Study On The Expression Of NLRX1 In C57BL/6 Mice Cochlear Hair Cells And Its Possible Relation To Aging- And Neomycin-induced Deafness

BackgroundSensory deafness is a common disease, which is mainly caused by hair cell damage, thereby leading to huge disadvantages to and the lower quality of life to the patients. The disease is closely related to the injury of hair cells, resulting in the defect of sensation of sound. The research and recognition of deafness-related genes are really important for the intervention and cure of deafness through specific treatment plan and drugs depending on the mechanism revealed. We assumed that the member of nucleotide-binding domain-and leucine-rich-repeat-containing family (NLRs), NLRX1, may be involved in the process of hair cell damage, being a new target on cure of sensory deafness.NLRX1, which is also named NoD5, NoD9, NOD26, DLNB26 or CLR11.3, is a cytoplasmic pattern recognition receptor located mainly to mitochondria. It is a highly conserved protein through different species and widely expressed in body, especially in heart and muscle, which possess high-level metabolism. The function of NLRX1 is complicated and it is important in the regulation of mitochondrial damage, reactive oxygen species (ROS) production, inflammation and apoptosis, which result from the interaction with proteins in mitochondria.A large number of papers have shown that sensory deafness is closely related to hair cell apoptosis. Many researches demonstrate that a variety of factors, such as noise, drugs, aging and the like, can cause hair cell apoptosis, indicating that the etiology of sensory deafness is highly heterogeneous with a broad-spectrum of pathological mechanisms. To date, however, no study on NLRX1 has been conducted in otology.ObjectiveThe purpose of this work was designed to investigate whether NLRX1 exists in cochlear hair cells of C57BL/6 mice, and, if so, to study the possible correlations between NLRX1 and hearing alteration.Methods1. Wild type C57BL/6 mice were divided into five groups according to their age, postnatal day 4 (P4), day 15 (P15),1 month (M1),3 months (M3) and 9 months (M9);2. The location and dynamic expression of NLRX1 were investigated by immunofluorescence, real-time PCR and Western blotting.3. We divided the M9 mice into two groups according to their hearing thresholds and measured the levels of certain factors relevant to apoptosis (p-JNK, caspase-3) and inflammation (NF-kB, IRF-3, IL-6, IFN-β). We also detected the p-JNK and caspase-3 in neomycin-treated M3 mice.Results1. Data showed that NLRX1 expressed in cytoplasm of C57BL/6 cochlear hair cells, especially in the cilia.2. The expression of NLRX1 in hair cells increased as the mice grew up, and, decreased as they aged.3. The activation of NF-kB or IRF-3 inflammatory signal pathway was undetectable in aged mice hair cells.4. Additionally, the activated apoptotic JNK pathway and increase of NLRX1 were detected in M9 mice with worse-hearing and M3 mice treated with neomycin.Conclusion1. The expression of NLRX1 in C57BL/6 mice cochlear hair cells made it possible to research the function and mechanism of NLRX1 in otology;2. The alteration of NLRX1 expression level in C57BL/6 mice life span indicates that NLRX1 may be related to hearing formation and maintenance;3. The up-regulation of NLRX1 expression and activation of JNK signaling pathway in mice with aging-or neomycin-induced hearing loss indicated that, NLRX1 might triger hair cell apoptosis through JNK pathway to lead to sensory deafness.