NEUROPROTECTIVE MECHANISMS OF ORIENTIN AGAINST HYDROGEN PEROXIDE-INDUCED OXIDATIVE DAMAGE IN SH-SY5Y CELLS

Abstract

The current treatment for neurodegenerative diseases (ND) is only the medications which treat the symptoms rather than the cause. Such medications for examples donepezil, galantamine and rivastigmine that used in treating Alzheimer’s diseases resulted in various adverse effects to the patients. Therefore, natural products or plants derivatives might be a better alternative to prevent or to treat ND. This study targets on one of the flavonoid compounds, orientin, which is a water-soluble flavonoid C-glycoside isolable from Ocimum sanctum. It has been suggested to have antioxidant, anti-inflammation, cardioprotective, antiviral, antibacterial, radioprotective, neuroprotective, anti-adipogenesis and anti-nociceptive properties. All these properties, particularly the antioxidant ability of orientin has portrayed the potential of orientin in combating ND. As oxidative stress has been highly implicated in the progression of ND, hence, this leads to the study on the involvement of Nrf2/Keap1 redox signalling, PI3K/Akt survival and MAPK/ERK apoptosis pathways in the hydrogen peroxide (H2O2)-induced oxidative damage in SH-SY5Y neuroblastoma cells. Generally, the cells were treated with 10 μM and 20 μM of orientin, which are the pre-determined half maximum non-toxic dose (½ MNTD) and maximum non-toxic dose (MNTD) for 24 h at 37ºC and subsequently with 150 μM of H2O2 oxidative induction. Cells were then subjected to the measurement of nitric oxide (NO) and intracellular calcium (Ca2+) levels utilising Griess reagent assay and Molecular Probes® Fluo-4 NW Calcium Assay Kit, respectively. The mitochondrial membrane potential (MMP) and annexin V/propidium iodide apoptosis assays of the treated and untreated cells were also determined through flow cytometer. The regulation of Nrf2/Keap1 redox signalling, PI3K/Akt survival, MAPK/ERK apoptosis pathways was analysed by Western blotting. Results showed that ½ MNTD of orientin significantly reduced the NO levels. Similarly, the intracellular Ca2+ level was also significantly reduced by MNTD and 1/2 MNTD of orientin by 33.49% and 28.20%, respectively. Meanwhile, the treatment with orientin at MNTD and ½ MNTD restored the loss of MMP by 30.66% and 34.21% when compared with the H2O2 treatment alone. Pre-treatment of ½ MNTD of orientin reduced 27.36% of early apoptotic cells, 37.22% of late apoptosis cells and 46.37 % necrotic cells where the MNTD of orientin reduced 33.21 % of late apoptotic cells. In studying the molecular mechanisms, orientin was found to up-regulate Nrf-2/Keap-1 and PI3K/Akt pathways by increasing the expressions of Nrf-2, HO-1, p-PI3K-p85, p-Akt whilst down-regulate the MAPK/ERK pathway through inhibition of c-Raf. Taking all the findings into consideration, it could be concluded that orientin is able to exert its neuroprotection effects via the reduction of NO and intracellular Ca2+ levels, restoring the loss of MMP, preventing mostly late apoptotic and necrotic cells, up-regulating PI3K/Akt survival and Nrf2/Keap1 redox signalling pathways while down-regulating MAPK/ERK apoptosis pathway. The findings from this study will be useful for the better targeted effective therapies and the prevention of ND in the near future.