Protective Effects of Safranal Against Spike Protein-Induced Mitochondrial Dysfunction and Inflammation in Peripheral and Central Immune Cells

Background: Saffron (Crocus sativus L.) contains bioactive molecules with antioxidant, anti-inflammatory, and neuroprotective properties. Growing evidence indicates that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) promotes neuroinflammation and mitochondrial dysfunction contributing to neuro-coronavirus disease. Objectives: The aim of this study is to evaluate the antioxidant, anti-inflammatory, and neuroprotective effects of 3 saffron derivatives, picrocrocin, 4-hydroxysafranal, and safranal, in peripheral immune cells and microglia, and to test the hypothesis that these compounds, especially safranal, counteract Spike protein 1(S1)-induced inflammation and mitochondrial dysfunction. Methods: An immortalized murine microglial cell line (BV2) and human peripheral blood mononuclear cells (PBMCs) from healthy donors were treated with saffron derivatives at nontoxic concentrations (0.05–0.5 mM). Cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-5-(3‑carboxymethoxyphenyl)-2-(4‑sulfophenyl)-2H‑tetrazolium (MTS) assay), antioxidant capacity [2,2-diphenyl-1-picrylhydrazyl (DPPH)], intracellular reactive oxygen species (ROS; 2,7-dichlorodihydrofluorescein diacetate), cytokine expression (enzyme-linked immunosorbent assay and quantitative polymerase chain reaction), and mitochondrial membrane potential (5,5′,6,6′‑tetrachloro‑1,1′,3,3′‑tetraethylbenzimidazolylcarbocyanine iodide (JC-1) assay) were assessed. Lipopolysaccharide (LPS) served as an inflammatory control, whereas S1 was used to model SARS-CoV-2-mediated neuroinflammation and mitochondrial damage. Results: All saffron derivatives showed antioxidant activity, with safranal demonstrating the strongest DPPH radical scavenging effect and the most pronounced reduction of intracellular ROS. In LPS-stimulated BV2 cells, safranal significantly decreased inducible nitric oxide synthase expression. In PBMCs, saffron compounds attenuated LPS-induced interleukin-1 beta (IL-1β) release, with safranal showing the greatest decrease. S1 increased IL-1β and tumor necrosis factor-alpha expression in BV2 microglia. Co-treatment with safranal reduced these cytokines by ∼38% and 44%, respectively. S1 induced a loss of mitochondrial membrane potential, which was effectively restored by safranal, as confirmed by JC-1 fluorescence analysis. Conclusions: These findings identify safranal as a promising neuroprotective candidate for preventing or mitigating SARS-CoV-2-associated neurological damage and other disorders involving microglial activation and mitochondrial impairment.