Influence of Panax quinquefolium saponins on increased intracellular Ca2+ in PC12 cells

摘要

BACKGROUND: Previous studies have demonstrated that intracellular Ca2+ ([Ca2+]i) overload,excitotoxicity, free radical injury, and nitric oxide toxicity are involved in mechanisms of neuronal death in the ischemic brain.OBJECTIVE: To investigate the influence of Panax quinquefolium saponins (PQS) on multiple factors-induced Ca2+ overload in the rat pheochromocytoma (PC12) cell line.DESIGN, TIME AND SETTING: Intergroup comparison, in vitro study. The experiment was performed at the Heilongjiang Key Laboratory of Anti-fibrosis Biotherapy, Mudanjiang Medical University between November 2007 and April 2008.MATERIALS: In vitro cultured PC12 cells in the logarithmic phase were assigned into blank control, model, and drug treatment groups (10 μmol/L nimodipine; 40 μg/L, 100 μg/L, and 250 μg/L PQS). Nimodipine was purchased from Jiangsu Yangtze River Pharmacy Group Co.,China; PQS (purity > 95%, HLPC grade) was provided by School of Basic Medical Sciences, Jilin University. Caffeine, Na2S204, L-glutamic acid (Glu), Fura-2/AM, and calcium ionophore A23187 were purchased from Sigma, USA.METHODS: PC12 cells in the model and drug treatment groups were separately incubated in glucose-free Hank's buffered saline solution + Na2S2O4, (2 mmol/L) for 6 hours, Glu (200 μ mol/L)plus A23187 (0.05 μmol/L) for 6 hours, KCI (50 mmol/L) for 1 hour, and caffeine (5 mmol/L) for 3 hours to establish models of intracellular Ca2+ overload induced by oxygen and glucose deprivation, Glu, A23187, high K+, or caffeine. In addition, control cells were incubated in high-glucose DMEM culture medium.MAIN OUTCOME MEASURES: [Ca2+]i changes in PC12 cells exposed to oxygen-glucose deprivation, Glu, A23187, high K+, or caffeine were detected using spectrofluorometer.RESULTS: PQS blocked the [Ca2+]i increase induced by oxygen-glucose deprivation, Glu,A23187, high K+, or caffeine. In particular, high-dose PQS was most effective (P < 0.01). PQS significantly inhibited Glu- or caffeine-induced [Ca2+]i increases in the absence of extracellular Ca2+, but nimodipine did not.CONCLUSION: PQS blocked intracellular Ca2+ overload induced by oxygen-glucose deprivation,Glu, A23187, high K+, or caffeine. This mechanism might be involved in the attenuation of neuronal apoptosis following ischemic brain injury.