SIRT1 Activation Reduces LPS-Induced Human Middle Ear Epithelial Cell Injury via Promoting Autophagy

Abstract

Background: Otitis media (OM) is a prevalent pervasive illness among the pediatric population. Research on sirtuin 1 (SIRT1), a deacetylase sensitive to nicotinamide adenine dinucleotide (NAD(+)), has increasingly identified its pivotal role in various inflammatory conditions. Our study aims to elucidate the role of SIRT1 in inflammatory injury in OM and its potential action mechanism. Methods: Human middle ear epithelial cells (HMEECs) were stimulated with lipopolysaccharide (LPS) to establish in vitro models of OM. Western blot was used to assess SIRT1 expression. Following the addition of SIRT1 activator SRT1720, with or without the autophagy inhibitor 3-Methyladenine (3-MA), the Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell viability. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was used to assess cell apoptotic levels. Enzyme-linked immunosorbent assay (ELISA) was used to estimate inflammatory levels and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining was used to estimate reactive oxygen species (ROS) activity. Western blot was used to analyze the content of apoptosis-, inflammatory response-, autophagy-, and nuclear factor-kappaB (NF-κB) signaling-associated proteins. Results: SIRT1 expression was attenuated in LPS-treated HMEECs (p < 0.05). SRT1720 pretreatment significantly enhanced cell viability (p < 0.05), attenuated apoptosis (p < 0.05), suppressed the inflammatory response (p < 0.05), and reduced ROS production (p < 0.05). Additionally, it inhibited NF-κB signaling (p < 0.05) while inducing autophagy (p < 0.05) in HMEECs exposed to LPS in a concentration-dependent manner. Furthermore, 3-Methyladenine (3-MA) partially counteracted the suppressive effects of SRT1720 in LPS-induced viability loss, apoptosis (p < 0.05), inflammatory response (p < 0.05), NF-κB signaling activation (p < 0.05), and ROS generation (p < 0.05) in HMEECs. Conclusions: In summary, the activation of SIRT1 may mitigate the inflammatory response in LPS-induced HMEECs through the modulation of apoptosis, inflammatory signaling, and autophagic processes.