Bilirubin Oxidation End Products (BOXes) Induce Neuronal Oxidative Stress Involving the Nrf2 Pathway

Figure 2

BOX A and BOX B do not affect cell viability and apoptosis. (a and b) Relative viability of primary cortical neurons (PCNs, ) cultured 11 days in vitro (DIV) (a) and neuroblastoma cells (top, SH-SY5Y, ; bottom, Neuro2a, ) (b), for the indicated concentrations of BOX A (red) and BOX B (blue). Vehicle (0.1% DMSO) was applied in the control experiments (pale blue). (c) A representative TUNEL staining of 13 DIV PCNs treated with BOX A or BOX B (30 μM) for 72 h. The apoptotic PCNs were stained with Apo-FITC (green) and counterstained with Hoechst33342 (blue). Bar: 200 μm. (d) The statistical analysis of the calculated apoptosis (%) of the PCNs as shown in (c). The ratio of green labeled cells to the blue ones was taken as a measure for apoptosis (%) in 7 ~ 9 random photographed field. Data were presented as (), ANOVA with post Bonferroni’s test. ns: no significant. (e) Representative immunoblots of caspase 3 of 11 DIV cultured PCNs treated with BOX A or BOX B (30 μM each) for 48 h. One representative blot of 3 independent experiments. The antibody against caspase 3 (#T40044, Abmart Inc.) used here could detect both caspase 3 and cleaved caspase 3 (c-Caspase3).