Research Paper Volume 12, Issue 24 pp 25035—25059

Figure 1. DOR activation attenuated hypoxic and/or MPP⁺ insults induced mitochondrial membrane potential depolarization and mitochondrial dysfunction. (A) PC12 cells were exposed to hypoxia at 1% O₂ for 48 hrs, the mitochondrial membrane potential was measured using TMRM reagent. 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. H+N: PC12 cells were treated with DOR antagonist naltrindole alone in hypoxic condition. N=3 in each group. ^**p<0.01 vs. C; ^Δp<0.05 vs. H; Note that hypoxia significantly decreased the red fluorescent intensity compared to the control, suggesting the collapse of mitochondrial membrane potential. The application of DOR agonist UFP-512 attenuated these changes and the flow cytometer results were consistent with the fluorescence microscope observation. (B) PC12 cells were exposed to 1.0 mM MPP⁺ for 24 hrs. 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⁺. M+N: PC12 cells were exposed to naltrindole along with MPP⁺. N=3 in each group. ^**p<0.01 vs. C; ^Δp<0.05, ^ΔΔp<0.01 vs. M. Note that MPP⁺ insults also caused a depolarization of mitochondrial membrane potential, while activating DOR using UFP-512 significantly reversed these destructive changes induced by MPP⁺ insults. In contrast, applying DOR antagonist naltrindole alone further aggravated the collapse of mitochondrial membrane potential under MPP⁺ insults. The results measured by flow cytometer were consistent with the florescence observation. (C) PC12 cells were exposed to 1% O₂ for 48 hrs or 1.0mM MPP⁺ for 24 hrs. N=3 in each group. ^**p<0.01 vs. C; ^Δp<0.05, ^ΔΔp<0.01 vs. H or M. Note that hypoxia and/or MPP⁺ caused a significant decrease in ATP generation, while DOR activation restored the capacity of mitochondria in ATP production.