Resveratrol Reduces Cisplatin-induced Cochlear Hair Cell Pyroptosis by Inhibiting the mtROS/TXNIP/NLRP3 Pathway
Authors: Peng Andi , Peng Jiahui , Lai Ruosha , Liu Wei , Chen Xubo , Hu Bing , Xu Yingying , Li Lihua 
Published: January 2025

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.

result:
Cisplatin exacerbated cell apoptosis, suppressed cell proliferation, and up-regulated NLRP3, pro-Caspase 1, cleaved-Caspase 1, GSDMD, GSDMD-N, and TXNIP expressions. Concurrently, cisplatin resulted in increased pyroptotic cells and heightened IL-1β, IL-18, IL-6, and TNF-α levels. These effects were mitigated through TXNIP knockdown. Furthermore, cisplatin led to elevated ROS, decreased MMP and inhibited the opening of MPTP. Cisplatin reduced the co-localization and interaction between TRX and TXNIP while enhancing those between TXNIP and NLRP3. MitoQ reversed these changes. Resveratrol displayed similar effects 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.