Neuroinflammation, which is mainly triggered by microglia, is a key contributor to multiple neurodegenerative diseases. Natural products, and in particular Cannabis sativa L., due to its richness in phytochemical components, represent ideal candidates to counteract neuroinflammation. We previously characterized different C. sativa commercial varieties which showed significantly different chemical profiles. On these bases, the aim of this study was to evaluate essential oils and aqueous distillation residues from the inflorescences of three different hemp varieties for their anti-neuroinflammatory activity in BV-2 microglial cells. Cells were pretreated with aqueous residues or essential oils and then activated with LPS. Unlike essential oils, aqueous residues showed negligible effects in terms of anti-inflammatory activity. Among the essential oils, the one obtained from ‘Gorilla Glue’ was the most effective in inhibiting pro-inflammatory mediators and in upregulating anti-inflammatory ones through the modulation of the p38 MAPK/NF-κB pathway. Moreover, the sesquiterpenes (E)-caryophyllene, α-humulene, and caryophyllene oxide were identified as the main contributors to the essential oils’ anti-inflammatory activity. To our knowledge, the anti-neuroinflammatory activity of α-humulene has not been previously described. In conclusion, our work shows that C. sativa essential oils characterized by high levels of sesquiterpenes can be promising candidates in the prevention/counteraction of neuroinflammation.

Neuroinflammation is a critical aspect of various neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. This study investigates the anti-neuroinflammatory properties of oleocanthal and its oxidation product, oleocanthalic acid, using the BV-2 cell line activated with lipopolysaccharide. Our findings revealed that oleocanthal significantly inhibited the production of pro-inflammatory cytokines and reduced the expression of inflammatory genes, counteracted oxidative stress induced by lipopolysaccharide, and increased cell phagocytic activity. Conversely, oleocanthalic acid was not able to counteract lipopolysaccharide-induced activation. The docking analysis revealed a plausible interaction of oleocanthal, with both CD14 and MD-2 leading to a potential interference with TLR4 signaling. Since our data show that oleocanthal only partially reduces the lipopolysaccharide-induced activation of NF-kB, its action as a TLR4 antagonist alone cannot explain its remarkable effect against neuroinflammation. Proteomic analysis revealed that oleocanthal counteracts the LPS modulation of 31 proteins, including significant targets such as gelsolin, clathrin, ACOD1, and four different isoforms of 14-3-3 protein, indicating new potential molecular targets of the compound. In conclusion, oleocanthal, but not oleocanthalic acid, mitigates neuroinflammation through multiple mechanisms, highlighting a pleiotropic action that is particularly important in the context of neurodegeneration.

Neuroinflammation is a hallmark of numerous neurodegenerative diseases and is characterized by profound inflammatory, metabolic, and redox alterations in activated microglia. Although lipopolysaccharide (LPS)-stimulated BV-2 cells represent one of the most widely used in vitro models of neuroinflammation, a comprehensive understanding of the coordinated molecular adaptations underlying this model remains incomplete. The aim of this study was to characterize the proteomic remodeling of BV-2 cells exposed to LPS 100 ng/mL for 24 h and investigate the relationship between antioxidant and inflammatory responses across increasing levels of LPS exposure. Proteomic analysis identified 89 differentially expressed proteins involved in cytoskeletal organization, clathrin-mediated endocytosis, lysosomal function, immunometabolism, and redox regulation. Validation experiments confirmed changes in cytoskeletal and endocytic proteins, including Vimentin, Gelsolin, Disabled homolog-2, and clathrin light chain A, together with modulation of proteins involved in the lysosomal pathways and marked induction of the immunometabolic enzyme aconitate decarboxylase 1. Functional enrichment analysis identified Nuclear factor erythroid 2-related factor 2 (NRF2) as the most significantly activated upstream regulator, a prediction consistent with engagement of an adaptive antioxidant program. Interestingly, analysis of increasing LPS concentrations revealed that antioxidant responses were not proportional to the inflammatory stimulus. Expression of NRF2-controlled antioxidant enzymes progressively increased for LPS concentrations up to 500 ng/mL, but declined at 1000 ng/mL LPS, whereas pro-inflammatory mediators remained continuously upregulated. This pattern is consistent with the attenuation of antioxidant adaptation under the highest inflammatory burden tested. Together, these findings demonstrate that the BV-2/LPS model is characterized by a dynamic interplay between inflammatory, redox, metabolic, lysosomal, endocytic, and cytoskeletal pathways, providing a more comprehensive characterization to support the interpretation and experimental use of this neuroinflammatory model.