[Protective effect of NAD

Objectives: To investigate the protective effect of nicotinamide adenine dinucleotide (NAD⁺) against noise-induced cochlear damage and preliminarily explore its underlying transcriptional and metabolic regulatory mechanisms. Methods: During the study period (January 2023-February 2025), an oxidative stress model was established using House Ear Institute-organ of Corti 1 (HEI-OC1) cells, and cell viability was assessed using the Cell Counting Kit-8 (CCK8) assay. Flow cytometry was employed to analyze cell apoptosis. A mouse model of noise-induced hearing loss was developed, and the mice were divided into three groups: a noise-exposed saline group, a noise-exposed NAD⁺ intervention group, and a noise-free control group. Hearing protection effects were evaluated by auditory brainstem response (ABR) and immunofluorescence. Metabolomics and transcriptomics were used to analyze the regulatory effects of NAD+on transcription and metabolism in mouse cochlea. Enzyme-linked immunosorbent assay, quantitative real-time PCR, and western blot were used to verify the differential transcription and metabolic molecules and their functions. Data were statistically analyzed with GraphPad Prism 9.3.0. Results: NAD+at concentrations ranging from 10-80 μM effectively restored cell viability and reduced apoptosis induced by H₂O₂ in HEI-OC1 cells. NAD⁺ intervention significantly improved 16-32 kHz ABR thresholds after noise exposure (P<0.05), reduced outer hair cell loss rates (P<0.05), and attenuated ribbon synapse damage (P<0.000 1). Metabolomics analysis revealed a significant downregulation in the glycerophospholipid metabolism pathway, with decreased levels of lysophosphatidic acid (LPA) and its related metabolites. ELISA results showed that LPA levels in the NAD⁺ intervention group were significantly lower (P<0.05). LPA inhibitor (ATX inhibitor 1) exhibited a cell protective effect similar to that of NAD⁺. Transcriptomics analysis indicated a significant upregulation of key genes related to potassium ion channels, such as Kcnq4. qPCR and Western blot further confirmed the significant upregulation of Kcnq4 and its encoded protein in the NAD⁺ intervention group (P<0.05). In the presence of the KCNQ4 inhibitor (ML252), the protective effect of NAD⁺ was inhibited. Conclusions: NAD⁺ exerts effective protective effects against noise-induced cochlear injury. Its protective mechanism may be closely related to the inhibition of LPA metabolic pathway and the up-regulation of KCNQ4 channel function.