| Objective: Dopaminergic neurons of PC12 cells were treated with arsenic or luteolin alone and combined.The morphology of cell apoptosis,the level of reactive oxygen species(ROS),the apoptosis markers of Caspase3 activity,the DNA damage index of ?-H2 AX and the expression of ?-synuclein(?-Syn)were measured under different treatment conditions.To investigate the protective effect of luteolin on arsenic induced apoptosis and its potential mechanism,and reveal the antagonistic mechanism of ?-Syn on arsenic induced apoptosis.Methods: PC12 cells were treated with different concentrations of arsenic or luteolin,cell viability was detected by MTT assay and the appropriate treatment dose were selected for subsequent experiments.The morphology of cell apoptosis were observed by Hoechst33258 staining.The percentage of apoptotic cells and the apoptosis level were observed by acridine orange/ethidium bromide staining(AO/EB)and flow cytometry,respectively.Meanwhile,the intracellular ROS levels,Caspase3 activity and the expression levels ?-H2 AX and the expression of ?-synuclein(?-Syn)were also measured.siRNA technology was used to reduce the expression of ?-Syn,and to investigate the antagonistic effect of ?-Syn on the apoptosis of PC12 cells induced by arsenic.Results: PC12 cells were treated with different concentrations of arsenic(0.1,0.25,0.5,1,2.5,5,10,20 and 40 M)for 24 h,our results found that ? 1 ?M arsenic treatment could lead to cell activity decreased significantly,of which 2.5 M after arsenic exposure the cell survival rate was(71.12+7.15)%.However,different doses of luteolin(5,10,20 M)had no significant effect on cell viability.2.5 M arsenic combined with different concentrations of luteolin showed significant protective effect,thus 2.5 M of arsenic and 20 M luteolin combined treatment were used for subsequent experiments.The results showed that the apoptosis of the arsenic and luteolin combined group were significantly lower than those of arsenic treated alone,while the cell viability of combination group was higher than that of single arsenic-treated group(P<0.05).Moreover,the ROS content,Caspase 3 activity,?-H2 AX and?-Syn expression in the combined group were significantly lower than those in the arsenic treated alone group(P<0.05).Downregualtion of ?-Syn by siRNA remarkably enhanced the benefit effect of luteolin against arsenite-induced apoptosis and cytotoxicity,the results showed that the cell activity,ROS content and apoptosis level were significantly improved than that of non-?-Syn intervention group.Conclusion: Luteolin could reduce the ROS level and Caspase 3 activity by reducing the accumulation of ?-Syn,alleviate the damage of DNA,and then antagonize the apoptosis induced by arsenic.The results suggest that luteolin could be used as an effective antioxidant for the prevention and treatment of Parkinson's disease... |
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