Characterization of a dietary supplement in neuroinflammation: modulation of microglial reactivity and blood-brain barrier's properties.

Neuroinflammation is a protective acute response to CNS injury or infection. If the inflammation becomes chronic, it can encourage pathological conditions like epilepsy or migraine and can also lead to the progression of several neurodegenerative disorders like Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. Microglia play a key role in neuroinflammation, in the pathophysiological process of pain transmission and in the development of neuropathic pain and migraine. Here we tested a bioactive compound, formulated with substances known in literature for their anti-inflammatory ability, but whose combined activity in contrasting an inflammatory insult on microglial cells have not been described yet. In this study, the impact of the bioactive compound described was tested on a human derived microglial cell line, HMC3. The inflammation was induced with an inflammatory stimulus: LPS 0.1 µg/ml + TNF-α 50 ng/ml (LPT). HMC3 cells exposed to LPT showed more than 20% reduction in their metabolic activity and viability, associated with significant increase in the production of reactive oxygen species (ROS), when compared to control. These effects where mitigated when cells were co-treated with the bioactive compound at two different concentrations. The ability of the two selected concentrations to resolve LPT damage in HMC3 cells were also assessed exposing cells to the bioactive compound either before or after the LPT insult. Confirming that the studied compound can prevent as well as resolve the damage induced by LPT. We further investigated the ability of the studied formulation to modulate the main inflammatory pathways, the NF-κB pathway and the NLRP3 inflammasome, as well as the expression of inflammatory cytokines. Western Blot and real time-PCR experiments confirmed that LPT induced on microglial cells the expression of NLRP3 and phosphorylated IκB, and upregulated selected cytokines like interleukine-6 or interleukine-1β, effects prevented by the exposure to the bioactive compound. Finally, the diet supplement was also tested to evaluate if microglial exposure to the bioactive compound may modulate endothelial response to inflammatory insults, setting a co-culture of HMC3 cells and the human microvascular endothelial cell line, TY-10. Endothelial cells are part of the BBB, whose integrity, necessary to maintain CNS homeostasis, is characterized by low and selective permeability, kept by tight junctional proteins, like Claudin-5 and low expression of adhesion molecules, such ICAM-1, reducing leukocytes access. Co-cultures exposure to LPT induced increased endothelial ICAM-1 expression, effect prevented by the exposure to the diet supplement. Interestingly, HMC3 pre-treated with the diet supplement could highly recover endothelial expression of claudin-5, also if the endothelial cells were not directly exposed to the treatments. These findings highlight the positive effects of a balanced combination of various bioactive compounds in the same formulation with potential ability to manage inflammatory disorders by modulating microglial activation.