| Objective:Parkinson’s disease (PD) is a progressive neurodegenerative disorder with a prevalence of1-2%in people over the age of50. a-Synuclein is a major protein component of Lewy bodies and Lewy neuritis that are involved in the pathology of PD. Overexpression of a-synuclein in cells has been suggested to cause elevation of mitochondrial oxidant radicals and mitochondrial structural and functional abnormalities. But the detailed mitochondrial mechanism involved in the onset and advanced stage in PD is not clear. Asiatic acid (AA), a triterpenoid, is an antioxidant and used for depression treatment, our previous results showed that pretreatment of AA can prevent PD-like damage, but the therapeutical effect of PD and mechanism is unclear.Methods:In rotenone induced mitochondrial mild dysfunctional model, mitohormesis was assessed by counting the number of mitochondria using Mitotrack red fluorescent dye, determining protein expression of PGC-1α and its regulator-SIRTl by western blot, and measuring oxygen consumption by Clark Oxygen electrode. ROS production was detected by DCFHDA fluorescent dye and levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined by kit. In acute or chronic rotenone-induced mitochondrial dysfunctional model, cell viability was detected by MTT assay. ROS generation and mitochondrial membrane potential were measured by DCFHDA and JC-1fluorescent dye respectively and the expression of voltage-dependent anion channel (VDAC), PGC-la and Cytochrome C (Cyto C) were detected by western blot. VDAC oligomerization was investigated by chemical cross-link experiment. And then the effects of AA against acute rotenone-induced mitochondrial dysfunction in SH-SY5Y cells were investigated by MTT assay. In acute or chronic rotenone-induced mitochondrial dysfunction in SH-SY5Y cells, ROS generation, mitochondrial membrane potential, expressions of VDAC, PGC-1α, Cyto C and a-syn were detected. VDAC oligomerization were investigated by chemical cross-link experiment. In an a-syn transgenic drosophila Parkinsonism model, the neuroprotective effects of AA on improving motor dysfunction and lifespan were studied. The levels of MDA and GSH were studied by kit. In a-syn damaged isolated mouse brain mitochondria, mitochondrial function and the levels of Cyto C and a-syn were detected to analyze the effect of AA against a-syn induced neural injury. At last, we analyzed the possible target of AA by molecular modeling and fluorescent AA.Results:In mild rotenone induced mitochondrial dysfunctional model, compared with control group, rotenone (12.5-25nmol/L) significantly increased the amount of oxygen consumption and PGC-la, whereas rotenone at>25nmol/L has no effect on cells. GSH contents and GSH/GSSG were also significantly enhanced by rotenone at12.5nmol/L. Meanwhile quantity of mitochondria, ROS production and SIRT1protein expression increased in a dose-dependent way. These data indicate that low concentrations of rotenone can induce mitohormesis, which may be attributed to ROS production. On the other hand, in rotenone induced acute or chronic model, rotenone induced cells and mitochondrial functional injury through down-regulation expression and oligomerization of VDAC.On the other hand treatment of AA (0.01-100nmol/L) protected cells against the toxicity induced by rotenone. In addition, mitochondrial membrane potential dissipation and ROS elevation following the exposure of rotenone could be blocked by AA treatment. More interestingly, administration of AA inhibited BAX up-regulation and VDAC down-regulation induced by rotenone (100nmol/L) and prevented VDAC oligomeration. In5nmol/L rotenone induced chronic cellular injury model, AA can protect mitochondria, inhibit a-syn overexpression and mitochondrial translocation. In the Drosophila model,0.5-2mg/100g AA diet could significantly improve climbing ability and extended life-span and such effects might be attributed to its antioxidant properties. On isolated mitochondrial model, AA can attenuate the mitochondrial membrane potential decline induced by a-syn. Consequently, AA maintained the membrane integrity and ATP production. Moreover, AA also suppressed ROS production and apoptosis. Finally we demonstrated that AA achieved its protection by blocking the translocation of a-syn into mitochondria. The direct target of AA may related to VDAC.Conclusion:In the onset of PD, mitochondrial mild damage induced ROS production, and then stimuli mitohormesis, however, the acute and chronic injury in mitochondria induced mitochondrial dysfunction and down-regulation and oligomeration of VDAC. On the other hand, it is suggested that AA have neuroprotective effects against mitochondrial dysfunctional injury through preventing VDAC oligomeration, and the suppressing a-syn mitochondrial translocation, maintaining mitochondrial membrane integrity, inhibiting Cyto C released and against cells’apoptosis. Our results suggest that mitochondria may play crucial role in PD and AA is an excellent candidate for Parkinson’s disease (PD) prevention and therapy. |
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