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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.
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Objective To detect the expression of BclGL in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) and to investigate its significance.Methods Peripheral blood was obtained from 20 patients with active SLE (A-SLE),18 patients with inactive SLE (Ⅰ-SLE) and 30 healthy controls.Flow cytometry was performed to calculate the number of PBMCs,flow cytometry combined with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining to determine the early apoptotic rates of PBMCs,fluorescence-based quantitative PCR and Western blot to measure the expression of BclGL mRNA and protein,respectively.The serum level of interferon (IFN)-α was determined by enzyme-linked immunosorbent assay (ELISA).Data were analyzed by using the software SPSS13.0.Mann-Whitney U-test or Kruskal-Wallis test was used for group comparisons.Pearson correlation coefficient test was applied to evaluate the relationship of BclGL expression with cell apoptotic rate and some clinical parameters.Results The number of PBMCs was significantly lower in patients with A-SLE than in those with Ⅰ-SLE and healthy controls ((0.16 ± 0.06) × 109/L vs.(0.27 ± 0.14) × 109/L and (0.34 ± 0.23) × 109/L,both P ＜ 0.01).Increased apoptotic rate of PBMCs was observed in patients with A-SLE compared with those with Ⅰ-SLE and healthy controls ((22.6 ± 1.1)％ vs.(16.4 ±0.9)％ and (10.1 ± 0.4)％,both P ＜ 0.01),and in patients with Ⅰ-SLE compared with the healthy controls (P ＜0.01).The mRNA and protein expressions of BclGL in PBMCs were both significantly higher in patients with ASLE than in those with Ⅰ-SLE and healthy controls (all P ＜ 0.01).A significant increase was observed in serum IFN-α level in the patients with SLE compared with the healthy controls ((32.5 ± 2.2) μg/L vs.(15.5 ± 1.3) μg/L,P ＜ 0.01).The mRNA expression of BclGL in PBMCs from patients with SLE was positively correlated with the apoptotic rate in PBMCs (r =0.486,P ＜ 0.01),SLE disease activity index score (r =0.496,P ＜ 0.01),titers of antinuclear antibodies (r =0.516,P ＜ 0.01) and serum IFN-o level (r =0.535,P ＜ 0.01),but was negatively correlated with complement C3 level (r =-0.515,P ＜ 0.01).Conclusions The increased expression of BclGL in PBMCs may contribute to the abnormal apoptosis in PBMCs,which in turn takes part in the pathogenesis of SLE.