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Journal of Otology logoLink to Journal of Otology
. 2025 Nov 13;20(4):211–218. doi: 10.26599/JOTO.2025.9540033

SIRT3 knockout aggravates LPS-induced eustachian tube dysfunction

Shimin Zong 1,2,3,†, Huimin Zhang 1,2,3,†, Ting Li 1,2,3, Xi Lian 1,2,3, Wei Tang 1,2,3, Tianyi Liu 4, Wenting Yu 1,2,3,*, Xuan Yu 1,2,3,*, Hongjun Xiao 1,2,3,*
PMCID: PMC12647948  PMID: 41311539

Abstract

Purpose

Acute otitis media caused by gram-negative bacteria is a common otological condition among pediatric patients. Eustachian tube dysfunction (ETD) plays a pivotal role in the delayed resolution of acute otitis media, whereas the precise contribution of SIRT3 in this mechanism remains uncertain. This study aims to reveal the involvement of SIRT3 in murine ETD induced by LPS.

Results

Histological analysis showed no baseline differences in ET structure between WT and SIRT3 knockout (SIRT3-KO) mice. However, LPS exposure led to increased goblet cell proliferation and MUC5AC mucus secretion in both genotypes, with SIRT3-KO exacerbating these effects. The SIRT3-KO group displayed reduced cilia length. Functionally, SIRT3-KO mice showed a significantly higher initial POP and decreased MCC compared to the WT group after LPS exposure. Additionally, the active clearance of negative pressure (ACNP) was significantly reduced in SIRT3-KO mice, indicating compromised ET function.

Conclusions

SIRT3-KO increased resistance to ET opening in mice exposed to LPS, and this effect may be related to the upregulated MUC5AC expression, the increased surface tension of the luminal fluid and the impaired MCC function in mice exposed to LPS.

Keywords: Sirt3, eustachian tube dysfunction, LPS, MUC5AC

1. Introduction

The prevalence of bacterial infections leading to otitis media is remarkably high in both pediatric and adult populations, making it a prevalent infectious inflammatory disease in the field of otology. Additionally, dysfunction of the eustachian tube (ET) may occur during acute inflammation in the middle ear(Bluestone, 1983). Failure to promptly repair ET function can result in prolonged inflammation of the middle ear, ultimately progressing into chronic otitis media, which poses significant challenges for treatment(Bluestone and Doyle, 1988). Therefore, investigating morphological and functional changes of the ET during the acute stage of otitis media and elucidating its potential mechanisms are crucial for optimizing rehabilitation strategies and preventing chronicity. Clinically, more than half of suppurative otitis media cases are caused by Gram-negative bacterial infection(Alam et al., 2022), with lipopolysaccharide (LPS) effectively inducing tissue inflammatory responses(Maldonado et al., 2016). Thus, this study aimed to investigate the impact of specific genes on ETD using a mouse model of Gram-negative bacteria-associated acute otitis media through intratympanic LPS injection.

The wall of the ET is composed of cartilage, bone, muscle, fibrous connective tissue, adipose tissue, and other components. Due to its inherent elasticity and the pressure exerted by surrounding tissues as well as the traction effect from the pharynx, the pharyngeal orifice remains typically closed. However, during actions such as yawning, swallowing, sneezing and similar activities, it opens to regulate equilibrium between air pressure in the tympanic cavity and that of the external atmosphere. This ensures proper functioning of the middle ear's sound transmission apparatus. The mucus secreted by goblet cells and mucus glands in the mucosa of the ET facilitates expulsion of pathological substances from the tympanic cavity to nasopharynx through ciliary movement of epithelial lining. Impairment of these functions during acute suppurative otitis media, also known as eustachian tube dysfunction (ETD), is closely associated with delayed inflammation within the tympanic cavity.

Sirtuin3 (SIRT3) is an NAD+-dependent deacetylase predominantly localized within mitochondria(Lombard et al., 2007). Emerging research suggests that the induction and activation of SIRT3 play a pivotal role in regulating cellular bioenergy, controlling inflammatory responses, and mitigating the severity of lipopolysaccharide (LPS)-mediated lung, kidney, and nerve injuries(Ahmedy et al., 2022; Jian et al., 2023; Jin et al., 2023). It is hypothesized that the expression status of the SIRT3 gene may be associated with ET function. However, no previous studies have reported on the specific involvement of genes such as SIRT3 in ETD production. In this study, we utilized SIRT3 knockout (SIRT3-KO) mice to establish animal models of LPS-induced acute otitis media, in order to elucidate the roles of SIRT3 in inflammation and dysfunction of the ET during the acute phase of LPS-induced otitis media.

2. Material and methods

2.1. Animal model

The experimental animals used in this study were 6-week-old SIRT3-KO with a C57BL/6 background, as well as age-matched wild-type (WT) mice obtained from Cyagen (Cyagen, China). Ethical approval for the study was granted by the institutional animal ethics committee (IACUC No.3510), and all experimental procedures were conducted in accordance with the guidelines of the Committee on Animal Research of Tongji Medical College, Huazhong University of Science and Technology. Mouse genotyping was performed by PCR amplification of genomic DNA extracted from tail blood samples, using primers whose sequences are provided in Table A.1 and Fig. A.1.

SIRT3-WT and SIRT3-KO mice were utilized to establish separate models of LPS-induced acute otitis media. Mice were anesthetized via intraperitoneal injection with a 0.3% solution of pentobarbital sodium. Prior to injection, electro-otoscopy was performed on all mice to ensure the normality and integrity of the tympanic membranes, as well as the absence of inflammation or effusion in the tympanic cavity. The infection group (left ear) was administered a 7 μL solution containing LPS at a concentration of 1 mg/ml, while the control group (right ear of the same mouse) received an injection of a 7 μL PBS solution. Subsequent experiments were conducted on mice after 72 hours of administration. To validate the success of the model, we conducted comprehensive validation through histological observation, immunohistochemistry, and functional assessments. The histological images related to this validation are shown in Fig. A.2.

2.2. ET ventilation function assessment

2.2.1. ET forced-response test

The ET forced response test system, as proposed by Ebert et al., has been enhanced based on the principle of ET passive opening pressure measurement introduced by Flisberg et al., in order to achieve a more precise and visually comprehensive assessment of pressure fluctuations in the tympanum(Flisberg, 1967; Ebert et al., 2002). Briefly, a substantial perforation was created in the tense region of the tympanic membrane (TM) in mice firstly. Then, a hermetically sealed external auditory canal (EAC) of the mouse was connected to a three-way tube system. This system facilitated connection between the EAC and an injection pump (used for applying positive pressure) as well as a pressure meter (linked to a computer for real-time visualization of pressure within the system) (Fig. 2A). Air was gradually infused into the enclosed system at a constant slow velocity. Once reaching a specific threshold pressure value, rapid declines in pressure were observed due to sudden opening of the ET. This peak value represents ET passive opening pressure (POP). Upon reaching another distinct pressure value with no further decrease, it stabilizes at this plateau level representing ET passive closure (PCP). Due to the fact that the initial POP and PCP often exhibited higher values compared to subsequent repeats, separate statistical analyses were performed on the initial POP/PCP and average POP/PCP (Fig. 2B).

Figure 2.

Figure 2

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The difference of the ET POP and PCP of WT and SIRT3-KO mice following administration of either PBS or LPS. (A) Schematic representation of the mouse ET POP/PCP detection system. (B) Representative image illustrating the ET forced response test results in mice. The results of paired t-tests to compare the initial POP (C&D), average POP (E&F), initial PCP (G&H), and average PCP (I&J), respectively. (* p < 0.05, ** p < 0.01). (K) The representative images of ET forced-response in the WT-PBS, WT-LPS, SIRT3-KO-PBS, and SIRT3-KO-LPS groups.

2.2.2. The assessment of the ET's capacity to active clearance of negative pressure

The mouse EAC, syringe, and pressure meter are interconnected via a three-way tube to form a closed system, similar to the ET forced response assessment system (Fig. 3A). A constant negative pressure of approximately -30cmH2O is generated using the syringe. Swallowing action is initiated by applying oral touch to the mouse epiglottis with a cotton swab. This swallowing action results in the opening of the ET and subsequent reduction or elimination of negative pressure within the EAC. The difference in pressure before and after swallowing reflects the ET ability of active clearance of negative pressure (ACNP). (Fig. 3B)

Figure 3.

Figure 3

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The difference of the ACNP of WT and SIRT3-KO mice following administration of either PBS or LPS. (A) Schematic representation of the mouse ET ACNP detection system. (B&C) Representative image illustrating the normal (B) and damaged (C) ACNP in mice. (D) The results of unpaired t-tests to compare the ACNP among WT-PBS, WT-LPS, SIRT3-KO-PBS, and SIRT3-KO-LPS groups (n = 10 in each group, **: p < 0.01, ***: p < 0.001, ns: no statistical significance). (E&F) The results of paired t-tests to compare the ACNP between the PBS side and the LPS side in WT (E) and SIRT3-KO (F) mice. (G) The representative images of ACNP in the WT-PBS, WT-LPS, SIRT3-KO-PBS, and SIRT3-KO-LPS groups.

2.2.3. The assessment of mucociliary clearance (MCC) of the ET

Fluorescein (7 μL, 1% fluorescent tracer fluorescein (MCE, Monmouth Junction, NJ, USA) aqueous solution) was injected into the Tympanum of anesthetized mice. After a 2-minute period, the neck was disbranched，and the mandible and tongue were promptly removed. Subsequently, position the remaining skull specimens in a prone orientation within a fluorescence imager for conducting fluorescence and X-ray imaging. The distance traveled by fluorescein along the ET from its tympanic ostium represents the MCC capacity of the ET.

2.3. H&E, AB-PAS staining

The mice body tissue was fixed by perfusion with 4% paraformaldehyde through the cardiovascular system. The ET and its surrounding tissue were dissected and subsequently fixed in 4% paraformaldehyde at 4 °C overnight. To prepare sections of the ET, the tissues underwent decalcification with disodium ethylenediaminetetraacetic acid for a period of 3-5 days, followed by dehydration in a gradient alcohol sequence and embedding in paraffin. The paraffin blocks were then sectioned at a thickness of 4 μm. Paraffin slices were dewaxed to water and subjected to histological examination using hematoxylin and eosin (H&E) staining as well as Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining separately according to the operational guidelines. Images were captured using a laser scanning confocal microscope (Nikon, Tokyo Japan) at ×60 magnification.

2.4. Scanning Electron Microscopy (SEM)

The ET and its surrounding tissue were promptly isolated and fixed in electron microscopy fixative at room temperature for 2 hours, followed by transfer to 4°C for preservation. Prior to usage, the tissues were rinsed with 0.1 M PBS (pH 7.4) and subsequently immersed in 1% OsO4 solution at room temperature for a duration of 1-2 hours. After thorough washing with PBS, the samples underwent dehydration and drying using a Critical Point Dryer. To enhance conductivity, carbon stickers were applied as coating before sputter-coating with gold for a period of 30 seconds. Finally, observation and image capture were conducted utilizing SEM.

2.5. Immunohistochemistry

The paraffin sections of ET tissue were deparaffinized and subjected to high temperature and high pressure for 30 minutes for antigen retrieval using EDTA (pH=9.0). After cooling, the sections were blocked with 10% goat serum at room temperature for 45 minutes. Subsequently, the sections were incubated with Muc5ac antibody (1:500, CST, #61193) overnight at 4 °C. The subsequent steps followed the instructions provided by the commercial secondary antibody kit (Beijing Zhong Shan-Golden Bridge Biological Technology CO., LTD, Cat# PV-6000D). Finally, after dehydration with gradient alcohol, the slices were air-dried and sealed. Observation was performed using a fully automated microscopic scanning system (NanoZoomer S360, Japan). Images of all sections were acquired under identical parameters.

2.6. Statistical analysis

The data were presented (as means ± SD), statistically analyzed and visualized by GraphPad Prism (Version 8.4.0, GraphPad Software Inc., La Jolla, CA, USA). Unpaired t-tests were used to compare the data of different mouse (WT-PBS vs. SIRT3-KO-PBS & WT-LPS vs. SIRT3-KO-LPS). Paired t-tests were performed to compare the data from the same mouse (WT-PBS vs. SIRT3-KO-PBS, & WT-LPS vs. SIRT3-KO-LPS). P < 0.05 was considered statistically significant.

3. Results

3.1. Morphological characteristics of ET and its changes in response to LPS in WT and SIRT3-KO mice

As shown in Figure 1, the mucosal epithelium of the ET is mainly composed of ciliated epithelium, which is rich in goblet cells. The histological examination revealed no significant differences in ET histology between WT and SIRT3-KO mice. However, upon exposure to LPS, both WT and SIRT3-KO mice exhibited a notable increase in goblet cells and goblet cell secretion within the ET lumen (Fig. 1).

Figure 1.

Figure 1

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The representative images of the ET mucosa of WT and SIRT3-KO mice following administration of either PBS or LPS. (A-D) HE staining and AB-PAS staining. Scales bar: 100 μm. (E) SEM images of the ET mucosa of WT and SIRT3-KO mice after intratympanic injection of PBS or LPS. Red asterisk: goblet cells in the secretory phase. Black arrows: goblet cells in the quiescent phase.

As shown in Figure 1E, compared to the WT+PBS group, the cilia in the SIRT3-KO+PBS group exhibited a noticeable reduction in length. In the SIRT3-KO+PBS group, the cilia were observed to be slightly shorter, with some exhibiting signs of atrophy and collapse. Building upon this, the SIRT3-KO+LPS group demonstrated a pronounced area of cilia loss as well as goblet cell infiltration.

3.2. The Difference in ET forced-response of WT and SIRT3-KO mice following tympanic injection of PBS or LPS.

The initial and average POPs of WT mice treated with LPS were not significantly different from those of WT mice treated with PBS (Fig. 2C, E, n = 12 in each group, p = 0.2211, p = 0.1905, respectively). However, both the initial and average POPs of SIRT3-KO mice treated with LPS were higher than those of SIRT3-KO mice treated with PBS. Paired t-tests revealed significant differences (Fig. 2D, F, n = 14 in each group, p = 0.0452, p = 0.0070 respectively). There was no statistically significant difference in PCPs between the WT-PBS and WT-LPS groups or between the SIRT3-KO-PBS and SIRT3-KO-LPS groups (Fig. 2G–J).

3.3. The Difference in ET ACNP of WT and SIRT3-KO mice following tympanic injection of PBS or LPS.

Compared to the WT-PBS group, the ACNP in SIRT3-KO-PBS group was significantly reduced (unpaired t-test, n = 13, p = 0.0057), while there was no difference in ACNP between the WT-LPS and SIRT3-KO-LPS groups (unpaired t-test, n = 13, p = 0.1487) (Fig. 3D). The ACNP on the WT-LPS side was significantly decreased compared to the WT-PBS side (paired t-test, n = 13, p < 0.0001, Fig. 3E). However, in SIRT3-KO mice, there was no significant difference in ACNP between the PBS side and the LPS side of the ET (paired t-test, n = 13, p = 0.3835, Fig. 3F).

3.4. The difference in ET MCC ability of WT and SIRT3-KO mice following tympanic injection of PBS or LPS.

Neither the SIRT3 knockout alone nor the intratympanic injection of LPS alone resulted in significant impairment of the MCC ability of the ET (WT-PBS vs. SIRT3-KO-PBS, unpaired t-test, n = 8, p = 0.1542; WT-PBS vs. WT-LPS, unpaired t-test, n = 8, p = 0.5723). However, when SIRT3 knockout and intratympanic injection of LPS were combined, there was a notable decrease in the MCC ability of the ET compared to either SIRT3 knockout or LPS injection alone (WT-LPS vs. SIRT3-KO-LPS, unpaired t-test, n = 8, p = 0.0425; SIRT3-KO-PBS vs. SIRT3-KO-LPS, unpaired t-test, n = 8, p = 0.0003) (Fig. 4).

Figure 4.

Figure 4

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Representative images MCC ability of the ET in WT and SIRT3-KO mice following tympanic injection of PBS or LPS. (A, B) Representative images showing simultaneous fluorescence imaging (A) and X-ray imaging (B) of the furthest distance reached by MCC. (C) Mice skulls were prepared by removing the mandible and tongues. (D) The statistical graph illustrating the distance of MCC within 2 minutes between WT-PBS, WT-LPS, SIRT3-KO-PBS, and SIRT3-KO-LPS groups (n = 8 in each group, * p < 0.05).

3.5. SIRT3-KO exacerbated the LPS-induced hypersecretion and MUC5AC expression in goblet cells of the ET.

As shown in Figure 5, LPS induced a significant augmentation in the quantity of goblet cells exhibiting positive expression of MUC5AC within the ET mucosa, both in WT and SIRT3-KO mice. Furthermore, a substantial accumulation of mucus containing MUC5AC was observed in the ET lumen of the SIRT3-KO-LPS group (Fig. 5).

Figure 5.

Figure 5

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Representative image of MUC5AC expression in the WT-PBS, WT-LPS, SIRT3-KO-PBS and SIRT3-KO-LPS groups. Brown represents MUC5AC positive expression. The red arrow indicates the mucus accumulated in the lumen of the ET. Semi-quantitative analysis of immunohistochemistry revealed a positive correlation between MUC5AC protein expression levels and the integrated optical density (IOD) of specific regions. The expression of MUC5AC in the SIRT3-KO + LPS group was significantly higher than that in the WT + LPS group (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (between-group comparisons).

4. Discussion

The forced-response of the ET primarily reflects the resistance that needs to be overcome during opening. However, the exact source of this resistance remains incompletely understood. We speculate that it may be associated with the compliance of the tissue in the ET wall and the surface tension of fluid within its lumen. In our study, "initial POP" represents the maximum resistance required for ET opening, while "initial PCP" may indicate a state where natural retraction force from the ET wall balances with gas pressure injected into its lumen. We found no significant difference in "initial PCP" and "average PCP" between groups, suggesting that increased ET opening resistance caused by SIRT3-KO is mainly due to elevated luminal fluid surface tension. During our experiment on ET forced-response, changes in natural retraction force of the ET wall and alterations in both quantity and composition of fluid within its lumen might have occurred after initial passive opening of the ET. Therefore, we believe that "initial POP" better reflects resistance to ET opening compared to "average POP". In our study, LPS did not significantly affect POP in WT mice. However, intratympanic injection of LPS in SIRT3-KO mice resulted in a significant increase in POP compared with contralateral side (intratympanic injection of PBS). These findings suggest that SIRT3-KO may enhance resistance to ET opening in response to LPS, and this increased resistance is likely derived from increased surface tension of liquid within the ET lumen.

Compared with "the ET forced-response test," "the ET's capacity to actively clear negative pressure" is more physiologically consistent and better reflects the ability of the ET to eliminate negative pressure in the tympanic cavity. The primary factors influencing ACNP may include the contractility of ET-related muscles (tensor veli palatini and levator veli palatini), elasticity of the ET wall, and surface tension of fluid within the ET cavity. Our results demonstrate that SIRT3-KO alone or LPS alone significantly reduce ACNP, while their combination does not have a significant effect on ACNP compared to each factor individually. In the ET forced-response test, opening of the ET occurs due to continuously increasing gas pressure in the tympanic cavity, regardless of resistance within the ET. However, in ACNP test, opening force for the ET originates from muscle contraction, which has its limitations. The resistance against ET opening caused by SIRT3-KO or LPS alone may have approached the limitations of these muscles, and adding a second factor does not significantly impact the ACNP compared to one factor alone.

It should be pointed out that how does SIRT3-KO alone (WT-PBS vs. SIRT3-KO-PBS) increase resistance to ET opening and reduce ACNP? In the ET forced-response test, LPS increased POP in SIRT3-KO mice (SIRT3-KO-PBS vs. SIRT3-KO-LPS). However, there was no significant difference in POP between WT and SIRT3-KO mice (WT-LPS vs. SIRT3-KO-LPS, Fig. A.3) after intratympanic LPS injection. The above phenomena have a common feature, that is, they are all comparisons between different individuals (WT-PBS vs. SIRT3-KO-PBS, WT-LPS vs. SIRT3-KO-LPS). Compared with the results of the same individual's own control (left vs. right) (WT-PBS vs. WT-LPS, SIRT3-KO-PBS vs. SIRT3-KO-LPS), there are confounding factors such as individual differences. Thus, the results from different individual's comparison may be less powerful than those from the same individual's own control (WT-PBS vs. WT-LPS, SIRT3-KO-PBS vs. SIRT3-KO). We only take the results of comparison between different individuals as a reference, and our conclusions are more from the results obtained from the same individual's own control.

Similar to the findings of "The ET forced-response test," LPS did not cause changes in MCC in WT mice, but it caused a significant decrease in MCC in SIRT3-KO mice. In other words, neither SIRT3-KO nor LPS alone exhibited a significant impact on the MCC function of the ET. However, when combined, SIRT3-KO and LPS significantly diminished the MCC function of the ET compared to individual factors. The primary factors influencing MCC function may lie in two aspects: 1) cilia morphology, number, and oscillatory ability; 2) composition of the mucus blanket. The cilia of ET mucosa in the SIRT3-KO-LPS group were sparse, shorter and less than those in the other groups (Fig. 1E). This morphological change may be related to the impairment of MCC function.

MUC5AC is the main solid component of airway mucus, and its increased expression is closely related to a variety of respiratory diseases such as COPD, asthma and cystic fibrosis(Hill et al., 2022; Abrami et al., 2024). Increased MUC5AC expression is a major cause of airway mucus obstruction(Pangeni et al., 2023). Our study revealed that the SIRT3-KO-LPS group displayed a notable increase in secretion of mucin MUC5AC by goblet cells in the ET. These results suggest that SIRT3-KO induces hyperfunctioning of goblet cells in ET and incensement of mucin MUC5AC synthesis and secretion. The formation of luminal mucus plugs in the SIRT3-KO-LPS group may predict the occurrence of ET obstruction, which may be responsible for the significant decrease in MCC function of the Eustachian tube in the SIRT3-KO-LPS group. On the other hand, an increase in the solid component of mucus significantly increases the surface tension of the fluid, so the deposition of Muc5AC-rich mucus plugs in the ET lumen in the SIRT3-KO-LPS group may also be responsible for the increased resistance to ET opening.

SIRT3 is a histone deacetylase widely distributed in tissues and localized in mitochondria(Shi et al., 2005). Recent studies have found that SIRT3 plays a deacetylation role in the nucleus and cytoplasm, and has a wide range of downstream biological effects in addition to the regulation of energy metabolism(Scher et al., 2007; Osborne et al., 2016). Inhibition of tissue inflammation is one of the newly discovered downstream effects of SIRT3. It plays a significant anti-inflammatory role in heart, lung, brain, kidney and other tissues through its deacetylation of histone H3(Kim et al., 2018; Tyagi et al., 2018; Kurundkar et al., 2019; Palomer et al., 2020). The mechanism underlying the regulation of MUC5AC expression in the airway mucosa by SIRT3 remains elusive. Only one study has reported that upregulation of SIRT3 in mice can downregulate MUC5AC expression in airway mucus through the NRF2/GPX4 pathway, ultimately attenuating COPD(Wang et al., 2023). Due to the intricate composition and challenging anatomy of ET tissue, extracting ET mucosa for molecular biological research is still arduous, as is accurately intervening with ET tissue in animal experiments at a molecular level. Currently, employing knockout animals to investigate the role of specific genes or molecules in ETD represents the only viable approach. This study also stands as one of the few endeavors utilizing genetically engineered animals to explore the pathogenesis of ETD.

Based on our findings, we postulated that SIRT-KO upregulated MUC5AC expression in ET mice subjected to LPS treatment, resulting in luminal obstruction of the ET, impaired MCC function, and enhanced resistance to ET opening. Consequently, this exacerbated the LPS-induced dysfunction of the ET.

5. Conclusions

In summary, this study highlights the critical role of SIRT3 in regulating ET function under conditions of inflammation induced by LPS. The absence of SIRT3 exacerbates ET dysfunction by increasing the POP, impairing mucociliary clearance, and promoting goblet cell hypersecretion with elevated MUC5AC expression. These findings suggest that SIRT3 is a key mediator in maintaining ET homeostasis and mitigating the pathophysiological processes underlying acute otitis media and ETD. Future research should explore the molecular mechanisms by which SIRT3 regulates epithelial and mucociliary functions in the ET, especially under inflammation. Additionally, targeting SIRT3 with pharmacological agents to restore or enhance its function may offer new therapeutic strategies for managing ETD and otitis media.

Acknowledgements

We are grateful to the Institute of Otorhinolaryngology, Tongji Medical College, Huazhong University of Science and Technology.

Acknowledgments

Informed consent

Not applicable.

Conflict of interest

We declare that we have no conflict of interest.

Data availability

The data are available upon the reasonable request to the corresponding author.

Author contributions

Shimin Zong, Hongjun Xiao designed the experiments and provided funding support. Shimin Zong wrote the main manuscript; Shimin Zong, Xuan Yu and Huimin Zhang carried out most of the experiments and prepared figures. Wenting Yu, Ting Li, Xi Lian, Wei Tang and Tianyi Liu participated in some research work. All authors have reviewed the manuscript.

Ethical approval

All animal procedures were performed according to the guidelines of the Committee on Animal Research of Tongji Medical College, Huazhong University of Science and Technology ([2022] IACUC No. 3510), and were consistent with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used and to prevent their suffering.

Funding Statement

This research was supported by the National Natural Science Foundation of China (Grant NO. 82071057, 82101229), and National Key Research and Development Program of China (Grant NO.2023YFC2508001).

Contributor Information

Wenting Yu, Email: ywt_ent@hust.edu.cn.

Xuan Yu, Email: yuxuan5226@163.com.

Hongjun Xiao, Email: xhjent_whxh@hust.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data are available upon the reasonable request to the corresponding author.

Articles from Journal of Otology are provided here courtesy of Chinese PLA General Hospital

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