Study on the protective effects and mechanisms of DMDD isolated from Averrhoa carambola L. root on differentiated SH-SY5Y cell damage induced by α-Synuclein PFFs

Objective To explore the protective effects and mechanisms of different concentrations of Averrhoa carambola L. root extract 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) on α -Syn PFFs-induced differentiated SH-SY5Y cell damage. Methods The SH-SY5Y cells differentiated by retinoic acid were treated with different concentrations of DMDD, followed by detecting the cell viability by MTS to determine the low, medium, and high concentrations of DMDD for use in these cells. The cells were treated with various concentrations of DMDD in an established model of cell damage induced by 5 μg/mL α-Syn PFF. Hoechst 33342 staining and nitric oxide kit were used to detect the number of cells with nuclear damage and intracellular nitric oxide production, respectively. The protein expression levels of tyrosine hydroxylase (TH), polyadenylate diphosphate (PAR) and polyadenylate diphosphate ribozyme-1 (PARP-1) were measured by western blotting. Immunofluorescence was used to detect the number of cells positive for phosphorylated serine 129 α-synuclein (pS129-α-Syn), phosphorylated histone H2AX (γH2AX), and nuclear translocation of apoptosis-inducing factor (AIF). Results Hoechst 33342 staining demonstrated that the number of cells with nuclear damage was significantly decreased in all DMDD treatment groups compared with the α-Syn PFFs model group (P < 0.001). The nitric oxide assay results showed that nitric oxide production was significantly reduced in the DMDD treatment groups compared with the α-Syn PFFs model group (P < 0.01 or P < 0.001). Western blotting revealed that compared with the α-Syn PFFs model group, TH protein expression level was significantly increased in the high DMDD concentration treatment group (P < 0.05), while PAR protein expression levels were significantly decreased in the medium and high DMDD concentration treatment groups (P < 0.05), and the protein expression levels of PARP-1 and Cleaved PARP-1 were significantly decreased in the high DMDD concentration treatment group (P < 0.05). Immunofluorescence results showed that compared with the α-Syn PFFs model group, the number of pS129-α-Synpositive cells was decreased significantly after treatment with medium and high concentrations of DMDD (P < 0.05 or P < 0.01), while the number of γH2AX-positive cells and AIF nuclear translocation cells was decreased significantly in all DMDD treatment groups (P < 0.01 or P < 0.001). Conclusion DMDD may attenuate the damage in differentiated SH-SY5Y cells induced by α-Syn PFFs through inhibition of PARP-1-dependent cell death mediated by the PARP-1/AIF pathway.