Cilnidipine mediates a neuroprotective effect by scavenging free radicals and activating the phosphatidylinositol 3-kinase pathway

초록

We investigated the neuroprotective effect and mechanisms of action of cilnidipine, a long-acting, second-generation 1,4-dihydropyridine inhibitor of L- and N-type calcium channels, in PC12 cells that were neuronally differentiated by treatment with nerve growth factor (nPC12 cells). To evaluate the effect of cilnidipine on viability, nPC12 cells were treated with several concentrations of this drug before performing viability assays. Free radical levels and intracellular signaling proteins were measured with the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate and western blotting, respectively. Cell viability was not affected by low concentrations of cilnidipine up to 150 mu M, but it was slightly decreased at 200 mu M cilnidipine. Following H2O2 exposure, the viability of nPC12 cells decreased significantly; however, treatment with cilnidipine increased the viability of H2O2-injured nPC12 cells in a concentration-dependent manner. Treatment with H2O2 resulted in a concentration-dependent increase in free radical levels in nPC12 cells, and cilnidipine treatment reduced free radical levels in H2O2-injured nPC12 cells in a dose-dependent manner. Cilnidipine treatment increased the expression of p85aPI3K (phosphatidylinositol 3-kinase) phosphorylated Akt, phosphorylated glycogen synthase kinase-3 (pGSK-3 beta), and heat shock transcription factor (HSTF-1) which are proteins related to neuronal cell survival, and decreased levels of cytosolic cytochrome c, activated caspase 3, and cleaved poly (ADP-ribose) polymerase (PARP), which are associated with neuronal cell death, in H2O2-injured nPC12 cells. These results indicate that cilnidipine mediates its neuroprotective effects by reducing oxidative stress, enhancing survival signals (e.g., PI3K, phosphorylated Akt, pGSK-3 beta, and HSTF-1), and inhibiting death signals from cytochrome c release, caspase 3 activation, and PARP cleavage.

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