Hesperidin Attenuates H₂O₂‐Induced Neurotoxicity via Modulation of Inflammatory Pathways and MMP Activity in Differentiated SH‐SY5Y Cells: In Vitro and In Silico Models

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ABSTRACT Chronic inflammation, often aggravated by oxidative stress, is a key contributor to the pathogenesis of neurodegenerative diseases. Molecular mediators such as NF‐κB, COX‐2, pro‐inflammatory cytokines, and matrix metalloproteinases (MMP‐2/MMP‐9) disrupt blood‐brain barrier (BBB) integrity and promote neuronal damage. Hesperidin, a natural citrus flavonoid with low cytotoxicity and high antioxidant capacity, has shown promise as a neuroprotective agent. This study aimed to evaluate its anti‐inflammatory and neuroprotective effects in differentiated SH‐SY5Y neuroblastoma cells exposed to hydrogen peroxide (H₂O₂), a well‐established inducer of oxidative neurotoxicity. The protective effects of Hesperidin (75 and 100 µM for 48 h), administered as both pre‐ and post‐treatment, were evaluated through cell viability assays, assessment of oxidative stress parameters, and expression analysis of inflammatory mediators (TNF‐α, IL‐1β, IL‐6, NF‐κB, COX‐2), and MMP‐2/MMP‐9, using ELISA and RT‐qPCR. In silico molecular docking analyses were also conducted using CB‐Dock2 Tools to support the experimental findings. In our results, Hesperidin significantly increased cell viability ( p < 0.001) and reduced morphological damage. It also downregulated TNF‐α, IL‐1β, and IL‐6 at both mRNA and protein levels ( p < 0.05–0.005), while markedly suppressing NF‐κB, COX‐2, MMP‐2, and MMP‐9 expression ( p < 0.01–0.005) and decreasing oxidative stress ( p < 0.005). Molecular docking revealed strong binding affinities of Hesperidin to key inflammatory targets, particularly COX‐2 (–12.2 kcal/mol) and TNF‐α (–11.6 kcal/mol). These findings indicate that Hesperidin exerts potent neuroprotective effects against H 2 O 2 ‐induced oxidative stress and neuroinflammation by modulating pro‐inflammatory signaling pathways, matrix metalloproteinase activity, and redox homeostasis. These results highlight Hesperidin's potential as a therapeutic candidate for neurodegenerative disorders.