Resveratrol Reduces Cisplatin-induced Cochlear Hair Cell Pyroptosis by Inhibiting the mtROS/TXNIP/NLRP3 Pathway.

Abstract

Background: Cisplatin is an effective anti-cancer drug with limited clinical applications due to ototoxicity. Resveratrol, known for its antioxidant and anti-inflammatory properties, has been reported to mitigate these adverse effects, although the underlying mechanism remains under-researched.

Objective: This study aimed to investigate the effects and underlying mechanisms of resveratrol on cisplatin-induced ototoxicity.

Methods: Ototoxicity was modeled in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells by cisplatin exposure, followed by interventions using thioredoxin-interacting protein (TXNIP) siRNA transfection, MitoQ, or resveratrol. Apoptosis and proliferation were quantitatively assessed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and Ki67 immunostaining. Quantitative real-time PCR (qRT-PCR) and western blotting were used to measure the changes in mRNA and protein levels. Flow cytometry and enzyme- linked immunosorbent assay (ELISA) were used to analyze pyroptotic cells and inflammatory responses. Reactive oxygen species (ROS) production was tracked using 2', 7'- dichlorofluorescein diacetate (DCFH-DA) staining and flow cytometry. Mitochondrial Membrane Potential (MMP) and mitochondrial permeability transition pore (MPTP) opening levels were analyzed through tetramethylrhodamine ethyl ester (TMRE) staining and specific reagent kits, respectively. Lastly, immunofluorescence staining and co-immunoprecipitation were employed to investigate the co-localization and interactions between TXNIP and thioredoxin (TRX)/NOD-like receptor family pyrin domain-containing 3 (NLRP3) proteins.

Results: Cisplatin exacerbated apoptosis, suppressed cell proliferation, and upregulated NLRP3, pro-Caspase-1, cleaved Caspase-1, Gasdermin D (GSDMD), GSDMD-N, and TXNIP expression. Concurrently, cisplatin resulted in increased pyroptotic cells and increased interleukin-6 (IL-6), IL-18, IL-1β, and tumor necrosis factor-α (TNF-α) levels. These effects were mitigated by TXNIP knockdown. Furthermore, cisplatin led to elevated cellular ROS and mitochondrial ROS (mtROS), decreased MMP, and inhibited MPTP opening. Cisplatin reduced the colocalization and interaction between TRX and TXNIP while enhancing those between TXNIP and NLRP3. These changes were attenuated by MitoQ. Resveratrol displayed effects similar to those of TXNIP knockdown and MitoQ treatment.

Conclusion: Resveratrol alleviated the toxic effects of cisplatin on cochlear hair cells by inhibiting cell pyroptosis process mediated by the mtROS/TXNIP/NLRP3 pathway.