Neuroprotection by association of palmitoylethanolamide with luteolin in experimental Alzheimer's disease models: the control of neuroinflammation.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Its neuropathological hallmarks include deposition of beta amyloid (Aβ) fibrils in senile plaques. Numerous biochemical events, leading to Aβ neurotoxicity in AD, have been proposed and it seems that neuroinflammation plays a prominent role among these. Thus, since inflammatory processes and oxidative stress are considered to play an important role in neuroinflammatory disorders and in AD pathology, in the present work we decided to test a new composite, which is a formulation constituted of an anti-inflammatory compound such as palmitoylethanolamide (PEA) and the well recognized antioxidant flavonoid luteolin (Lut), subjected to an ultra-micronization process, here designated co-ultraPEALut. We investigated the effect of co-ultraPEALut in both an in vitro and ex vivo organotypic model of AD. For the in vitro model, we used human neuronal cells, obtained by differentiating SH-SY5Y neuroblastoma cells into sustainable neuronal morphology. These well differentiated cells express features specific to mature neurons, such as synaptic structures and functional axonal vesicle transport, making this new concept for in vitro differentiation valuable for many neuroscientific research areas, including AD. Differentiated SH-SY5Y cells were pre-treated with co-ultraPEALut (reference concentrations: 27, 2.7 and 0.27 µM PEA) for 2 h. AD features were induced by Aβ₁₋₄₂ stimulation (1 µM). Twenty-four hours later cell vitality was evaluated by the colorimetric MTT assay, whereas the neuroinflammation underling AD was observed by Western blot analysis for IκBα degradation and nuclear factor-κB traslocation, as well as glial fibrillary acidic protein expression. For the organotypic model of AD, hippocampal slice cultures were prepared from mice at postnatal day 6 and after 21 days of culturing the slices were pre-treated with co-ultraPEALut (reference concentrations: 27, 2.7 and 0.27 µM PEA) for 2 h and then incubated with Aβ₁₋₄₂ (1 µg/ml) for 24 h. Pre-treatment with co-ultraPEALut significantly reduced inducible nitric oxide synthase and glial fibrillary acidic protein expression, restored neuronal nitric oxide synthase and brainderived neurotrophic factor and reduced the apoptosis. Taken together our results clearly showed that co-ultraPEALut is able to blunt Aβ-induced astrocyte activation and to exert a marked protective effect on glial cells. These findings suggest that the association of co-ultraPEALut may provide an effective strategy for AD.