Work

Studies of chitosan-Prussian blue nanozyme in auditory protection: from cellular mechanisms to in vivo validation

Year: 2026

Type: article

Abstract: Oxidative stress and inflammation are interconnected drivers of cellular damage in pathologies ranging from neurodegenerative disorders to sensorineural hearing loss. We aimed to develop a chitosan-Prussian blue nanozyme (CS-PB) to target these processes in sensorineural hearing loss. CS-PB (35 μg/mL) pretreatment of H 2 O 2 -injured HEI-OC1 cochlear cells for 4 h markedly decreased the levels of reactive oxygen species (ROS) inside cells (from 3.8 to 2.4 relative fluorescence intensity) by approximately 37% ( P < 0.001)-and concomitantly significantly decreased expression of the oxidative damage markers 4-HNE and 3-NT (both P < 0.001). The rate of apoptosis decreased from 27.5% in the H 2 O 2 -treated group to 14.1% following CS-PB treatment (P < 0.01). This reduction was accompanied by a significant downregulation of the pro-apoptotic proteins Bax (P < 0.05) and Cleaved-caspase-3 (P < 0.001), as well as an upregulation of the anti-apoptotic protein Bcl-XL (P < 0.01).Western blot analysis confirmed that CS–PB significantly downregulated TLR4 expression and inhibited downstream phosphorylation of p-P65/P65, while upregulating p-IκBα/IκBα protein (all P < 0.01) associated with reduction in the production of inflammatory cytokines like TNF-α, IL-1β, and IL-6. Treatment with the specific TLR4 antagonist TAK-242 (2 μM) mimicked the effect of CS-PB, with coadministration showing no additional ant-inflammatory effect (although HEI-OC1 viability was additionally increased), indicating that the anti-inflammatory properties of CS-PB were facilitated via the TLR4/NF-κB signaling pathway. In vivo , CS-PB pretreatment (2 mg/mL, 2 μL) was administered by carefully applying the solution to the round window membrane (RWM) using a microsyringe in a noise-induced hearing loss rat model, which significantly curtailed the noise-induced activation of the TLR4/NF-κB pathway in the cochlea. After 24 hours, the levels of p-P65/P65 and TLR4 decreased by 54% and 50%, respectively. ( P < 0.001), and levels of the inflammatory cytokines IL-6, TNF-α and IL-1β were decreased by 53%, 48%, and 51% ( P < 0.001). Furthermore, the degree of pathway inhibition showed a strong correlation with cytokine reduction (r = 0.87–0.89, all P < 0.001). CS-PB showed no significant cytotoxicity in vitro or in vivo , suggesting that its combined ROS-scavenging and TLR4/NF-κB–modulating properties may represent a potential therapeutic strategy, pending further mechanistic and translational validation.

Source: Frontiers in Immunology

DOI: 10.3389/fimmu.2026.1758392