Effects of Bcl-2/adenovirus E1B 19kDa-interacting protein 3-like on mitophagy in PC12 cells / 中华创伤杂志

ABSTRACT

Objective:To explore the possible mechanism of Bcl-2/adenovirus E1B 19kDa-interacting protein 3-like (also known as NIX) mediating mitophagy in PC12 cells.Methods:The PC12 cells (rat adrenal pheochromocytoma cells) were cultured in a hypoxic incubator with a volume fraction of 1% O 2 to establish hypoxic injury models. The cells were divided into normoxia group and hypoxia groups at 6, 12, 24 and 48 hours after cells were exposed to hypoxic conditions. Afterwards, the expression levels of NIX, microtubule-associated protein 1 light chain 3 (LC3), translocase of outer mitochondrial membrane 20 (TOMM20), and cyclooxegenase 4 (COX4) were determined by Western blot analysis. Electron microscopy was used to observe the formation of autophagosomes after 24 hours of hypoxia. The mitochondria-NIX-LC3-autophagosome complexes were detected by confocal microscopy after the overexpression of NIX for 48 hours. The interaction between NIX and LC3 was verified by Co-immunoprecipitation (CoIP). After downregulation of NIX, the changes in mitochondria morphology were detected by confocal microscopy. The PC12 cells were divided into normoxia group, normoxia+ NIXshRNA group, hypoxia group and hypoxia+ NIXshRNA group, then the expression levels of NIX, LC3, TOMM20 and COX4 in each group were detected via Western blotting. Results:Compared to normoxia group, hypoxia group showed up-regulated expressions of NIX and LC3 [(0.44±0.03)∶(0.21±0.01), (1.04±0.03)∶(0.32±0.01)], and down-regulated expressions of TOMM20 and COX4 [(0.78±0.07)∶(1.46±0.08), (0.52±0.04)∶(0.98±0.06)] after 24 hours of hypoxia ( P<0.05). Autophagosomes containing mitochondria were detected by electron microscopy after 24 hours of hypoxia. The formation of the mitochondria-NIX-LC3-autophagosome complex were detected by confocal microscopy after the overexpression of NIX for 48 hours. CoIP demonstrated an interaction between NIX and LC3. Furthermore, inhibition of NIX preserved the integrity of the mitochondria compared with hypoxia group. Western blot analysis showed decreased expressions of NIX and LC3 in hypoxia+ NIXshRNA group [(0.90±0.04)∶ (1.30±0.19), (0.55±0.03)∶(0.75±0.03)] and increased expressions of TOMM20 and COX4 [(0.78±0.06)∶( 0.69±0.08), (0.81±0.07)∶( 0.81±0.07) in comparison to hypoxia group ( P<0.05). Conclusions:NIX can interact with LC3 to mediate mitophagy in PC12 cells. Therefore, the inhibition of NIX can preserve the integrity of the mitochondria and decrease the level of mitophagy, thus provide a protective effect.