive correlation with OGD time prolongation, and significant difference appeared after 12 hours OGD (P<0.05). The results showed that the expression of HIF-1α and COX2 were up-regulated in OGD groups with time prolongation, and significant difference appeared after 12 hours OGD (P<0.05). The LC3 was labelled by green-fluorescent protein in OGD groups with number increasing, and fluorescence intensity enhancement. The results of Western blotting revealed that Beclin-1 was positive relevant with OGD time, and significant difference appeared after 6 hours OGD (P<0.05). Conclusion Oxygen glucose deprivation can induce PC12 cells oxidative stress response and autophagy activation.

Key words

Ischemia / Hypoxia / Oxygen-glucose deprivation / Oxidative stress response / Western blotting / PC12 cell

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LI Yong-qiang WANG Meng WANG Lai LIU Bin SHI Zhen-yu DENG Jin-bo. Effects of permanent ischemia and hypoxia on PC12 cells autophagy[J]. Acta Anatomica Sinica. 2017, 48(3): 242-253 https://doi.org/10.16098/j.issn.0529-1356.2017.03.001

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