Korean J Physiol Pharmacol.  2016 Jul;20(4):399-406. 10.4196/kjpp.2016.20.4.399.

Preventive effects of imperatorin on perfluorohexanesulfonate-induced neuronal apoptosis via inhibition of intracellular calcium-mediated ERK pathway

• Lee E
• Choi SY
• Yang JH
• Lee YJ

• KMID: 2371057
• DOI: http://doi.org/10.4196/kjpp.2016.20.4.399

Abstract

Early life neuronal exposure to environmental toxicants has been suggested to be an important etiology of neurodegenerative disease development. Perfluorohexanesulfonate (PFHxS), one of the major perfluoroalkyl compounds, is widely distributed environmental contaminants. We have reported that PFHxS induces neuronal apoptosis via ERK-mediated pathway. Imperatorin is a furanocoumarin found in various edible plants and has a wide range of pharmacological effects including neuroprotection. In this study, the effects of imperatorin on PFHxS-induced neuronal apoptosis and the underlying mechanisms are examined using cerebellar granule cells (CGC). CGC were isolated from seven-day old rats and were grown in culture for seven days. Caspase-3 activity and TUNEL staining were used to determine neuronal apoptosis. PFHxS-induced apoptosis of CGC was significantly reduced by imperatorin and PD98059, an ERK pathway inhibitor. PFHxS induced a persistent increase in intracellular calcium, which was significantly blocked by imperatorin, NMDA receptor antagonist, MK801 and the L-type voltage-dependent calcium channel blockers, diltiazem and nifedipine. The activation of caspase-3 by PFHxS was also inhibited by MK801, diltiazem and nifedipine. PFHxS-increased ERK activation was inhibited by imperatorin, MK801, diltiazem and nifedipine. Taken together, imperatorin protects CGC against PFHxS-induced apoptosis via inhibition of NMDA receptor/intracellular calcium-mediated ERK pathway.

Keyword

Apoptosis; Calcium; Cerebellar granule cell; Imperatorin; Perfluorohexane sulfonate

MeSH Terms

Animals
Apoptosis*
Calcium
Calcium Channel Blockers
Caspase 3
Diltiazem
Dizocilpine Maleate
In Situ Nick-End Labeling
MAP Kinase Signaling System*
N-Methylaspartate
Neurodegenerative Diseases
Neurons*
Neuroprotection
Nifedipine
Plants, Edible
Rats
Calcium
Calcium Channel Blockers
Caspase 3
Diltiazem
Dizocilpine Maleate
N-Methylaspartate
Nifedipine

Figure

• 

  Fig. 1 Effects of imperatorin on cell viability and PFHxS-increased caspase-3 activity in CGC.(A) Cells were treated with different concentrations of imperatorin (0~1000 nM) for 24 h. Cell viability was determined by MTS assay. (B) Cells were pretreated with different concentrations of imperatorin (10~1000 nM) or PD98059 (50 µM) for 1 h and treated with either PFHxS (300 µM) or DMSO as a vehicle control for 3 h. Then, cells were incubated in fresh media for 21 h. Caspase-3 activity was measured. Data (fold increase) are mean±SEM of three independent experiments. **p<0.01, ***p<0.001 vs. DMSO. #p<0.05, ###p<0.001 vs. corresponding Control-treated cells. (IPT, imperatorin; PD, PD98059).

• 

  Fig. 2 Effects of imperatorin on CGC apoptosis induced by PFHxS.Cells were pretreated with imperatorin (500 nM), MK801 (1 µM) or AC-DEVE-CHO (10 µM) for 1 h and treated with either PFHxS (300 µM) or DMSO as a vehicle control for 3 h. Then, cells were incubated in fresh media for 21 h. The apoptotic cells were stained with TUNEL (green) and all cells were counterstained with PI (red). (A) TUNEL- and PI-positive cells were monitored by fluorescence microscopy. Representative microscopic images from three independent experiments are presented (magnification, x200). (B). TUNEL– and PI-positive cells were counted. The number of TUNEL-positive cells was expressed as a percentage of the total number of cells. Data (% TUNEL-positive cells) are mean±SEM of three independent experiments. *p<0.05, ***p<0.001 vs. DMSO. ###p&0.001 vs. corresponding Control-treated cells (IPT, imperatorin).

• 

  Fig. 3 Effects of imperatorin on PFHxS-induced NMDA receptor activation and Ca2+ influx.(A) Cells were pretreated with MK801 (1 µM), DTZ (10 µM) or NFD (10 µM) and then stimulated with 300 µM of PFHxS or DMSO as a vehicle control for 3 h. Then, the cells were incubated in fresh media for 21 h to detect caspase-3 activity. (B) Cells were pretreated with imperatorin (100 and 500 nM), MK801 (1 µM), DTZ (10 µM) or NFD (10 µM) and then stimulated with 100 µM NMDA or DMSO as a vehicle control for 15 min. Then, the cells were incubated in fresh media for 24 h to detect caspase-3 activity. (C) Cells were treated with 300 µM PFHxS for different times (0~24 h). The level of intracellular Ca2+ ([Ca2+]i) was measured. (D) Cells were pretreated with imperatorin (100 and 500 nM), MK801 (1 µM), DTZ (10 µM) or NFD (10 µM) and then stimulated with 300 µM of PFHxS for 1 h to detect intracellular [Ca2+]. Data (fold increase) are represented as the mean±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. DMSO. ###p<0.001 vs. corresponding Control-treated cells (IPT, imperatorin; DTZ, diltiazem; NFD, nifedipine).

• 

  Fig. 4 Effects of imperatorin on PFHxS-induced ERK activation.Cells were treated with 300 µM PFHxS or DMSO as a vehicle control for 30 min in the presence or absence of (A) imperatorin (50, 100, 500 nM), (B) MK801 (1 µM), DTZ (10 µM), NFD (10 µM) or PD (50 µM). The levels of phosphorylatedand total protein of ERK1/2 were detected by Western blot analysis. The blots were reprobed with GAPDH. The blots represent three independent experiments. The densities of bands were measured and the fold increase in ratio pERK/ERK was presented as mean±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. DMSO. #p<0.05, ##p<0.01, ###p<0.001 vs. corresponding Control-treated cells (Con, control; IPT, imperatorin; DTZ, diltiazem; NFD, nifedipine; PD, PD98059).

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