trans-Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

Abstract

An in vitro model of ischemic cerebral stroke [oxygen-glucose deprivation (OGD) for 6 h followed by 24 h reoxygenation (R)] with PC12 cells increases Ca(2+) influx by upregulating native L-type Ca(2+) channels and reactive oxygen species (ROS) generation. This reactive oxygen species generation and increase in intracellular Ca(2+) triggers the expression of hypoxic homeostasis transcription factors such as hypoxia induced factor-1 alpha (HIF-1α), Cav-beta 3 (Cav β3), signal transducer and activator of transcription 3 (STAT3), heat shock protein 27 (hsp-27), and cationic channel transient receptor potential melastatin 7 (TRPM7). OGD insulted PC12 cells were subjected to biologically safe doses (5, 10, and 25 μM) of trans-resveratrol in three different treatment groups: 24 h prior to OGD (pre-treatment); 24 h post OGD (post-treatment); and from 24 h before OGD to end of reoxygenation period (whole-treatment). Here, we demonstrated that OGD-R-induced neuronal injury/death is by reactive oxygen species generation, increase in intracellular calcium levels, and decrease in antioxidant defense enzymes. trans-Resveratrol increases the viability of OGD-R insulted PC12 cells, which was assessed by using MTT, NRU, and LDH release assay. In addition, trans-resveratrol significantly decreases reactive oxygen species generation, intracellular Ca(2+) levels, and hypoxia associated transcription factors and also increases the level of antioxidant defense enzymes. Our data shows that the whole-treatment group of trans-resveratrol is most efficient in decreasing hypoxia induced cell death through its antioxidant properties.

Efficacy of prophylactic flavan-3-ol in permanent focal ischemia in 12-mo-old mice

“…For example, it is possible that endothelial cell death and subsequent disruption of the neurovascular unit at early time points initiates a series of apoptotic signaling events that lead to delayed neurodegeneration, possibly through hypoxia-inducible factor 1␣. Such events are known to occur after cerebral ischemia (3,12,33) and would fit with both the injury profile and the effects of EC observed in the present study. Notably, resveratrol has been shown to exert beneficial effects on endothelial cell function, which in turn promotes vascular function (60,62,63).…”

Resveratrol protects PC12 cells against OGD/R-induced apoptosis via the mitochondrial-mediated signaling pathway

“…For instance, administration of resveratrol to culture medium protects a wide variety of neuronal cell types from oxidative stress-induced injury [7][8][9]; dietary supplementation of resveratrol can ameliorate neuronal damage and death resulted from oxidative stress in rodents [10]. However, these studies have not fully explored the specific molecular mechanisms by which resveratrol acts at the cellular level.…”

“…Consequently, disruption of MMP triggers the pathological opening of MPTP and excessive MPTP opening leads to the further collapse of the MMP. This vicious circle may result in mitochondrial swelling, rupture of the outer mitochondrial membrane, and the consequent release of cytochrome c from mitochondria into cytosol, which ultimately activates caspase cascade and induces apoptosis [8]. In accordance with the previous experimental results [29], we also found that OGD/ R caused the disruption of MMP, the pathological opening of MPTP, and the release of cytochrome c from mitochondria, suggesting that mitochondrial-mediated signaling pathway may have a determinant role in OGD/R-induced neurotoxicity.…”

“…Then cells were allowed to grow in a hypoxia chamber (Thermo scientific, Waltham, USA) with a compact oxygen controller to maintain oxygen concentration at 1% by injecting a gas mixture of 94% N 2 and 5% CO 2 for 6 h. After hypoxia, the cells were then transferred back to normal DMEM medium containing 4.5 g/l glucose under an atmosphere of 95% air and 5% CO 2 , and incubated for 24 h for reoxygenation [8].…”

“…MTT assay was performed following the protocols of Agrawal et al [8] with some modification. In brief, cells (1 × 10 4 ) were seeded in 96-well microculture plates that had been pre-coated poly-L-lysine and cultured overnight in complete DMEM medium in a CO 2 incubator (95% air/5% CO 2 ).…”