D-Limonene protects PC12 cells against corticosterone-induced neurotoxicity by activating the AMPK pathway

The stress-induced hormone corticosterone initiates oxidative stress and inflammatory responses, culminating in cell apoptosis and neurological changes. We assessed the effects of D-Limonene on a PC12 cellular model of corticosterone-induced neurotoxicity, and whether these effects involved the AMP-activated protein kinase (AMPK alpha) pathway. PC12 cells were treated with corticosterone with or without D-limonene for 24 h. Western blots were performed to measure activation of AMPK pathway members [Silent mating type information regulation 2 homolog-1 (SIRT1), AMPK alpha, and nuclear factor (NF kappa B)], reactive oxygen species, inflammatory cytokines, and markers of apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was used to measure cell death after treatment. D-Limonene reversed the effects of corticosterone on PC12 cells: it decreased the levels of malondialdehyde (MDA) and nitric oxide (NO), activities of NADPH oxidase (p67-phox and p47-phox), expression of pro-inflammatory markers [inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF-alpha)], and expression of pro-apoptotic proteins [Bcl2 associated with X protein (Bax) and cleaved caspase-3)]. D-Limonene also increased levels of the antioxidant enzymes superoxide dismutase 1 (SOD1) and heme oxygenase 1 (HO-1) and the anti-apoptotic protein Bcl-2 while decreasing the number of TUNEL-positive cells. Dlimonene significantly activated AMPK alpha and suppressed NF-kappa B nuclear translocation through up-regulation of SIRT1. Addition of compound C, an AMPK inhibitor, severely weakened these neuroprotective effects of D-limonene. D-Limonene has a neuroprotective effect on corticosterone-induced PC12 cell injury induced by activating the AMPK alpha signaling pathway, and thereby inhibiting reactive oxygen species and inflammatory factors. These data suggest that D-limonene might protect against neuronal death to improve depressive symptoms.