Molecular Insights into the Nociceptive Modulation by Palmitoylethanolamide and Equisetum arvense Extract: An In Vitro Study Across the Blood–Brain Barrier

<b>Background:</b> The blood-brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) but also limits drug delivery. Insufficient knowledge of how the CNS promotes the onset and maintenance of peripheral neuropathic pain limits therapeutic methods for the treatment of persistent neuropathic pain. Thus, this study aimed to evaluate the ability of a novel combination of Palmitoylethanolamide (PEA) and <i>Equisetum arvense</i> L. (<i>Equisetum A.L.</i>) to cross the BBB and modulate nociceptive pathways. <b>Methods:</b> Using a humanised in vitro BBB tri-culture model, the permeability, cytotoxicity, and integrity of the barrier were assessed after exposure to two different PEA forms, PEA ultramicronized (PEA-um) and PEA80mesh, <i>Equisetum A.L.</i>, and a combination of the last two samples. The samples exhibited no cytotoxicity, maintained tight junction integrity, and efficiently crossed the blood-brain barrier (BBB), with the combination displaying the highest permeability. The eluate from the BBB model was then used to stimulate the co-culture of CCF-STTG1 astrocytes and SH-SY5Y neurons pre-treated with H₂O₂ 200 µM. <b>Results:</b> Treatment with the combination significantly increased cell viability (1.8-fold, <i>p</i> < 0.05), reduced oxidative stress (2.5-fold, <i>p</i> < 0.05), and decreased pro-inflammatory cytokines (TNFα, IL-1β) compared to single agents. Mechanistic analysis revealed modulation of key targets involved in pain pathways, including decreased FAAH and NAAA activity, increased levels of endocannabinoids (AEA and 2-AG), upregulation of CB2 receptor expression, enhanced PPARα activity, and reduced phosphorylation of PKA and TRPV1. <b>Conclusions:</b> These findings suggest that the combination of PEA and <i>Equisetum A.L.</i> effectively crosses the BBB and exerts combined anti-inflammatory and analgesic effects at the CNS level, suggesting a possible role in modulating neuroinflammatory and nociception responses.