2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione protects against MPP-induced neurotoxicity by ameliorating oxidative stress, apoptosis and autophagy in SH-SY5Y cells.

2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) is a cyclohexanedione compound extracted from the roots of Averrhoa carambola L. Several studies have documented its beneficial effects on diabetes, Alzheimer's disease, and cancer. However, its potential neuroprotective effects on Parkinson's disease (PD) have not yet been explored. The present study aimed to investigate the protective effects and underlying mechanisms of DMDD in a cellular model of PD. In this study, SH-SY5Y cells were incubated with or without DMDD following intoxication with the parkinsonian neurotoxin 1-methyl-4-phenylpyridine (MPP). Cell viability and apoptosis were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay and Hoechst 33,342 staining, respectively. The mitochondrial membrane potential (Δψm) was assessed through the JC-10 assay. The activities of superoxide dismutase (SOD) and the levels of reactive oxygen species (ROS) were measured using WST-8 and DCFH-DA assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore significant biological processes and pathways influenced by DMDD. Molecular docking was employed to predict the domains of potential protein targets interacting with DMDD. Western blotting was subsequently conducted to determine the protein expression levels of TH, Nrf2, Bax, Bcl-2, Caspase-3, Beclin-1, PARP, LC3-II, LC3-I, p-PI3K, PI3K, p-mTOR and mTOR. Our study showed that DMDD treatment significantly increased cell viability and reduced apoptosis in MPP-treated SH-SY5Y cells. In addition, DMDD treatment reversed the loss of TH expression and Δψm in MPP-exposed SH-SY5Y cells. Moreover, DMDD treatment reduced MPP+-induced ROS production by promoting SOD activity. Additionally, compared with those in the MPP group, the protein expression levels of Beclin-1, Caspase-3, and PARP and the LC3II/I ratio were significantly decreased, whereas the protein expression levels of Nrf2 and the Bcl-2/Bax, p-PI3K/PI3K, and p-mTOR/mTOR ratios were significantly increased in the DMDD-treated group. In conclusion, DMDD protects against MPP-induced cytotoxicity by mitigating oxidative stress, apoptosis, and autophagy. PI3K/mTOR signaling at least partly mediates the cytoprotective effect of DMDD.