Although both hypoxia–reoxygenation and SIN-1 induce oxidative stress, mechanistic components and injury propagation following these treatments are different. Following SIN-1 treatment, FBP preserves GSH concentrations from decreasing for at least 6 h after treatment, but FBP fails to alter the almost complete depletion of GSH following combined BSO and SIN-1 treatments.
Despite the durations of hypoxia, GSH concentrations are significantly higher in neurons when FBP is present in the medium. During hypoxia, the concentration of intracellular GSH decreases significantly after a short period of hypoxia and even further when the hypoxic period is prolonged. The latter findings are consistent with our previous observations that 8 h of hypoxia causes neuronal death when astrocytes are absent, and that the addition of FBP to the medium does not prevent cell death any more.
However, FBP fails to rescue neurons if the hypoxic episode is prolonged to 6 h when additional decrease of neuronal viability occurs. FBP also protects against another type of oxidative stress, hypoxia–reoxygenation, when the hypoxic period is short. While FBP does not alter neuronal viability when a mild injury is produced by application of SIN-1 itself, it protects neurons against more severe injury caused by pre-depletion of GSH prior to SIN-1 application.
Since in a co-culture of neurons and astrocytes FBP reduces neuronal death partly via an astrocyte-mediated increase in glutamate uptake, glutamine production, and increased glutamine synthetase activity, we utilized almost pure neuronal cultures to avoid astrocyte-dependent effects in the present study. The protective effect of FBP is associated with preservation of GSH metabolism in neurons that likely occurs in part via increased glutathione reductase activity with a consequent increased GSH regeneration from its oxidized form. The current study shows that FBP protects neurons against hypoxia–reoxygenation and reactive oxidants directly, not through effects mediated by astrocytes. Under these conditions, FBP did not preserve GSH from falling (3.2 6 0.8%). Exposure of cells with pre-depleted GSH to SIN-1 resulted in further decrease of GSH over the value post-BSO treatment alone (2.6 6 1.8 vs. The presence of FBP in the medium during the SIN-1 exposure lead to a significantly higher GSH level than in cells not treated with FBP (60.5 6 6.9%, P, 0.045). While GSH level was not significantly different from the control levels 1.5 h after SIN-1 exposure and was not changed by the presence of FBP during the SIN-1 exposure, GSH level declined by 6 h post-treatment (47.2 6 9.3%, P, 0.0001). Neuronal GSH level reduced over time following the SIN-1 treatment for 5 min ( Fig. 6 2.6% of control when FBP was present during hypoxic period ( P, 0.05).
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