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

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 H2O2-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 H2O2-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.