Neuroprotective Effects of Thymoquinone in Aβ 1–42 ‐induced Toxicity in SK‐N‐SH Neuronal Cells

Alzheimer's disease (AD) is a consequence of an imbalance between the generation and clearance of amyloid‐beta peptide, which may be involved in neuronal death in the brain. Thymoquinone (TQ) is a phytochemical compound obtained from the seeds of Nigella sativa (black cumin seed oil). Earlier, we have shown that TQ inhibited inflammation in primary microglia and human neuroblastoma cells (1). However, nothing is known about its effect on Aβ‐induced neurotoxicity. This study was designed to investigate neuroprotective effect of TQ in Aβ 1–42 treated SK‐N‐SH neuroblastoma cells. Cultured SK–N–SH cells were treated with TQ (2.5, 5 and 10 μM) prior to exposure to Aβ 1–42 (10 μM). Levels of prostaglandin E 2 (PGE 2 ) production was measured using enzyme immunoassay (EIA), while ELISAs were used to detect levels of pro‐inflammatory cytokines tumour necrosis factor‐alpha (TNFα), Interleukin‐1 beta (IL‐1β) and interleukin‐6 (IL‐6). Further experiments were carried out to evaluate the effect of TQ on intracellular reactive oxygen species (ROS) levels in Aβ‐treated SK‐N‐SH cells using the fluorescent 2′,7′‐dichlorofluorescin diacetate (DCFDA)‐cellular reactive oxygen species detection assay kit. The effect of TQ on neuronal DNA fragmentation was conducted using the DNA Fragmentation assay. Immunofluorescence experiments were carried out to detect NF‐kB‐p65 protein localisation and MAP2 expression. MTT assay was used to determine the effect of TQ on Aβ 1–42 ‐induced SK‐N‐SH viability, while LDH levels in supernatants were determined using the CytoTox 96® non‐radioactive cytotoxicity assay kit. Results showed that TQ (2.5, 5 and 10 μM) produced concentration‐dependent and significant inhibition of PGE 2 , TNFα, IL‐1β and IL‐6. ROS levels and DNA fragmentation was significantly increased by Aβ 1–42 (10 μM) in SK‐N‐SH cells, while the compound at 5 and 10 μM markedly inhibited cellular ROS and DNA fragmentation. These concentrations of the compound also reduced LDH levels in Aβ‐induced SK‐N‐SH cells, while MTT results showed that Aβ 1–42 ‐mediated neuronal death was significantly reversed by TQ (2.5, 5 and 10 μM). Immunofluorescence experiments showed that Aβ 1–42 (10 μM)‐induced localisation of NF‐kBp65 and suppression of neuronal marker MAP2. However, pre‐treatment with TQ (2.5–10 μM) resulted in suppression of nuclear accumulation of NF‐kB‐p65 and reversed the expression of MAP2. In conclusion, our study demonstrated neuroprotective effects of TQ against Aβ‐induced neurotoxicity in vitro, which may provide a potential therapeutic strategy for treating neurodegenerative disorders like AD. 1Thymoquinone produced significant inhibition of PGE 2 , TNFα, IL‐1β and IL‐6.2Immunofluorescence experiments on NF‐kBp65.3Immunofluorescence experiments on neuronal marker MAP24Experiments on cellular ROS and DNA fragmentation