Abstract
OBJECTIVE: The aim of this study was to investigate the potential role of Activated Protein C (APC) to inhibit caspase-3 and PARP activation in SHSY-5Y cells. BACKGROUND: With the recent interest of Activated Protein C (APC) as possible targets for the cerebrovascular injury, we tested whether Activated Protein C protect neuronal ischemic injury via inhibition of caspase-3 and PARP activation against oxygen-glucose-deprivation (OGD) induced ischemic injury in cultured SHSY-5Y cells. PARP is activated by DNA strand nicks and breaks, which is also induced by evironmental stimuli or reactive oxygen species. METHODS: Oxygen-glucose deprivation of SHSY-5Y cells were prepared in media deprived of glucose and fetal bovine serum and kept in a air-tight chamber containing 95[percnt] N2 and 5[percnt] CO2 for 4 hours with 24 hours reoxygenation. The ischemic injury was checked by measuring the release of lactate dehydrogenase (LDH) in to the medium and the cell viability was assessed by WST-8 assay. The expression of EPCR, PAR-1 & PAR-3 was assessed by flow cytometry. The expression of caspase-3 and PARP was assessed by western blotting and rtPCR respectively after 24 hours of reoxygenation. RESULTS: APC pre-treatment at a dose of 100 nM reduce the cell toxicity upto 35[percnt] (P<0.05); the expression of all three receptors EPCR, PAR-1 and PAR-3 was up-regulated significantly (p<0.05) in both hypoxia treatment and hypoxia and APC both compared to control normoxic condition. The quantitative caspase-3 and PARP expression analyses at 24 h of re-oxygenation revealed the expressions of caspase-3 and PARP downregulated upon APC treatment. CONCLUSIONS: We concluded that OGD induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway and could be inhibited by PARP inhibition via APC treatment.
Disclosure: Dr. Sriwastva has nothing to disclose. Dr. K has nothing to disclose. Dr. Prasad has nothing to disclose. Dr. Saxena has nothing to disclose. Dr. S has nothing to disclose.
Wednesday, April 22 2015, 2:00 pm-6:30 pm
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