Tanshinone IIA potentiates the therapeutic efficacy of glucocorticoids in lipopolysaccharide-treated HEI-OC1 cells through modulation of the FOXP3/Nrf2 signaling pathway

Abstract

Glucocorticoids (GCs) are commonly used to treat sudden sensorineural hearing loss (SSNHL), although some patients are resistant to this therapeutic approach. Clinical studies have demonstrated the efficacy of tanshinone IIA (TA) in combination with GC for managing various human ailments. However, it remains unclear whether TA can mitigate GC resistance in SSNHL. Our aim is to elucidate the role of NRF2-induced transcriptional regulation of HDAC2 in influencing GC resistance and investigate the involvement of TA-related molecular pathways in GC resistance. Here, HEI-OC1 cells are treated with lipopolysaccharide (LPS) to establish an in vitro model for SSNHL. The cells are subsequently treated with dexamethasone (DXE) or DXE + TA. RT-qPCR and western blot analysis are used to measure the mRNA and protein levels of Forkhead box P3 (FOXP3), nuclear factor erythroid 2-related factor 2 (NRF2), and histone deacetylase 2 (HDAC2). Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2’-deoxyuridine (EdU) assays are carried out to assess cell proliferation. Flow cytometry analysis is performed to evaluate apoptosis. Mechanistic studies involve chromatin immunoprecipitation (ChIP), luciferase reporter, and DNA pull-down assays. Our results show that treatment with TA + DEX significantly increases proliferation and suppresses apoptosis in LPS-treated HEI-treated OC1 cells. TA upregulates HDAC2 expression by activating NRF2-mediated transcription of HDAC2, with the NRF2-HDAC2 binding site located at bases 419–429 (ATGACACTCCA) in the promoter sequence of HDAC2. Furthermore, TA upregulates FOXP3 expression to activate NRF2 transcription, with the predicted FOXP3-binding site located at bases 864–870 (GCAAACA) in the promoter sequence of NRF2. In summary, these findings suggest that TA enhances the therapeutic effects of GC on the proliferation and apoptosis of HEI OC1 cells by increasing FOXP3/Nrf2 expression. These results indicate that TA may be promising for ameliorating GC resistance in patients with SSNHL.

1. Introduction

Sudden sensorineural hearing loss (SSHL) is a prevalent otological emergency. Previous studies have shown that SSHL affects 5–27 per 100,000 people per year, with nearly 66,000 new cases annually in the United States of America [1]. Sensory hearing loss often occurs due to damaged or deficient cochlear hair cells; hair cells may be abnormal at birth or damaged during an individual’s lifetime. The proposed etiologies of primary SSHL include virus infection, vascular insufficiency, autoimmune disorders, and stress theory [2,3]. There is no proven or recommended treatment or cure for SSHL. The treatment of SSHL remains one of the most challenging issues in contemporary otorhinolaryngology. Therefore, it is necessary to explore promising therapeutic strategies for patients with SSHL.

HEI-OC1 is one of the few mouse auditory cell lines available for research. Originally proposed as an in vitro system for screening ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanisms of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and the expressions of molecular channels and receptors [4]. Currently, the HEI-OC1 cell line is widely used in the field of hair cell research. To a certain extent, it can be used to speculate on in vivo changes and establish a hair cell injury and inflammation model.

Currently, glucocorticoids (GCs) are widely used to treat SSHL and various immune and inflammatory diseases [5–8]. Although most patients with SSHL respond well to GCs, approximately 20% of patients show no significant hearing improvement after GC treatment, indicating GC resistance in these patients [9,10]. Fortunately, traditional Chinese medicine has been combined with Western medicine to prevent and reverse drug resistance in cancer cells [11]. This approach has also been applied to the treatment of SSHL. For example, treating SSHL patients with a combination of GCs and breviscapine (a traditional Chinese medicine) has been proven to be more effective than using GCs alone [12]. Therefore, exploring the potential of traditional Chinese medicine for preventing and reversing GC resistance in SSHL is worthwhile.