Protective Effects Of Curcumin On Mouse Neuroblastoma Neuro-2A Cells Against Oxidative Damage

Neurodegenerative disorders are strongly connected with oxidative stress, which is caused by reactive oxygen species (ROS). Curcumin is a natural polyphenol antioxidant extracted from Curcuma longa. Yet until now, there are still few researches focused on the neuroprotective effects of curcumin and its underlying mechanisms have not been fully defined, especially at cellular level. In current study, H₂O₂-induced oxidative stress model in mouse neuroblastoma Neuro-2A cells was employed to research the neuroprotective effects and its potential mechanisms of curcumin.Neuro-2A cells were seeded into culture plates, cultured overnight at 37℃, and pretreated with curcumin at different concentrations for 1 h before 30μmol/L H₂O₂ exposure for 24 h. Cell viability was measured by MTT assay, and result showed that pretreatment with 25μg/ml curcumin for 1 h enhanced the percentage of viable cells by 8.5%. Cell apoptosis was assessed by nuclear staining, and result showed that the proportion of apoptotic cells was decreased by 15.1%, which indicated that curcumin could inhibit H₂O₂-induced apoptosis in Neuro-2A cells. The level of intracellular total antioxidant capacity, which was determined by colorimetric method, was boosted by 15.9% in 25μg/ml curcumin-pretreated group. Intracellular ROS accumulation, mitochondrial membrane potential loss, and Ca2+ influx were tested by flow cytometry, and results displayed that curcumin could be effect to suppress the augmentation of ROS level and intracellular calcium induced by H₂O₂, furthermore, curcumin also reduced the percentage of the Rhodamine 123 negative cells by 11.81%, which suggested that curcumin is conducive to inhibit the loss of mitochondrial membrane potential. The levels of two antiapoptotic proteins, poly(ADP-ribose) polymerase (PARP) and B cell lymphoma/lewkmia-2 (Bcl-2), which were measured by western blot analysis, were enhanced by 12.1%, and 12.0%, respectively, through pretreating with 25μg/ml curcumin for 1 h. In addition, nuclear and cytoplasmic protein extraction method, combined with western blot analysis, was applied to examine the activation of Nuclear factor-κ-gene binding (NF-κB) signaling pathway which is related to apoptosis and inflammation, and curcumin was found to block H₂O₂-mediate degradation of IκBαwhich was increased by 34.3%, and subsequent activation and relocation of NF-κB, thus inhibit the expression of its target gene Cyclooxygenase-2 (COX-2) which was decreased by 31.0%.In a word, the results of our study suggested that curcumin could significantly protect Neuro-2A cells against oxidative damage induced by H₂O₂. The specific neuroprotective mechanisms of curcumin included reinforcing the endogenous total antioxidant capacity, suppressing intracellular ROS accumulation, maintaining normal mitochondrial membrane potential and calcium homeostasis, improving the expression of antiapoptotic proteins, as well as blocking the activation of NF-κB signaling pathway. Taken together, we speculated that curcumin has great potential for preventing and treating the neurodegenerative diseases associated with oxidative stress.