Studies On Molecular Mechanisms Of Rotenone-induced Hydrogen Peroxide Inhibition Of Akt/XIAP Signaling Pathways Leading To Neuronal Apoptosis

The present study, using techniques and methods of cell culture, MTS assay, fluorescence staining and imaging, RNA interference, Western blotting, etc., and employing PC 12 and primary neurons as experiment objects, synthetically investigated the effects of oxidative stress H2O2 on Akt pathway and mechanisms of its regulation of related XIAP signaling pathway in the process of rotenone-induced neuronal apoptosis, and discussed the mechanisms of rotenone-induced H2O2 inhibition of Akt and XIAP pathways contributing to neuronal apoptosis. The results were summarized as follows:1 Rotenone induces H2O2-dependent neuronal apoptosisPC 12 cells and primary neurons were treated with 0-1 μM rotenone for 24 h, or treated with rotenone (0.5 and 1 μM) for 24 h following pretreatment of catalase (CAT, 350 U/ml) for 1 h. Specific fluorescent probe H2DCFDA was used to analyze intracellular H2O2, cell viability was evaluated by morphology and MTS assay, and expression of caspase-3 and PARP proteins was determined to evaluate apoptotic signal status by Western blotting. The results showed that rotenone significantly induced H2O2 elevation and viability reduction, as well as the increases of cleaved-caspase-3 and cleaved-PARP in a dose-dependent manner in PC12 cells and primary neurons; pretreatment with CAT improved rotenone-induced morphological changes and viability reduction in the cells, and attenuated rotenone-increased expression of cleaved-caspase-3 and cleaved-PARP. It suggested that rotenone-induced H2O2 generation elicits oxidative stress leading to apoptosis in neuronal cells.2 Rotenone induction of H2O2 inhibits Akt pathway leading to neuronal apoptosisPC 12 cells and/or primary neurons, or PC 12 cells infected with Ad-myr-Akt and Ad-GFP, respectively, were treated with 0-1 μM rotenone for 24 h, or treated with rotenone(0.5 and 1 μM) for 24 h, or treated with rotenone(0.5 and 1 μM) for 24 h following pretreatment of catalase (CAT,350 U/ml) for 1 h. Expressions of Akt and its downstream proteins were determined by Western blotting, fluorescent probe H2DCFDA was used to analyze intracellular H2O2 levels, cell viability was evaluated by MTS assay, and apoptotic cells were detected by DAPI staining. The results showed that rotenone inhibited phosphorylation of Akt, Foxo3a and GSK3 in a dose-dependent manner; CAT obviously prevented from rotenone-inhibited phosphorylation of Akt, Foxo3a and GSK3, implying that rotenone induction of H2O2 is involved in inhibition of Akt pathway. Overexpression of Akt diminished rotenone-induced cell viability reduction and apoptosis increase, and in particular, overexpression of Akt can significantly reduce the H2O2 generation, suggesting that Akt plays a key role in rotenone-induced H2O2-dependent neuronal apoptosis. Our data uncover that rotenone induction of H2O2 inhibits Akt pathway contributing to neuronal apoptosis.3 Rotenone induction of H2O2 inhibits Akt contributing to neuronal apoptosis via XIAP pathwayPC 12 cells and/or primary neurons, or PC 12 cells infected with Ad-myr-Akt and Ad-GFP, respectively, or PC 12 cells transfected with exogenous plasmid which overexpress XIAP, were treated with 0-1 μM rotenone for 24 h, or treated with rotenone(0.5 and 1 μM) for 24 h, or treated with rotenone(0.5 and 1 μM) for 24 h following pretreatment of catalase (CAT,350 U/ml) or XIAP inhibitor Embelin (100 ng/ml) for 1 h. Expressions of XIAP and its downstream proteins were determined by Western blotting, fluorescent probe H2DCFDA was used to analyze intracellular H2O2 levels, cell viability was evaluated by MTS assay, and apoptotic cells were detected by DAPI staining. The results showed that rotenone significantly inhibited phosphorylation of XIAP and its downstream Mdm2, as well as markedly increased p53 and inhibited survivin in a dose-dependent manner; CAT obviously prevented from rotenone-induced inhibited phosphorylation of XIAP and its downstream Mdm2, as well as increase of p53 and inhibition of survivin; Overexpression of XIAP inhibited rotenone-induced neuronal apoptosis; Embelin inhibition of XIAP strengthened H2O2 production and apoptosis in neuronal cells induced by rotenone; Overexpression of Akt blocked rotenone-inhibited XIAP phosphorylation, its downstream proteins such as Mdm2, p53 and survivin were successively improved as well. It suggested that rotenone induction of H2O2 inhibits Akt contributing to neuronal apoptosis via XIAP pathway.