Although many causes have been suggested, the concept of oxidative stress has often been put forward as one of the important causes of neurodegenerative diseases. Natural antioxidants such as flavonoid polyphenols are essential in protecting neuronal cells from undergoing lipid peroxidation and cell death. The present study was designed to elucidate the antioxidant mechanisms of rutin and isoquercitrin in 6-OHDA induced PC12 rat pheochromocytoma cells. Pre-treatment of PC12 cells with rutin or isoquercitrin markedly (p<0.05) reduced the toxic effects caused by exposure to 6-OHDA as this was found to increase the viability of the PC12 cells. In addition, rutin and isoquercetrin were also found to elevate the levels of the endogenous antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase, catalase as well as glutathione. Rutin and isoquercitrin attenuated lipid peroxidation caused by 6-OHDA with p<0.05. Gene expression study using the qPCR array technique showed rutin suppressed the expression of the Parkin genes such Park 5, Park 7 and pro-apoptotic gene Casp3 gene in the PC12 cells exposed to 6-OHDA whilst isoquercitrin pre-treatment down-regulated expression of the Park 5 and Park 7 genes in these cells. Moreover, pre-treatment with rutin stimulated the expression of the TH gene in the PC12 cells. Tyrosine hydroxylase, the protein coded by the TH gene is crucial for biosynthesis of dopamine. Thus, quercetin glycosides treatment may present a novel approach in improving neurodegenerative diseases, including Parkinson’s (PD) and Alzheimer’s (AD) diseases.

Several natural bioactive compounds have been recognised to mediate neuroprotective effects by modulating events associated with antioxidant, antiinflammatory and anti-apoptotic mechanisms. One of such natural compounds is tocotrienol, an unsaturated form of vitamin E. The studies on neuroprotective effects of tocotrienol and tocotrienol rich fractions have shown that these compounds are effective in alleviating oxidative stress induced apoptosis and neuroinflammation. In this study, the effects of tocotrienol isoforms (α-, δ-, γ- tocotrienols) and tocotrienol rich fractions on antioxidant and non-antioxidant pathways, as well as alterations in proteomic profiling and DNA methylation processes, were elucidated on a cell-based model. In phase 1 of the studies, a cellular model of Parkinson disease was developed by differentiating the SH-SY5Y neuroblastoma cells using low serum and retinoic acid for 6 days. The established cellular model was investigated for the expression of dopaminergic characteristics by evaluating the neuronal morphology, biochemical changes and expression of dopamine receptors. The differentiated SH-SY5Y cells were pre-treated with tocotrienol isoforms and tocotrienol rich fraction prior to exposure with 6-hydroxydopamine demonstrated a significant elevation of antioxidant enzymes and up-regulated the dopamine biosynthesis parameters. Strikingly, α-tocotrienol displayed a remarkable increase in superoxide dismutase enzymes and tocotrienol rich fraction was effective in the blocking of α-synuclein leakage and up-regulation of dopamine and tyrosine hydroxylase enzymes in differentiated SH-SY5Y cells. The quantitative label-free proteome profiling of tocotrienols treated differentiated SH-SY5Y cells revealed the up-regulation of proteins that play crucial roles in several protective pathways including spliceosome, systemic lupus erythematosus (SLE) and ribosome pathways. Tocotrienol isoforms (-α, -δ, -γ) demonstrated remarkable increases in the expression of critical proteins in differentiated SHSY5Y cells namely Neuroblast differentiation-associated protein (AHNAK) and vimentin (Vim). Finally, the genome-wide associated studies (GWAS) using Infinium EPIC DNA methylation uncovered the differential methylation patterns implicated by tocotrienol isoforms in 6-hydroxydopamine induced differentiated SH-SY5Y cells. The data provided evidence on tocotrienol implicated differentially methylated regions (DMR) in Parkinson’s disease-related genes. The GWAS showed that γ-tocotrienol pre-treatment resulted in the highest number of differentially methylated CpG sites followed by δ- and α-tocotrienols in comparison with 6-hydroxydopamine. We also found that tocotrienol isoforms effectively reversed the 6-hydroxydopamine induced DMRs including metallothionein-1F (MT1F), cellular communication network factor 2 (CTGF) and proteosome 20S subunit beta 9 (PSM9) genes, E3 ubiquitin-protein ligase (RNF135) and T-complex 1 (TCP1) genes. In a nutshell, this study has clearly exposed the novel pathways of tocotrienols in protecting the dopaminergic cells against 6-hydroxydopamine induced cytotoxicity.