Protective effect of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride on hypoxia-induced toxicity by suppressing microglial activation in BV-2 cells

Abstract

We recently reported the anti-inflammatory effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on the ATP-induced activation of the NFAT and MAPK pathways through the P2X7 receptor in microglia. To further investigate the underlying mechanism of KHG26792, we studied its protective effects on hypoxia-induced toxicity in microglia. The administration of KHG26792 significantly reduced the hypoxia-induced expression and activity of caspase-3 in BV-2 microglial cells. KHG26792 also reduced hypoxia-induced inducible nitric oxide synthase protein expression, which correlated with reduced nitric oxide accumulation. In addition, KHG26792 attenuated hypoxiainduced protein nitration, reactive oxygen species production, and NADPH oxidase activity. These effects were accompanied by the suppression of hypoxia-induced protein expression of hypoxia-inducible factor 1-alpha and NADPH oxidase-2. Although the clinical relevance of our findings remains to be determined, these data results suggest that KHG26792 prevents hypoxia-induced toxicity by suppressing microglial activation. [BMB Reports 2016; 49(12): 687-692].

Fig. 1

Effects of KHG26792 on cell viability in hypoxia-induced (HI) BV-2 microglial cells. (A) Cell viability was assessed by the MTT reduction assay. (B) TUNEL assay. Scale bars indicate 10 μm. Data are presented as means ± S.D. and are representative of three independent experiments. *indicates statistical significance between the hypoxia-induced group and hypoxia-induced group pretreated with KHG26792 (P < 0.01).

Fig. 2

Effects of KHG26792 on activity and protein level of caspase-3 in hypoxia-induced (HI) BV-2 microglial cells. KHG26792 significantly reduced hypoxia-induced caspase-3 activity (A) and protein levels (B) in BV-2 cells at 24 h after hypoxia. Data are presented as means ± S.D. and are representative of three independent experiments. *indicates statistical significance between the hypoxia-induced group and hypoxia-induced group pretreated with KHG26792 (P < 0.01).

Fig. 3

KHG26792 attenuates hypoxia-induced (HI) increases in NO (A), iNOS protein level (B), protein nitration (C), ROS production (D), and NOX activity (E) in BV-2 cells. Data are presented as means ± S.D. and are representative of three independent experiments. *indicates statistical significance between the hypoxia-induced group and hypoxia-induced group pretreated with KHG26792 (P < 0.01).

Fig. 4

Effects of KHG26792 on the protein expression levels of NOX2, HIF-1α, and NF-κB in hypoxia-induced (HI) BV-2 microglial cells. Equal amounts of crude extracts were immunoblotted using primary antibodies against each protein (A). Bar graphs showing the quantification of levels of NOX2/β-actin, HIF-1α/β-actin, and phospho-NF-κB/NF-κB were calculated using densitometry (B-D). Data are presented as means ± S.D. and are representative of three independent experiments. *indicates statistical significance between the hypoxia-induced group and hypoxia-induced group pretreated with KHG26792 (P < 0.01).