Hydrogen sulfide inhibits beta-amyloid peptide-induced apoptosis in PC12 cells and the underlying me

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BACKGROUND: Studies have demonstrated that hydrogen sulfide (H2S) levels are 55% lower in brains of Alzheimers disease (AD) patients than in age-matched normal individuals, which suggests that H2S might be involved in some aspects of AD pathogenesis.OBJECTIVE: To observe the protective mechanisms of varied concentrations of H2S against β -amyloid-peptide (A β) induced apoptosis in pheochromoytoma (PC12) cells, and to analyze the pathway of action.DESIGN, TIME AND SETTING: A controlled, observational, in vitro experiment was performed at Nenrophysiology Laboratory in Zhougshan Medical School, Sun Yat-sen University between July 2006 and May 2007.MATERIALS: PC12 cells were provided by the Animal Experimental Center of Medical School of Sun Yat-sen University. Glybenclamide, rhodamine123, and dihydrorhodamine123 were purchased from Sigma (USA).METHODS: PCI2 cells were incubated at 37℃ in a 5% CO2-enriched incubator with RPMI-1640 medium, supplemented with 5% horse-serum and 10% fetal bovine serum. Cells in logarithmic growth curves received different treatment: The PC12 cells were maintains at 37℃ with the original medium, then incubated in A β 25-35, sodium hydrosulfide (NariS), glybenclamide, NailS+ A β 25-35, or pretreated with glybenelamide 30 minutes prior to administration of and A β 25-35, respectively. MAIN OUTCOME MEASURES: (1) The survival rate of PC12 cells was detected by MTT assay and Hoechst staining. (2) The apoptosis rate of PC12 cells was detected utilizing flow cytometry with propidium iodide staining, and morphological changes of apoptotic cells were observed. (3) The mitochondrial membrane potential was detected by Rhodamine 123-combined flow cytometry. (4) The intracellular reactive oxygen species content was detected by dihydrorhodamine123-combined flow cytometry. RESULTS: A β 25-35 induced significantly decreased viability and increased percentage of apoptosis in PC 12 cells, as well as dissipated mitochondrial membrane potential expression and an overproduction of reactive oxygen species. When PC12 cells were co-treated with Nails and A β 25-35, the decreased cell viability induced by 20 μ mol/L A β 25-35 was concentration-dependently blocked by NariS (50, 100, and 200 p mol/L). NaHS (100 μ mol/L) obviously reduced the percentage of apoptotic PCI2 cells induced by 20 μ mol/L A β 25-35. In addition, 100 μ mol/L NariS inhibited mitochondrial membrane potential dissipation and reactive oxygen species overproduction. When the ATP-sensitive K channel (KATP) inhibitor, glybenclamide, was administered 30 minutes prior to NailS and A β 25-35 treatment, the NariS-dependent cellular protection was partly blocked. This resulted in reduced PC12 cell viability and increased the percentage of apoptosis, as well as significantly blocked mitochondrial membrane potential preservation and inhibited reactive oxygen species overproduction due to Naris treatment.CONCLUSION: NaHS protected PC 12 cells against A β 25-35-induced damage. NariS-dependent cellular protection was associated with mitochondrial membrane potential preservation and inhibition of reactive oxygen species overproduction. The KATP channel inhibitor, glybenclamide, significantly blocked the cellular protective effects of NariS, indicating that KATP channel activation plays an important role in NariS-induced protection of PC 12 cells to A β 25 35-induced damage.
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