Category: Preclinical Development
Purpose: Stroke kills about 140,000 Americans each year and is the leading cause of long-term disability. Eighty-seven percent of all stroke is ischemic stroke. Nitric oxide (NO), synthesized by nitric oxide synthase (NOS), has a dual role in the ischemic condition. Endothelial nitric oxide synthase (eNOS) derived NO regulates vascular tone in a way that it considered neuroprotective. In contrast, inducible nitric oxide synthase (iNOS) expression increases from 12 hours after the onset of ischemic stroke and can last for 1 week. When iNOS is induced, it can produce 100 to 1000 times more NO than eNOS, concentrations that are neurotoxic, leading to neuronal cell death1. Our lab has published that Triacsin C, a fungal metabolite which inhibits long chain fatty acyl CoA synthetase (ACSL), decreases eNOS palmitoylation and increases eNOS derived NO without upregulating eNOS transcription in cultured human coronary endothelial cells2. In addition, Triacsin C reduces the infarct volume and inhibits iNOS expression in the mouse middle cerebral artery occlusion (MCAO) model of stroke. In the current study, we wanted to see whether iNOS expression is modulated by ACSL activity.
Methods: In our qPCR experiment, cells were stimulated with cytokine mix, (TNFα, 60 ng/mL; IL-1β, 2 ng/mL; IFNγ, 100 units/mL), in the presence or absence of 5µM Triacsin C (Cayman Chemical) for 24 hours. RNA was extracted following the RNeasy Plus Mini Kit (Qiagen) instruction. The concentration and purity of RNA were determined by Spectrophotometer. 20 μl first strand cDNA was synthesized from 1μg total RNA for each sample using Superscript III First-Strand (Invitrogen). One μl cDNA was amplified using SYBR Green PCR Master Mix (Applied Biosystems) in the CFX96 Touch Real-Time PCR Detection System (BioRad). β-actin (cat 330001 PPM02945B), iNOS (cat 330001PPM02928B), and eNOS (cat 330001 PPM03801A) primers for bEnd.3 cells and β-actin (cat 330001 PPR06570C), iNOS (cat 330001 PPR44835A) and nNOS (cat 330001 PPR44930E) primers for C6 astrocytoma cells were purchased from Qiagen. Expression level in fold change was determined by the comparative threshold cycle method (2–ΔΔCt) with β-actin as the control gene. For western blot experiments, bEnd.3 cells were grown to confluence under standard conditions, then challenged with cytokine mixture in the presence or absence of Triacsin C. After 24 hours, cells were washed with ice-cold PBS buffer and protein extracted using RIPA buffer. Proteins were separated by 4-20% Tris-Glycine gel and transferred to a PVDF membrane then probed with an antibody against iNOS. After blots were visualized, membrane was stripped and re-probed for α-actin as a loading control. To determine NOS catalytic activity, C6 astrocytoma cells were grown to confluence under standard conditions, then challenged with cytokine mixture in the presence or absence of Triacsin C. After 24 hours, cells were processed for assay. Activity was assayed by mixing 10 μl of cell homogenate and 40 μl of NOS assay cocktail. After 10 min at 37 0C, the reaction was terminated by adding 400 μl of stop solution (50 mM HEPES, pH 5.5, 5 mM EDTA). The unreacted substrate was removed by Dowex 50WX8 (100-200 mesh) ion-exchange resin chromatography. Product formation was calculated as fmol product/min/ mg protein.
Results: Cytomix stimulated iNOS mRNA expression in the presence of Triacsin C was 72.9 ± 1.96 % of cytomix group (p = 0.0001, n = 3) in C6 astrocytoma cells and 45.6 ± 6.7 % of cytomix group (p = 0.0001, n = 6) in bEnd.3 cells. No change has been observed in nNOS and eNOS mRNA expression in any group compared to the control in C6 astrocytoma cells and bEnd.3 cells respectively. We also observed that ACSL inhibition with Triacsin C reduced cytokine-stimulated iNOS protein expression in bEnd.3 cells from our western blot analysis. Total nitric oxide synthase activity was measured by conversion of [14C] arginine to [14C] citrulline in the presence or absence of calcium in C6 cells. Cytomix stimulated iNOS activity in the presence of Triacsin C was 79.95 ± 6.04 % (p =0.0423, n = 3) and 81.02 ± 4.29 % of cytomix group (p = 0.0095, n = 3) in the presence or absence of calcium respectively.
Conclusion: Our data confirm that iNOS expression is modulated by ACSL activity, and support our hypothesis that the Triacsin C effect on stroke infarct volume is related to the suppression of iNOS expression.