| Parkinson’s Disease(PD)is the second most common neurodegenerative disease after Alzheimer’s disease(AD).PD patients are characterized by the degeneration of dopaminergic neurons in substantia nigra pars compacta(SNpc).Patients at early stage are characterized by non-motor symptoms such as anosmia,depression and cognitive dysfunction,while by motor symptoms such as slow movements,quiescent tremor and muscle stiffness at late stage.PD imposes a heavy burden on families and society.Therefore,it is urgent to find effective ways to prevent or reverse PD for governments and researchers.Under pathological conditions,it is prone to misfold and aggregate in neuronal cells to form intracellular contents named Lewy bodies(LBs),spread in central nervous system and eventually lead to the death of dopaminergic neurons.Targeting neurotoxicity of alpha-syn aggregates is an effective strategy for the prevention and treatment of PD.Small natural compounds have many advantages,such as their availability,small molecular weights,and low cytotoxicity,and most of them are food additives and traditional Chinese medicine components.Ginnalin A(GA)is a natural polyphenol compound extracted from red maple and Kujin tea,which has antiinflammatory,antioxidant and anticancer activities.Our previous work has proved that GA can activate the Nrf2-ARE signaling pathway to improve the antioxidant capacity of cells.GA can inhibit Aβ(1-42)aggregation,dissociate preformed fibrils and reduce cytotoxicity.GA was also found to inhibit α-syn aggregation and dislodge α-syn fibrils.However,whether GA can play a neuroprotective role by targeting the most toxic aggregates,soluble α-syn oligomers,has not been studied.We focus on the interaction between GA and soluble α-syn oligomers.First,circular dichroism(CD)was used to screen the formation time of soluble α-syn oligomers and to probe whether GA could change the typical conformation of theα-syn oligomers.The protective effect of GA on SH-SY5 Y cells was evaluated by CCK-8 cytotoxicity assay.Using the DCFH-DA probe,we tested the effect of GA on the reduction of intracellular reactive oxygen species(ROS)levels indued by soluble induced α-syn oligomers.Subsequently,the expression of Nrf2 and related antioxidant genes in SH-SY5 Y cells was analyzed by fluorescence quantitative PCR following treatments with GA and soluble α-syn oligomers.Finally,molecular docking simulation and isothermal titration calorimetry were used to analyze the interaction between andα-syn oligomers,to further investigate the underline mechanism of GA-targeting soluble α-syn oligomers.CD results showed that GA delayed the formation of soluble α-syn oligomers in a dose-dependent manner.CCK-8 cytotoxicity assay demonstrated that GA alleviated the cytotoxicity of soluble α-syn oligomers and protected nerve cells.DCFH-DA results showed that GA pretreatment could protect cells and reduce the intracellular ROS content induced by soluble α-syn oligomer.Fluorescence quantitative PCR results showed that the expression of Nrf2 and related antioxidant genes can be up-regulated by different treatments and GA pretreatment plays a protective role in the activation of NRF2-ARE antioxidant signaling pathway.Molecular docking simulation showed that GA can interact with α-syn oligomers(-5.36 kcal/mol)and the hydroxyl groups of GA form hydrogen bonds with amino acids in α-syn oligomers,which in turn disturb hydrogen bonds in α-syn oligomers and reduce the ability of α-syn to form β-sheet.ITC results again confirmed the binding of GA to α-syn oligomers(-6.45 kcal/mol).In conclusion,GA can target soluble α-syn oligomers in PD at multiple levels:(1)activating the intracellular antioxidant signaling pathway.(2)delaying the formation of soluble α-syn oligomers.(3)preventing binding of soluble α-syn oligomers to cell membranes.Therefore,GA is expected to be a desirable multifunctional drug candidate,providing more possibilities for the prevention and treatment of neurodegenerative diseases. |
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