Probenecid and N-Acetylcysteine Prevent Loss of Intracellular Glutathione and Inhibit Neuronal Death after Mechanical Stretch Injury In Vitro

Abstract

Probenecid and N-acetylcysteine (NAC) can preserve intracellular levels of the vital antioxidant glutathione (GSH) via two distinct biochemical pathways. Probenecid inhibits transporter-mediated GSH efflux and NAC serves as a cysteine donor for GSH synthesis. We hypothesized that probenecid and NAC alone would maintain intracellular GSH concentrations and inhibit neuronal death after traumatic stretch injury, and that the drugs in combination would produce additive effects. Sex-segregated rat primary cortical neurons were treated with probenecid (100 μM) and NAC (50 μM), alone and in combination (Pro-NAC), then subjected to mechanical stretch (10s^-1 strain rate, 50% membrane deformation). At 24 h, both probenecid and NAC inhibited trauma-induced intracellular GSH depletion, lactate dehydrogenase (LDH) release, and propidium iodide (PI) uptake in both XY- and XX-neurons. Combined Pro-NAC treatment was superior to probenecid or NAC alone in maintenance of intracellular GSH and neuronal death assessed by PI uptake. Interestingly, caspase 3 activity 24 h after mechanical trauma was more prominent in XX-neurons, and treatment effects (probenecid, NAC, and Pro-NAC) were observed in XX- but not XY-neurons; however, XY-neurons were ultimately more vulnerable to mechanical stretch-induced injury than their XX counterparts, as was evidenced by more neuronal death detected by LDH release and PI uptake. In addition, after stretch injury in HT22 hippocampal cells, both NAC and probenecid were highly effective at reducing oxidative stress detected by dichlorofluorescein fluorescence. These in vitro data support further testing of this drug combination in models of traumatic neuronal injury in vivo.

Keywords: adenosine triphosphate binding cassette transporter C1; gender; multidrug resistance associated protein 1; primary cortical neurons; spinal cord injury; traumatic brain injury.

FIG. 1.

Effects of N-acetylcysteine (NAC) (50 μM) and probenecid (100 μM), alone and in combination (Pro-NAC), on intracellular glutathione (GSH) stores and cell death when administered to sex-segregated primary cortical neurons immediately prior to mechanical stretch (10s⁻¹ strain rate, 50% membrane deformation) or sham injury. (A) Intracellular GSH (n = 4/sex/group). (B) Lactate dehydrogenase (LDH) release (n = 5-8/sex/group). (C) Caspase 3/7-like activity (n = 4/sex/group). (D) Propidium iodide (PI) staining measured by flow cytometry (n = 4/sex/group). XY neurons harvested from male rats, XX neurons harvested from female rats; mean ± SD; *p < 0.01 versus control; †p < 0.04 versus vehicle; ‡p < 0.03 versus Pro-NAC; ¶p < 0.02 versus XY neurons.

FIG. 2.

Effects of N-acetylcysteine (NAC) (50 μM) and probenecid (100 μM), alone and in combination (Pro-NAC), on intracellular reactive oxygen species (ROS) production detected by dichlorodihydrofluorescein (DCF) fluorescence in HT22 hippocampal cells 2 h after stretch injury (n = 3/group for treatments, n = 2 for control cells). The percentage of DCF positive cells was measured using flow cytometry (mean ± SD; †p < 0.05 vs. vehicle). Representative fluorescent images of HT22 cells for each group are shown.