Psilocybin suppresses inflammatory signalling and restores neuroplasticity in a dexamethasone-induced neuroinflammatory neurosphere model

Sterile neuroinflammation and impaired neuroplasticity are increasingly recognised as key biological processes underlying stress-associated brain dysfunction, including depression, anxiety, and cognitive decline. Damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) and S100 calcium-binding protein B (S100B) can trigger pattern-recognition receptors and promote assembly of the NLRP3 inflammasome, which has been proposed as a critical mechanistic link between innate immune signalling and synaptic alterations. Psilocybin, a serotonergic psychedelic acting primarily at 5-HT2A receptors, has recently attracted attention for its potential neuroplastic and anti-inflammatory properties; however, its cellular effects under defined neuroinflammatory conditions remain incompletely characterised, particularly in three-dimensional models that more faithfully recapitulate native cortical architecture. The aim of the present study was to evaluate the effects of psilocybin on inflammatory signalling and neuroplasticity in a dexamethasone-induced neuroinflammatory environment using a three-dimensional neurosphere platform. Primary cortical neurons isolated from embryonic rat cortex were cultured in a micro-spheroid device to generate uniform neurospheres of approximately 200 μm in diameter. Based on prior dose–response optimisation, dexamethasone (100 μM) was applied to induce a stress-associated neuroinflammatory condition. Psilocybin was initially screened at concentrations ranging from 0.1 to 100 μM to assess cytotoxicity, and mechanistic analyses were subsequently performed at 1 μM and 10 μM. Cell viability was evaluated using the MTT assay. Protein expression levels of HMGB1, S100B, NLRP3, nuclear factor kappa B (NF-κB), and brain-derived neurotrophic factor (BDNF) were analysed by western blot. Tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) were quantified by ELISA. Neurite outgrowth and dendritic complexity were assessed using microtubule-associated protein 2 (MAP2) immunofluorescence followed by Sholl analysis. Statistical comparisons were performed using one-way ANOVA followed by appropriate post hoc testing. Dexamethasone exposure reduced cell viability from approximately 100% to 85% of control and decreased BDNF expression to approximately 77% of control. In parallel, HMGB1, S100B, NLRP3, and NF-κB increased to approximately 124–136% of control. Pro-inflammatory cytokines were also elevated, with TNF-α rising from approximately 100 to 250 pg/mL and IL-1β from approximately 1,300 to 2,620 pg/mL. Neurite complexity, expressed as the area under the Sholl curve, decreased from approximately 28,700 to 24,000. Treatment with psilocybin (10 μM) restored BDNF expression to approximately 99% of control and reduced TNF-α and IL-1β to approximately 130 pg/mL and 970 pg/mL, respectively . NLRP3 expression decreased to approximately 70% of control, while HMGB1, S100B, and NF-κB were partially normalised. In Sholl analysis, psilocybin (1 μM) increased neurite complexity to approximately 30,100. Across the 0.1–10 μM range, psilocybin did not produce evidence of overt additional cytotoxicity.

In conclusion, psilocybin attenuated the DAMP–NLRP3–NF-κB–cytokine axis and restored BDNF expression and neurite complexity in a dexamethasone-induced neuroinflammatory neurosphere model. These findings suggest that psilocybin can simultaneously modulate inflammatory signalling and structural neuroplasticity under stress-associated inflammatory conditions. Further studies, including detailed statistical validation, receptor-level mechanistic dissection, and in vivo investigation, are required to determine the translational relevance of these observations for stress-related neuropsychiatric disorders.

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