Minor cannabinoids have pleiotropic effects on human brain microvascular endothelial activation and barrier function

Background Disruption of the blood–brain barrier is a key driver of neuroinflammation during systemic inflammatory states. While minor phytocannabinoids, including cannabinol (CBN), cannabigerol (CBG), and cannabidiol (CBD), are recognized for their potential vascular and immunomodulatory actions, their direct impact on human brain endothelial barrier function under cytokine stress has not been systematically evaluated. Methods Primary human brain microvascular endothelial cells (HBMEC) from two donors were treated with TNF-α or IL-1β (0.1 ng/mL) in the presence or absence of CBN, CBG, or CBD (0.01–10 µM). Barrier function was assessed between 0–10 h using transendothelial electrical resistance (TEER). The structural integrity of tight and adherens junctions was assessed using immunofluorescence for Zonula Occludens-1 (ZO-1), Vascular endothelial (VE)-cadherin, and F-actin. IL-6 and IL-8 levels were measured using ELISA. Results TNF-α and IL-1β each induced a rapid and sustained reduction in HBMEC TEER, consistent with barrier dysfunction and junctional disruption, as assessed by immunofluorescence microscopy of ZO-1, VE-cadherin, and F-actin. Across both donors, CBN attenuated TNF-α–induced reductions in TEER and suppressed TNF-α–stimulated IL-6 and IL-8 release (p < 0.05), and partially restored VE-cadherin organization towards baseline, but not ZO-1 or F-actin. In contrast, CBN showed minimal or donor-variable effect under IL-1β, with no reproducible effect on TEER. CBD had little or no effect in the TNF-α model, and at higher concentrations, exacerbated IL-1β-driven permeability of HBMEC from one donor (p < 0.05). CBD selectively reduced IL-1β–induced IL-8 secretion with limited impact on IL-6. CBG at 10 µM augmented reductions in TEER induced by IL-1β or TNF-α (p < 0.05), and variably affected IL-1β or TNF-α-induced IL-6 and IL-8 release. Conclusions CBN partially reverses TNF-α-induced HBMEC barrier dysfunction and IL-6 and IL-8 production. In contrast, CBD shows a narrow, cytokine-specific anti-inflammatory signal, with partial suppression of IL-1β–induced IL-8 without barrier protection. Finally, CBG potentiates IL-1β and TNF-α-induced permeability, and is pro-inflammatory at higher concentrations. Our study identifies CBN as the most promising of these minor cannabinoids for TNF-α–driven inflammatory states, with associated endothelial activation and dysfunction, such as sepsis. These findings underscore that cannabinoid actions on brain endothelial cells are both ligand- and cytokine-dependent and highlight the need to match cannabinoid choice and dosing to the prevailing inflammatory milieu.