077 | Activation of the Keap1–Nrf2 axis underlies the antioxidant astrocytes protective effects of Boswellia sacra ethanolic extract: in vitro evidences supported by in silico analysis

Graziella Serio¹, Alessia Bono¹, Roberto Chiarelli¹, Marta Amato¹, Mariangela Geraci¹, Adriano Trapani¹, Alessia Vermiglio¹, Lorenza La Rosa¹, Andrea Occhipinti², Gabriella Schiera¹, Antonino Lauria¹, Carla Gentile¹ | ¹Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Italy; ²Abel Nutraceuticals SRL, Turin, Italy.

Redox imbalance plays a key role in neurodegenerative disorders, where oxidative stress contribute to neuronal dysfunction and cell loss. Astrocytes represent pivotal components of neuronal defense systems through their involvement in blood–brain barrier organization, metabolic coupling with neurons, and antioxidant protection. In this context, the Keap1–Nrf2 signaling axis orchestrates the cellular antioxidant response while modulates inflammation through functional cross-talk with NF-κB. Boswellia plants, members of the Burseraceae family, are known for producing frankincense, a resin traditionally used in religious ritual and medicinal contexts, whose main bioactive components include boswellic acids and cembrene-type diterpenes. Preliminary data indicated that an ethanolic extract from Boswellia sacra gum (BSE) exerts multifactorial protective effects in murine astrocytes exposed to oxidative stress, involving anti-inflammatory, anti-apoptotic, and autophagy-modulating mechanisms. In the present study, we focused on the antioxidant properties of BSE and their contribution to astrocyte protection under ischemic-like conditions. First, the intrinsic antioxidant potential of BSE was characterized using in solution and cell-based assays. BSE displayed significant radical-scavenging (DPPH assay) and metal-reducing activities (FRAP assay), and effectively inhibited membrane lipid peroxidation (CAA assay), supporting its ability to counteract oxidative processes. On the basis of these preliminary findings, we next investigated whether BSE could protect primary murine astrocytes from oxidative damage. Astrocytes were pretreated for 24 h with non-cytotoxic concentrations of BSE and subsequently exposed for 4 or 24 h to combined nutrient deprivation (serum-free medium) and hydrogen peroxide–induced oxidative stress, conditions mimicking an ischemic-like environment. Exposure to oxidative stress under starvation conditions resulted in a marked reduction of cell viability, as assessed by MTT and TUNEL assays, whereas BSE treatment significantly improved astrocyte survival in a dose- and time-dependent manner. At the molecular level, the nutritional and oxidative stress induced iNOS expression and increased intracellular ROS, while BSE significantly reduced iNOS levels and intracellular ROS accumulation while increased NRF2 levels, consistent with attenuation of oxidative and nitrosative stress and activation of antioxidant defenses. To investigate potential molecular mechanisms underlying BSE-mediated antioxidant effects, an in silico Induced Fit docking (IFD) approach was applied to major BSE phytochemicals, targeting the BTB and Kelch domains of the Keap1–Nrf2 complex. Docking results highlighted a preferential interaction with the BTB domain, prompting molecular dynamics simulations to evaluate the stability of ligand–protein complexes. These analyses supported the ability of selected boswellic acids to form stable interactions with key BTB residues, suggesting a contribution to Nrf2 stabilization and activation of the cellular antioxidant response. Collectively, these findings indicate that activation of the Nrf2 pathway underlies the protective effects of BSE on astrocytes exposed to oxidative and nutritional stress, supporting a glia-centered neuroprotective mechanism relevant to neurodegenerative conditions.