Study On The Protective Effect And Mechanism Of Ferrostatin-1 Against Noise-induced Hearing Loss

Objective:To clarify whether ferroptosis is involved in the pathogenesis of NIHL through constructing an in vivo NIHL mouse model and in vitro cochlear hair cell oxidative damage models.To explore the protective effect of ferrostatin-1（Fer-1）against noise-induced auditory threshold elevation and cochlear structure injury.To investigate the possible mechanism of Fer-1 on hearing protection.To explore whether the Fer-1-loaded thermosensitive nanodelivery system is suitable for intratympanic injection.To investigate the protective effect of the Fer-1-loaded thermosensitive nanodelivery system against noise-induced hearing loss and cochlear structure injury.Methods:1.The perilymph was extracted 15min,1h,and 3h after intraperitoneal injection of 10mg/kg Fer-1.The concentrations of Fer-1 in the perilymph were detected.Mice were randomly divided into DMSO group,Fer-1 group,DMSO+Noise group and Fer-1+Nosie group.Mice were exposed to 2-20 k Hz broadband white noise of 100±1 d B SPL for 2 h.The protective effect of Fer-1 on hearing was determined by ABR tests after noise exposure.The protective effect of Fer-1 on cochlear structure was evaluated by immunofluorescence staining of cochlear samples.The protective effect of Fer-1 on mitochondria in cochlear cells was observed by transmission electron microscopy（TEM）.2.In the in vivo NIHL mouse model,the levels of oxidative stress and lipid peroxidation in cochlea were detected by immunofluorescence staining.The malondialdehyde（MDA）and glutathione（GSH）kits were used to detect MDA and GSH contents in cochlea of mice in each group.The changes of protein and m RNA in the signaling pathway of ferroptosis and apoptosis were detected by immunoblotting,immunofluorescence staining and q RT-PCR.In the in vitro cochlear hair cell oxidative damage models,tert-Butyl hydroperoxide（TBHP）was used to treat cochlear explants and HEI-OC1 cells.And they were randomly divided into Ctrl group,Fer-1 group,TBHP group and Fer-1+TBHP group.Apoptosis analysis was carried out with a TUNEL kit and a flow cytometry assay using an Annexin V-FITC/PI kit.Cell viability was detected by a CCK-8 kit.Intracellular ROS and lipid peroxidation levels were detected by DCFH-DA and Liperfluo fluorescence staining.The concentration of Fe²⁺in cells was detected by Ferro Orange fluorescence staining.The mitochondria in the HEI-OC1 cells were observed using TEM.3.Fer-1 was loaded with PLGA nanoparticles,and the polypeptide LS19 was coupled with the surface of PLGA nanoparticles.And then the nanoparticles were dispersed into the chitosan-glycerophosphate hydrogel to create the Fer-1-loaed thermosensitive nanodelivery system.Rhodamine B was added to the nanoparticles as a fluorescent indicator.According to whether LS19 or Fer-1 were loaded,4 groups of drug delivery systems were synthesized.We used a dynamic light scattering（DLS）detector to evaluate the hydrodynamic diameter and zeta potential of nanoparticles.The morphological characteristics of nanoparticles were detected using TEM.The morphological characteristics of gels were detected using scanning electron microscope（SEM）.Fourier transform infrared（FTIR）analysis was conducted to study the chemical structures of the four groups of gels.The in vitro drug release of nanoparticles and gels were evaluated by the dialysis method.A CCK-8 kit and TNF-αimmunofluorescence staining were used to determine the biocompatibility of the Fer-1-loaed thermosensitive nanodelivery system.4.Adult mice with normal hearing were randomly divided into 5 groups,namely blank control group and 4 Noise+thermosensitive nanodelivery system groups.Mice received a single intratympanic injection of thermosensitive nanodelivery system 3 d before noise exposure.The retention of gels in the middle ear was evaluated by an in vivo imaging system（IVIS）.Then mice were exposed to 2-20 k Hz broadband white noise of 105±1 d B SPL for 2 h.The protective effect of the Fer-1-loaed thermosensitive nanodelivery system on hearing was determined by ABR tests after noise exposure.The protective effect of the Fer-1-loaed thermosensitive nanodelivery system on cochlear structure was evaluated by immunofluorescence staining.Results:1.At 15 min,1 h,and 3 h after injection,the concentrations of Fer-1 in perilymph were2.077,0.301,and 0.122μg/L,respectively.Administration of Fer-1 significantly alleviated noise-induced auditory threshold elevation at 8 k Hz,16 k Hz and 32 k Hz.Treatment with Fer-1 significantly reduced the death of outer hair cells,the loss of inner hair cell ribbon synapses,and the degeneration of spiral neuron fibers caused by noise.Treatment with Fer-1 significantly alleviated noise-induced mitochondrial shrinkage,mitochondrial cristae reduction or disappearance,and mitochondrial vacuolation in mouse cochlear cells.2.In the in vivo NIHL mouse model,treatment with Fer-1 significantly reduced the noise-induced increase of 4-hydroxynonenal（4-HNE）,3-nitrotyrosine（3-NT）,MDA and dihydroethidium（DHE）levels in the cochlea.Fer-1 significantly inhibited the Tf R1expression and reduced the increase of P53 and AIFM2 induced by noise.But Fer-1 has no effect on GSH content and GPX4 and ACSL4 expression in cochlea.In the in vitro cochlear hair cell oxidative damage models,administration of Fer-1 significantly alleviated TBHP-induced cochlear hair cell apoptosis.Treatment with Fer-1 significantly reduced TBHP-induced ROS accumulation,lipid peroxidation and Fe²⁺accumulation in cochlear hair cells.Treatment with Fer-1 significantly alleviated TBHP-induced mitochondrial shrinkage,mitochondrial cristae reduction or disappearance,and mitochondrial vacuolation in cochlear hair cells.3.The mean hydrodynamic diameters of the nanoparticles in the 4 groups of drug delivery systems were 155.53 nm,159.31 nm,147.01 nm and 173.53 nm,respectively.The PDIs of these 4 groups of nanoparticles suggested that these 4 groups of nanoparticles displayed a narrow distribution,characterized by mean PDIs of 0.19,0.19,0.20 and 0.18.The zeta potential values of these 4 groups of nanoparticles were-12.97 m V,-11.11 m V,-14.26m V and-10.36 m V,respectively.According to the TEM results,the nanoparticles were spherical and had a smooth surface.The SEM results showed that these drug delivery systems had a three-dimensional porous network structure.According to the results of FTIR analysis,these 4 groups of drug delivery systems were polymerized successfully.Each group of drug delivery systems had no cytotoxicity,did not induce inner ear inflammation,and may cause little inflammatory response in the middle ear.4.According to the IVIS results,the thermosensitive nanodelivery system could achieve long-term drug retention（at least 7 d）in the middle ear,prolonging the administration time.Intratympanic injection of the Fer-1 loaded thermosensitive nanodelivery system significantly alleviated noise-induced auditory threshold elevation at 8 k Hz,16 k Hz,24k Hz and 32 k Hz.Intratympanic injection of the Fer-1 loaded thermosensitive nanodelivery system significantly reduced the death of outer hair cells,the loss of inner hair cell ribbon synapses,and the degeneration of spiral neuron fibers caused by noise.Conclusion:Ferroptosis is one of the pathogenic mechanisms of NIHL.The ferroptosis inhibitor Fer-1was demonstrated to enter the inner ear after intraperitoneal injection.Administration of Fer-1 significantly alleviated noise-induced auditory threshold elevation and reduced the death of outer hair cells,the loss of inner hair cell ribbon synapses,and the degeneration of spiral neuron fibers caused by noise through scavenging of ROS and inhibition of Tf R1-mediated ferroptosis and P53-AIFM2 signaling pathway-mediated apoptosis.The Fer-1 loaded thermosensitive nanodelivery system showed great biocompatibility,and achieved long-term drug retention in the middle ear after intratympanic injection.Intratympanic injection of the Fer-1-loaded thermosensitive nanodelivery system is a safe and effective way to attenuate NIHL.