Characterization of new bioactive molecules for the treatment of neuroinflammatory and neurodegenerative diseases

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and a significant global health concern. It is characterized by the accumulation of extracellular amyloid-β (Aβ) plaques, intracellular neurofibrillary tangles of hyperphosphorylated tau, and progressive neuronal and synaptic degeneration. These pathological changes lead to cognitive decline and dementia. A key feature of AD is the neuroinflammation, where chronic microglial activation promotes the release of pro-inflammatory cytokines and neurotoxic mediators, which exacerbate neuronal injury and enhance neurodegeneration. Due to the lack of effective treatments in AD, research efforts are increasingly focusing also on natural compounds as potential alternative therapeutics strategies to address AD pathology. The present study is part of National Biodiversity Future Center project, which aims to identify and characterize novel bioactive plant-derived extracts from Italian flora, evaluating their potential to modulate neuroinflammation and provide neuroprotection. Twenty-three natural extracts were first tested in vitro for cytotoxicity and biological activity using the murine SIM-A9 microglial cell line, primary microglia isolated from neonatal 3xTg-AD mice, and N2A neuroblastoma cells. Particularly, cytotoxicity was evaluated using the MTT assay, whereas the anti-inflammatory activity was assessed in LPS-stimulated microglia by measuring nitric oxide (NO) production using the Griess assay and determining the release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) using ELISA. The promising anti-inflammatory natural extracts were subsequently evaluated for their ability to reduce the cytokine release in primary microglia stimulated by Aβ peptides. Among the natural extracts tested, Althaea officinalis, Adenophora lilifolia, Dianthus superbus, and Succisa pratensis exhibited the most potent inhibitory effects on the production of TNF- and IL-6 in microglia stimulated by both LPS and Aβ. Additionally, the neuroprotective efficacy of these extracts was evaluated in N2A cells exposed to glutamate-induced excitotoxicity. Althaea officinalis and Dianthus superbus significantly enhanced cell viability in glutamate-exposed N2A neuroblastoma cells, indicating their potential to mitigate excitotoxic neuronal damage. These findings highlight new properties for Althaea officinalis and Dianthus superbus that have not been previously studied, showing a significant reduction in the production of pro-inflammatory mediators induced by LPS and Aβ, along with notable neuroprotective effects. Although further research is needed to confirm the effects of Althaea officinalis and Dianthus superbus in in vivo studies, our in vitro findings suggest that these plants may be promising therapeutic approaches to attenuate neuroinflammation and neuronal damage, particularly relevant for neurodegenerative disorders, like AD.