Role of mitochondrial calcium uniporter in mitophagy in SH-SY5Y cells subjected to oxygen-glucose deprivation and restoration

Abstract

Objective To evaluate the role of mitochondrial calcium uniporter (MCU) in mitophagy in SH-SY5Y cells subjected to oxygen-glucose deprivation and restoration (OGD/R). Methods SH-SY5Y cells were cultured in vitro, seeded in 96-well plates at a density of 2×105 cells/ml, and randomly divided into 4 groups (n=6 each) using a random number table method: control group (group C), group OGD/R, OGD/R plus MCU inhibitor group (group OGD/R + Ru360) and MCU inhibitor group (group Ru360). Cells were cultured in normal culture medium in group C. Cells were subjected to O2-glucose deprivation for 6 h followed by restoration of O2-glucose supply for 24 h in group OGD/R.In group OGD/R+ Ru360, Ru360 at a final concentration of 10 μmol/L was added at 30 min before O2-glucose deprivation, and the other treatments were similar to those previously described in group OGD/R.Ru360 was added at a final concentration of 10 μmol/L, and 30 min later cells were cultured under normoxic conditions in group Ru360.At 24 h of restoration of O2-glucose supply, cell counting kit-8 assay was used to detect the cell survival rate, JC-1 assay was used to detect mitochondrial membrane potential (MMP), the ultrastructure of cells was observed with a transmission electron microscope, and the expression of p62, Tom20 and Beclin-1 was detected by Western blot. Results Compared with group C, no significant change was found in each parameter in group Ru360 (P>0.05), the cell survival rate and MMP were significantly decreased, the expression of Tom20 and p62 was down-regulated, Beclin-1 expression was up-regulated (P<0.01), the mitochondria swelled, and mitochondrial autophagosomes were increased in group OGD/R.Compared with group OGD/R, the cell survival rate and MMP were significantly increased, the expression of Tom20 and p62 was up-regulated, Beclin-1 expression was down-regulated (P<0.01), the mitochondrial morphology kept well, and mitochondrial autophagosomes were decreased. Conclusion MCU is involved in the process of mitophagy in SH-SY5Y cells subjected to OGD/R. Key words: Mitochondria; Ion transport; Cell hypoxia; Autophagy