Diallyl Disulfide Inhibits Cytochrome c-Mediated Apoptosis in H2O2 Induced Death of Neuronal-differentiated PC12 Cells

BACKGROUND: The effects of diallyl disulfide (DADS), a garlic derived compound, on the viability and cell signaling- like the downstream signaling through cytochrome c, caspase-3, poly (ADP-ribose) polymerase (PARP) during an oxidative-stress induced injury were studied using H2O2 treated neuronal-differentiated PC12 cells by a nerve growth factor. METHODS: To evaluate the toxicity of the DADS itself, the viability of the differentiated PC12 cells treated with several concentrations of DADS was evaluated with 3, (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. To evaluate the protective effect of the low concentration of DADS from oxidative stress, the viability of the cells (DADS pretreated vs. not pretreated) was evaluated following the exposure to 100 micro M H2O2. Additionally, the expression of caspase-3, PARP, and cytochrome c was examined using western blot analyses. RESULTS: The viability was not affected at low concentrations of DADS, up to 20 micro M, but, over this concentration, it was decreased. Compared with the cells treated with only 100 micro M H2O2, the pretreatment with low concentrations of DADS before exposure to 100 micro M H2O2 increased the viability and induced the inhibition of caspase-3 activation, PARP cleavage, and cytochrome c release. CONCLUSIONS: These results show that low concentrations of DADS shows neuroprotective effects by affecting the downstream signaling through cytochrome c, caspase-3, and PARP pathway and may be a new potential therapeutic strategy for neurodegenerative diseases associated with oxidative injury.

Effect of Poly (ADP-ribose) Polymerase Inhibitor Against the Ischemia/Reperfusion Injury in Ischemic Stroke Model

BACKGROUND: Nuclear enzyme poly (ADP-ribose) polymerase (PARP) activated by DNA damage participates in DNA repair. However, overactivation of PARP could be an important pathogenic mechanism of ischemic cell death. We investigated the protective effect of an inhibitor of PARP, 3-aminobenzamide (3-AB), against ischemia/reperfusion injury in ischemic stroke model. METHODS: Occlusion of left middle cerebral artery (MCA) was done by intraluminal filament technique in 24 rats weighing from 315 g to 358 g, and reperfusion was done at 2 hours after occlusion. To evaluate the effect of PARP inhibitor in ischemic stroke, 3-AB was administered to 12 rats (3-AB group) 10 minutes before artificial occlusion of left MCA. Infarct area was confirmed by using 2, 3, 5-triphenyltetrazolium chloride stain. The immunoreactivities of poly (ADP-ribose) reflecting activity of enzyme PARP and activated caspase-3 were compared in infarct, peri-infarct and normal zones in 3-AB group and 12 controls. RESULTS: The volume of infarction was decreased about 34% in 3-AB group compared with controls. In 3-AB group, immunoreactivities of PAR were significantly reduced in ischemic regions, especially peri-infarct zone, but those of activated caspase-3 were significantly increased in same region. CONCLUSIONS: These results suggest that treatment of PARP inhibitor can reduce the infarct volume by converting necrotic cell death into apoptosis. PARP inhibition can be another potential neuroprotective strategy in ischemic stroke.

Cytoprotective Effect of Epigallocatechin gallate (EGCG) in Oxidative-stressed PC12 Cells Following H2O2 Exposure-Effect of EGCG on Phosphoinositide 3-kinase/Akt and Glycogen Synthase Kinase-3 Pathway

BACKGROUND: Neurodegenerative diseases (ND) are associated with oxidative stress, and antioxidants including epigallocatechin gallate (EGCG) have been tried as potential therapeutic regimens of an experimental model of ND. We performed this study to determine the neuroprotective role of EGCG on up stream and down stream signals in oxidative-stress-injured PC12 cells by exposing them to H2O2. METHODS: Following 100 microM H2O2 exposure, the viability of PC12 cells (not pretreated vs EGCG or z-VAD-fmk pretreated) was evaluated by using a MTT assay. Immunoreactivity (IR) of cytochrome c, caspase-3, poly (ADP-ribose) polymerase (PARP), PI3K/Akt and GSK-3 was examined by using a Western blot. RESULTS: EGCG or z-VAD-fmk pretreated PC12 cells showed increased viability. Dose-dependent inhibition of caspase-3 activation and PARP cleavage was demonstrated by the pretreatment of both agents. However, the inhibition of cytochrome c release was only detected in EGCG pretreated cells. On the pathway through PI3K/Akt and GSK-3, however, the result of a western blot in EGCG pretreated cells showed decreased IR of Akt and GSK-3 and increased IR of p85a PI3K, phosphorylated Akt and GSK-3, and contrasted with that in z-VAD-fmk pretreated cells showing no changes. CONCLUSIONS: These data show that EGCG affects apoptotic pathways through upstream signals including PI3K/Akt and GSK-3 pathways as well as downstream signals including cytochrome c and caspase-3 pathways. Therefore, these results suggest that EGCG mediated activation of PI3K/Akt and inhibition GSK-3 could be a new protective mechanism on the pathogenesis of ND.