The Association of Palmitoylethanolamide with Luteolin Decreases Neuroinflammation and Stimulates Autophagy in Parkinson’s Disease Model

Abstract

Parkinson's disease (PD) is a disorder resulted by degeneration of dopaminergic neurons. To counteract the neuroinflammation and oxidative stress of PD, we decided to test a new composite constituted by palmitoylethanolamide (PEA) and luteolin (Lut), in a mass ratio of 10:1, respectively (co-ultraPEALut). In this study the neuroprotective property of the new compound was investigated. For the in vivo model of PD, mice received four injections of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). Starting 24 h after the first administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we treated animals with co-ultraPEALut daily until 7 days. On day 8, brains were processed for Western blotting and immunohistochemical analysis. Treatment with co-ultraPEALut reduced the specific markers of PD (tyrosine hydroxylase immunopositive), and the increased levels of activated astrocytes and pro-inflammatory cytokines as well as inducible nitric oxide synthase. Further, the possible association of autophagy with the beneficial effects of coultraPEALut. Western blot analysis and immunofluorescence staining showed that co-ultraPEALut administration increased autophagy process. These data were confirmed by an in vitro model, using SH-SY5Y neuroblastoma cells. Western blot analysis showed that co-ultraPEALut pre-treatment maintained high Beclin-1 and p62 expression, while continued to inhibit the p70S6K expression. Altogether, these results put forward that treatment with co-ultraPEALut is able to modulate both the neuroinflammatory process and the autophagic pathway involved in PD, actions which may underlie its neuroprotective effect.

The Neuroprotective Effect of Dimethyl Fumarate in an MPTP-Mouse Model of Parkinson's Disease: Involvement of Reactive Oxygen Species/Nuclear Factor-κB/Nuclear Transcription Factor Related to NF-E2

“…Brain tissues were taken 8 days after the MPTP injection. Tissue sections were stained with hematoxylin/eosin and studied by using light microscopy connected to an imaging system (AxioVision; Zeiss, Milan, Italy) as previously described (62). Histological assessment was made by blinded observation, and slides were scored for severity of pathological profiles after hematoxylin/eosin staining by using a semiquantitative 5-point rating: 0 = no pathology; 1 = mild pathology; 2 = moderate pathology; 3 = severe pathology; 4 = more severe pathology (63).…”

“…Sections were processed for immunofluorescence staining as previously described (63). Sections were incubated with rabbit anti-Nrf-2 (1:100; Santa Cruz Biotechnology) or mouse monoclonal anti-neuronal nuclei (anti-NeuN) (1:100; Merck-Millipore) in a humidified chamber overnight at 37°C.…”

“…Neuroblastoma SH-SY5Y cells (ATCC Ò CRL-2266Ô) were cultured as previously described (62). In preliminary experiments to assess cell viability, 3 · 10 4 cells were plated in a volume of 150 ll in 96-well plates.…”

“…Cells were then preincubated for 30 min with 1 lM TR, an Nrf-2 inhibitor, as previously described (55) before treatment with DMF (30 lM). The concentration of MPTP (3 mM) used was based on previous in vitro studies (62). After 24 h, cell lysates were prepared for Western blot analysis.…”

“…Western blot analysis was performed as previously described (62). The membrane was incubated overnight at 4°C with: anti-iNOS (1:500; Transduction Labs) and anti-Mn-SOD (1:500; Merck-Millipore).…”