Research Paper Volume 12, Issue 24 pp 25035—25059

Figure 2. DOR activation strongly protected PC12 cells from the oxidative injury induced by MPP⁺, and inhibited the cytochrome c release with a mild effect on the cells exposed to hypoxia. (A) PC12 cells were exposed to 1% O₂ for 48 hrs. The mitochondrial superoxide was detected by MitoSOX Red Mitochondrial Superoxide Indicator. C: normoxic control. H: hypoxia. H+U: DOR was activated using UFP-512 in hypoxic conditions. H+U+N: PC12 cells were treated with UFP-512 plus naltrindole at the same time in hypoxic conditions. N=3 for each group. ^**p<0.01 vs. C. Note that hypoxia significantly increased the mitochondrial superoxide appearing as a sharp increase in red fluorescent intensity, incubation the cells with DOR specific agonist UFP-512 showed no appreciable difference. (B) PC12 cells were treated with 1.0mM MPP⁺ for 24hrs. C: control. M: MPP⁺. M+U: DOR was activated using UFP-512 and exposed to MPP⁺. M+U+N: PC12 cells were treated with UFP-512 plus naltrindole and exposed to MPP⁺. N=3 for each group. ^**p<0.01 vs. C; ^ΔΔp<0.01 vs. M. Note that MPP⁺ up-regulated mitochondrial superoxide in PC12 cells. DOR activation significantly attenuated the superoxide generation with a remarkable decrease in red fluorescent density under MPP⁺ insults, while the addition of naltrindole plus UFP-512 reversed the effects induced by DOR activation. (C) PC12 cells were exposed to 1% O₂ for 48 hrs or treated with 1.0mM MPP⁺ for 24 hrs, the protein from cytosol were extracted and analyzed by Western blot. N=3 for each group. ^*p<0.05, ^**p<0.01 vs. C; ^Δp<0.05 vs. H; ^ΔΔp<0.01 vs. M. Note that hypoxia and MPP⁺ caused a rise of cytochrome c in the cytoplasm, whereas DOR activation remarkably inhibited the release of cytochrome c from the mitochondria to the cytoplasm against MPP⁺ insults with a mild effect on the cells exposed to hypoxia.