The Aminopyridinol Derivative BJ-1201 Protects Murine Hippocampal Cells against Glutamate-Induced Neurotoxicity via Heme Oxygenase-1

Abstract

Glutamate is the major excitatory neurotransmitter in the brain. It can cause neuronal cell damage in the context of oxidative stress. BJ-1201 is a derivative of the compound aminopyridinol, which is known for its antioxidant activity. In this study, we examined the effect of BJ-1201, a 6-(diphenylamino)-2,4,5-trimethylpyridin-3-ol compound, on neuroprotection in HT22 cells. Our data showed that BJ-1201 can protect HT22 cells against glutamate-induced cell cytotoxicity. In addition, BJ-1201 upregulated heme oxygenase-1 (HO-1) to levels comparable to those of the CoPP-treated group. BJ-1201 treatment induced phosphorylation of JNK, but not p38-MAPK or ERK. It also increased the signal in the reporter assay based on β-galactosidase activity driven by the nuclear transcription factor erythroid-2 related factor 2 (Nrf2) promoter harboring antioxidant response elements (AREs) and induced the translocation of Nrf2. These results demonstrate that BJ-1201 may be a good therapeutic platform against neurodegenerative diseases induced by oxidative stress.

Keywords: aminopyridinol HT22; aminopyridinol compound BJ-1201; heme oxygenase-1; neuroprotection; nuclear transcription factor erythroid-2 related factor 2.

Figure 1

Effects of BJ-1201 on glutamate-induced oxidative neurotoxicity. (a) The structure of BJ-1201; (b) HT22 cells were incubated for 72 h with various concentration of BJ-1201; (c) HT22 cells were pre-treated with BJ-1201 for 12 h and then incubated for 12 h with glutamate (10 mM). Cell viability was measured by MTT assay; (d) Exposure of HT22 cells to glutamate increased ROS production. Each bar represents the mean ± S.D. of three independent experiments. * p < 0.05 compared with glutamate. Trolox (100 μM) was used as the positive control.

Figure 1

Effects of BJ-1201 on glutamate-induced oxidative neurotoxicity. (a) The structure of BJ-1201; (b) HT22 cells were incubated for 72 h with various concentration of BJ-1201; (c) HT22 cells were pre-treated with BJ-1201 for 12 h and then incubated for 12 h with glutamate (10 mM). Cell viability was measured by MTT assay; (d) Exposure of HT22 cells to glutamate increased ROS production. Each bar represents the mean ± S.D. of three independent experiments. * p < 0.05 compared with glutamate. Trolox (100 μM) was used as the positive control.

Figure 2

Effects of BJ-1201 on HO-1 expression in HT22 cell. (a) Cells were incubated for 18 h with sample (1–20 μM). Expression of HO-1 was determined by western blot analysis; (b) HO activity was determined via bilirubin formation at 12 h after treatment with various concentrations of BJ-1201. Each data represents the mean ± S.D. of three independent experiments are shown. CoPP (10 μM) was used as the positive control. * p < 0.05 compared with control group.

Figure 3

Effects of BJ-1201-induced MAPK activation in HT22 cells. Cells were treated with 20 μM BJ-1201 for the indicated times. Cell extracts were analyzed by western blot with antibodies specific for (a) phosphorylated ERK1/2 (p-ERK); (b) phosphorylated JNK (p-JNK); or (c) phosphorylated p38 (p-p38). Membranes were stripped and re-probed for the total form of each MAPK as a control, and representative blots of three independent experiments are shown. Each data represents the mean ± S.D. of three independent experiments are shown. * p < 0.05 compared with control group.

Figure 4

Effects of BJ-1201 on MAPK dependent HO-1 expression and cytoprotection in HT22 cells. (a) Cells were pretreated for 1 h with the specific inhibitor PD98059 (40 μM), SB203580 (20 μM), and SP600125 (25 μM), and then treated with 20 μM BJ-1201 for 18 h. Western blotting was then performed with HO-1 antibody; (b) Cells were treated with 20 μM BJ-1201 in the presence or absence of each specific inhibitors and SnPP (50 μM) for 12 h were exposed to 5 mM glutamate for 12 h. Data represent the mean ± S.D. of three independent experiments. * p < 0.05 compared with control group. ^# p < 0.05 compared with BJ-1201. ** p < 0.05 compared with glutamate (10 mM). ^$ p < 0.05 compared with glutamate + BJ-1201.

Figure 5

Effects of BJ-1201 on Nrf2 nuclear translocation in HT22 cells. (a) Cells were treated with 30 μM BJ-1201 for 0.5, 1, and 1.5 h. Nrf2 protein was detected by western blot analysis; (b) Cells were transfected with ARE-luciferase of control vector were incubated for 1 h with the indicated concentrations of BJ-1201. Cells were assayed for luciferase activity as the fold induction by normalizing the transfection efficiency and dividing the values from each experimental sample relative to the control. Data represent the mean ± S.D. of three independent experiments. * p < 0.05 compared with control group.