Ototoxicity of polystyrene nanoplastics in mice, HEI-OC1 cells and zebrafish.

Polystyrene nanoplastics are a novel class of pollutants. They are easily absorbed by living organisms, and their potential toxicity has raised concerns. However, the impact of polystyrene nanoplastics on auditory organs remains unknown. Here, our results showed that polystyrene nanoplastics entered the cochlea of mice, HEI-OC1 cells, and lateral line hair cells of zebrafish, causing cellular injury and increasing apoptosis. Additionally, we found that exposure to polystyrene nanoplastics resulted in a significant elevation in the auditory brainstem response thresholds, a loss of auditory sensory hair cells, stereocilia degeneration and a decrease in expression of Claudin-5 and Occludin proteins at the blood-lymphatic barrier in mice. We also observed a significant decrease in the acoustic alarm response of zebrafish after exposure to polystyrene nanoplastics. Mechanistic analysis revealed that polystyrene nanoplastics induced up-regulation of the Nrf2/HO-1 pathway, increased levels of malondialdehyde, and decreased superoxide dismutase and catalase levels in cochlea and HEI-OC1 cells. Furthermore, we observed that the expression of ferroptosis-related indicators GPX4 and SLC7A11 decreased as well as increased expression of ACLS4 in cochlea and HEI-OC1 cells. This study also revealed that polystyrene nanoplastics exposure led to increased expression of the inflammatory factors TNF-α, IL-1ß and COX2 in cochlea and HEI-OC1 cells. Further research found that the cell apoptosis, ferroptosis and inflammatory reactions induced by polystyrene nanoplastics in HEI-OC1 cells was reversed through the pretreatment with N-acetylcysteine, a reactive oxygen species inhibitor. Overall, our study first discovered and systematically revealed the ototoxicity of polystyrene nanoplastics and its underlying mechanism.

MiR-29a-deficiency causes thickening of the basilar membrane and age-related hearing loss by upregulating collagen IV and laminin.

Age-related hearing loss (ARHL) is the most common sensory degenerative disease and can significantly impact the quality of life in elderly people. A previous study using GeneChip miRNA microarray assays showed that the expression of miR-29a changes with age, however, its role in hearing loss is still unclear. In this study, we characterized the cochlear phenotype of miR-29a knockout (miR-29a-/-) mice and found that miR-29a-deficient mice had a rapid progressive elevation of the hearing threshold from 2 to 5 months of age compared with littermate controls as measured by the auditory brainstem response. Stereocilia degeneration, hair cell loss and abnormal stria vascularis (SV) were observed in miR-29a-/- mice at 4 months of age. Transcriptome sequencing results showed elevated extracellular matrix (ECM) gene expression in miR-29a-/- mice. Both Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the key differences were closely related to ECM. Further examination with a transmission electron microscope showed thickening of the basilar membrane in the cochlea of miR-29a-/- mice. Five Col4a genes (Col4a1-a5) and two laminin genes (Lamb2 and Lamc1) were validated as miR-29a direct targets by dual luciferase assays and miR-29a inhibition assays with a miR-29a inhibitor. Consistent with the target gene validation results, the expression of these genes was significantly increased in the cochlea of miR-29a-/- mice, as shown by RT-PCR and Western blot. These findings suggest that miR-29a plays an important role in maintaining cochlear structure and function by regulating the expression of collagen and laminin and that the disturbance of its expression could be a cause of progressive hearing loss.

Ferroptosis is involved in PGPS-induced otitis media in C57BL/6 mice.

Otitis media (OM) is a common disease that can cause hearing loss in children. Currently, the main clinical treatment for OM is antibiotics, but the overuse of antibiotics might lead to bacterial resistance, which is a worldwide public health challenge. Studying the pathogenesis of OM will help us develop new effective treatments. Ferroptosis is one type of programmed cell death characterized by the occurrence of lipid peroxidation driven by iron ions. Many studies have shown that ferroptosis is associated with infectious diseases. It is presently unclear whether ferroptosis is involved in the pathogenesis of OM. In this study, we explored the relationship between ferroptosis and OM by PGPS-induced OM in C57BL/6 mice and treating the induced OM with ferroptosis inhibitors deferoxamine (DFO), Ferrostatin-1 (Fer-1), and Liperoxstatin-1 (Lip-1). We examined the expression of ferroptosis-related proteins acyl-CoA synthetase long chain family member 4 (ACSL4) and prostaglandin-endoperoxide synthase 2 (Cox2), glutathione peroxidase 4 (GPX4) protein as well as lipid peroxidation markers 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). The results showed that in PGPS-induced OM model mice, several ferroptosis-related proteins including ACSL4 and Cox2 were up-regulated compared to mice treated with saline. Meanwhile, a ferroptosis-related protein GPX4 was down-regulated upon PGPS treatment. The DFO treatment in PGPS-inoculated mice effectively inhibited the development of OM. The inhibitors treatment caused a significant decrease in the expression of ACSL4, Cox2, 4 HNE, MDA, reduction in free iron. Meanwhile, the ferroptosis inhibitors treatment caused increase in the expression of inflammation-related factors tumor necrosis factor-α (TNF-α) and antioxidant protein GPX4. Our results suggest that there is a crosstalk between ferroptosis signaling pathway and the pathogenesis of OM. Ferroptosis inhibition can alleviate PGPS-induced OM.

Autophagy Contributes to the Rapamycin-Induced Improvement of Otitis Media.

Otitis media (OM) is a pervasive disease that involves hearing loss and severe complications. In our previous study, we successfully established a mouse model of human OM using Tlr2tm1Kir (TLR2-/-) mice with middle ear (ME) inoculation of streptococcal peptidoglycan-polysaccharide (PGPS). In this study, we found that hearing loss and OM infections in OM mice were significantly alleviated after treatment with rapamycin (RPM), a widely used mechanistic target of RPM complex 1 (mTORC1) inhibitor and autophagy inducer. First of all, we tested the activity of mTORC1 by evaluating p-S6, Raptor, and mTOR protein expression. The data suggested that the protein expression level of p-S6, Raptor and mTOR are decreased in TLR2-/- mice after the injection of PGPS. Furthermore, our data showed that both the autophagosome protein LC3-II, Beclin-1, ATG7, and autophagy substrate protein p62 accumulated at higher levels in mice with OM than in OM-negative mice. The expression of lysosomal-associated proteins LAMP1, Cathepsin B, and Cathepsin D increased in the OM mice compared with OM-negative mice. Rab7 and Syntaxin 17, which is necessary for the fusion of autophagosomes with lysosomes, are reduced in the OM mice. In addition, data also described that the protein expression level of p-S6, mTOR and Raptor are lower than PGPS group after RPM treatment. The accumulation of LC3-II, Beclin-1, and ATG7 are decreased, and the expression of Rab7 and Syntaxin 17 are increased significantly after RPM treatment. Our results suggest that autophagy impairment is involved in PGPS-induced OM and that RPM improves OM at least partly by relieving autophagy impairment. Modulating autophagic activity by RPM may be a possible effective treatment strategy for OM.