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

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 H2O2-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.