Exploring the anti-inflammatory and protective properties of Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson in brain endothelial cells.

(P6) Achillea erba-rotta subsp. moschata (Asteraceae), an alpine endemic plant is traditionally used to treat gastrointestinal diseases. An ethnobotanical survey conducted in Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy) between 2019 and 2023 (Bottoni et al, 2022; Bottoni et al., 2024), highlighted its traditional uses, including digestive, antiinflammatory and pain-relieving properties, as well as hypotensive characteristics. Despite its widespread use, limited studies have been conducted to validate its bioactivity. In literature, the anti-inflammatory activity of this species is demonstrate on the gastrointestinal tract (Bottoni et al., 2024); furthermore, different in vivo studies have shown beneficial effects of congeneric species in neuroinflammation (Mozafari et al, 2021; Elmann et al, 2011). This study aims to investigate the effects of A. erba-rotta subsp. moschata extract and its potential role in mitigating inflammation and restoring blood-brain barrier (BBB) integrity. The decoction was prepared as documented during the ethnobotanical fieldwork, following the dosages and indications provided by the informants as previously described by Bottoniet al., 2022. For the laboratory analysis, the filtered decoction was freeze-dried. For in vitro experiments, immortalized human brain microvascular endothelial cells (ihBMEC) were treated with the extract at different doses (20 and 200 μg/ml) under inflammatory conditions induced by lipopolysaccharide stimulation (LPS, 200 μg/ml). Potential toxicity of the extract was assessed using XTT assay. Expression of inflammatory cytokines, matrix metalloproteinase and adhesion molecules were evaluated using real-time PCR. Lastly, tight junction protein levels were analyzed through western blot analysis. The A. erba-rotta subsp. moschata extract at 200 μg/ml significantly reduced the expression of pro-inflammatory cytokines IL-6 and IL-1β after 6 hours of treatment compared to the LPS-treated group, in ihBMEC cells. Both concentrations, 20 and 200 μg/ml, have been shown to significantly reverse the increased permeability of BBB, caused by LPS stimulus, by recovery Occludin levels. Additionally, the extract at both doses effectively restores matrix metalloproteinase 2 (MMP-2) and intercellular ashesion molecule 1 (ICAM-1) levels, following their LPS-induces increase. Lastly, no cytotoxic effects were observed at any concentration. These findings provide the first evidence that an aqueous extract of A. erba-rotta subsp. moschata, prepared according to traditional use, exerts anti-inflammatory effects and promotes BBB integrity. These effects are partially mediated through the modulation of inflammatory molecules, including ICAM-1 and MMP-2, which contribute to protection and restoration of tight junctions. This highlights its potential therapeutic application in conditions associated with BBB dysfunction and neuroinflammation.