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. 2024 Dec 27;62(5):6166–6178. doi: 10.1007/s12035-024-04675-7

CD38 Coordinates with NF-κB to Promote Cochlear Inflammation in Noise-Induced Hearing Loss: the Protective Effect of Apigenin

Da Liu 1,2,3,4,5,#, Na Sai 1,2,3,4,5,#, Ying Zhou 1,2,3,4,5,#, Ning Yu 1,2,3,4,5, Qing-qing Jiang 1,2,3,4,5, Wei Sun 6, Wei-ju Han 1,2,3,4,5,✉, Weiwei Guo 1,2,3,4,5,✉
PMCID: PMC11953141  PMID: 39725836

Abstract

Noise exposure is one of the most common causes of sensorineural hearing loss. Although many studies considered inflammation to be a major contributor to noise-induced hearing loss, the process of cochlear inflammation is still unclear. Studies have found that activation of the NF-κB signaling pathway results in the accumulation of macrophages in the inner ear plays an important role in hair cell damage. In this study, tandem mass tag (TMT) technique was used to analyze the changes in basilar membrane proteome expression before and after acoustic injury. After noise exposure, the nicotinamide adenine dinucleotide (NAD) metabolism level was decreased, and the NF-κB signaling pathway was activated. The expression of CD38, the main NAD hydrolase in mammals, may directly lead to inflammation onset. Then, anakinra, an IL-1 receptor blocker, and apigenin, a CD38 inhibitor, were administered to animals to protect against noise-induced hearing loss. Our results showed that anakinra had little influence on the hearing threshold shift, while apigenin significantly reduce the threshold shift of hearing by inhibiting the expression of NF-κB and CD38 can be a promising target for protecting against noise-induced hearing loss.

Keywords: CD38, NF-κB, Hearing loss, Apigenin

Introduction

Noise-induced hearing loss (NIHL) is caused by multiple components, including direct mechanical damage of hair cells, and dysfunction of cochlear microvascular circulation metabolic homeostasis [1, 2]. An increasing number of researchers have considered cochlear inflammation to be one of the crucial causes of hair cell damage [3]. NF-κB signaling pathway, which is related to immune cell activation and inflammatory factor release, is one of the inflammatory pathways involved in cochlear inflammation. Previously, it is reported that NLRP3 inflammasome is related to inflammatory factors in noise-induced hearing loss [4]. Several studies suggested that the activation of macrophages played a vital part of cochlear inflammation [5–7].

It is still unclear how the macrophages work in cochlear. Lang reported that the resident immune cells of the cochlear are rooted in hematopoietic stem cells [8] and the innate immune system of the cochlear plays an important role in maintaining the normal structure and function of the cochlear [9]. CD38 is expressed on the surface of lymphocytes, monocytes, macrophages, dendritic cells, and natural killer cells is considered as the signal of immune cell activation [10]. It has a chemotactic effect on leukocyte aggregation [11]. Recent studies found that CD38 is the main NAD hydrolase in mammals, which can transform NAD into adenosine diphosphate ribose (ADPR), cyclic adenosine diphosphate ribose (cADPR), and nicotinamide. CD38 can also degrade NAD precursors and NADP [12, 13]. However, the function and role of CD38 in cochlear have not been reported.

Apigenin is a flavonoid with 4, 5, and 7-trihydroxyflavone which has been found in vegetables (parsley, celery), fruits, and tea [14]. Studies have shown that apigenin can increase NAD + levels by inhibiting CD38 expression. Therefore, apigenin can be used to regulate NAD/NADH and treat metabolic syndrome [15] and reduce mitochondrial oxidative stress [16]. We speculated that apigenin may downregulate the expression of CD38 in the cochlear. So far, there is no report on using the apigenin in NIHL.

The purpose of this study is to explore the expression of CD38 and the role of macrophages in noise-induced cochlear inflammation, and if apigenin can be used as a therapeutic agent against NIHL.

Materials and Methods

Animals

Healthy C57/BL6J mice (6–8 weeks, male, weighing 18 ~ 22 g) were purchased from SPF (Beijing) biotechnology company. All of the mice went through an auditory brainstem response (ABR) test to ensure normal hearing. The animals were divided into control group, noise exposure group, and noise + drug group by using a random number table. Each group contained fifteen animals. This study was approved by the Institutional Animal Care and Use Committee of Chinese PLA General Hospital.

Auditory Brainstem Response Testing

ABR tests were performed before, and in brief, animals were anesthetized using pentobarbital sodium (0.8 mg/kg). Three stainless-steel electrodes were placed under the vertex and postauricular skin respectively. Sound delivery device tube was placed into the external auditory canal of the tested ear, to make sure each ear was stimulated independently. Tucker Davis Technologies (TDT System III, USA) system and Biosig software (TDT, RX6, Alachua, FL, USA) were used for ABR testing. The sound was produced with a multifunction processor (MEDUSA4Z, TDT). Click and tone bursts at 2 kHz, 4 kHz, 8 kHz, 16 kHz, and 24 kHz were used to elicit ABRs. The ABR threshold was defined as the lowest sound stimulus intensity induced a repeatable response.

Drug Administration

Apigenin (Sunshine Yingrui, LA6510) was given via oral gavage at a dose of 25 mg/kg body weight based on previous study [16]. Anakinra (MCE, HY-108841) was injected intraperitoneally (i.p.) at a dose of 20 mg/kg body weight (Fig. 1). In the pre-experiment, no evidence of hearing impairment was found during apigenin and anakinra administration.

Fig. 1.

Fig. 1

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Schedule of ABR tests and drug administration. Baseline hearing threshold were evaluated 3 days before the noise exposure. ABR tests was performed at day 4, day 7 and day 14. Apigenin and anakinra were given from day − 3 to day 14. ABR: auditory brainstem response

Noise Exposure

Animals of the NE group, NE + Ana group and NE + Api group were placed in the mesh cases and were exposed to 4 h white noise at 120 dB SPL on 2 consecutive days. The noise signal was routed to loudspeaker (Aijie Audio Equipment Factory) through a power amplifier (MF-1201 MOSTET, ATech). The intensity of the sound field was calibrated using a sound level meter (Brüel & Kjær, 2250L, Denmark) during the exposure. The control group did not receive noise exposure. The noise exposure was proved to cause stable threshold shifts [17].

Tandem Mass Tags Experiment

The animals were sacrificed by spinal dislocation under anesthesia after experiment. Both sides of the cochlear were dissected and placed in a cool PBS solution for further studies. A hundred and eighty animals were equally divided into two groups.

The basilar membrane tissue of the control group and noise exposure group was extracted using a protein extraction kit (Bangfei Bioscience), then capsuled in EP tubes marked Ctrl 1, Ctrl 2, Ctrl 3 (control group) and NE 1, NE 2, NE 3 (noise exposure group). Each tube contains membrane tissues collected from 30 mice. The actual protein concentration was quantified by microplate reader. The tissue of each group was catalyzed by trypsin (Promega, V5113) for 16 h, then the peptide was labeled according to the tandem mass tags (TMT) kit instructions. Each sample was separated by high-performance liquid chromatography (HPLC) liquid phase system at a nanoliter flow rate, and then loaded into mass spectrometry precolumn and the analytical column. A total of six samples were pooled dissolved respectively in 2% acetonitrile solution and loaded onto the reverse-phase column. Then eluted the sample with 98% acetonitrile solution using a step linear elution program at a rate of 0.7 ml/min for half an hour. The samples were collected every 1.5 min during the elution procedure [18].

Bioinformatics Analysis

According to the protein quantitative results of the samples, protein difference was screened out between the NE group and the control group (Student T test, P < 0.05). Proteins with FC (fold change) ≥ 1.2 and P < 0.05 were defined as upregulated, and proteins with FC ≤ 0.833 and P < 0.05 were defined as downregulated. The differential proteins were compared with all protein backgrounds by searching the online database (http://www.geneontology.org), and the biological functions related to the differential proteins were inferred based on the significantly enriched GO functional items. Differential proteins were mapped to each item in the Gene Ontology database, and the number of proteins contained in each item was calculated using the hypergeometric distribution.

Immunofluorescence

The cochlear basilar membrane was prepared with the surface preparation method, antibodies used in this experiment were rabbit anti-NF-κB p65 polyclonal antibody (Abcam, ab32536), rat anti-F4/80 polyclonal antibody (Abcam, ab6640), and mouse anti-CD38 monoclonal antibody (Proteintech, 60,006–1-lg). Rinse thoroughly and seal the tablet with DAPI-containing glycerin (Abcam, ab228549). The specimens were observed by excitation light at 405 nm (blue), 488 nm (green), and 555 nm (red) with a Zeise confocal microscope. The specimens were scanned with a thickness of 0.26 μm. The images were superimposed by orthogonal projection using ZEN (Blue 2.3SP) software.

Western Blot

Western blot was performed to quantitatively analyze CD38 and NF-κB (p65) expression in the cochlear of each group. Animals were sacrificed at day 1 (noise exposure) and day 7 (drug administration), and the whole cochlear protein was extracted. After polyacrylamide gel electrophoresis, membrane transfer, and 5% skim milk powder sealing, primary antibodies were incubated at 4 ℃ overnight. The next day, corresponding secondary antibodies were treated at room temperature for 1 h. The primary antibody used in this study were anti-CD38 (Abcam, ab216343), anti-NF-kappaB (p65) (Abcam, ab32536) and anti-β-actin (Immunoway, YM3028). The protein band was visualized in darkroom on photographic film. Image J software (v.1.52a) was used to measure the gray value of the band, and the gray value ratio of the target protein to the internal reference protein was used as the relative content for statistical analysis.

Statistical Analysis

SPSS 21.0 software was used for statistical analysis. The mean ± standard deviation was used to express data. Student T test was performed for comparison between the NE group and the control group. One-way ANOVA and Turkey test were performed for comparison of multiple group results. P < 0.05 was considered to be statistically significant.

Results

TMT Experiment Result

A total of 12,003 kinds of peptides and 3124 kinds of proteins were identified in this TMT experiment. The protein molecular weight is mainly concentrated in 10–120 kDa (Fig. 2A). And 131 differential proteins were screened out from the two groups, including 37 upregulated proteins and 94 downregulated proteins. The frequency histogram is drawn by the screening results. Statistical analysis was conducted on the screened differential proteins. As shown in the volcano map (Fig. 2D), the black dots represent proteins with no significant change between the control group and the NE group, the red dots represent upregulated proteins while the green dots represent downregulated proteins.

Fig. 2.

Fig. 2

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Identification of basilar membrane proteins. A Molecular weight of identified proteins. The horizontal axis (kDa) represents the molecular weight range of proteins, and the left vertical axis (red) shows the number of proteins in the molecular weight range. The right ordinate (percentage of molecular, %) corresponds to the cumulative curve (blue) showing the percentage of the accumulated protein below the molecular weight to the total protein. B Correlation analysis between the control and the NE group protein. C The ordinate represents the number of proteins, and the abscissa represents the quantitative ratio (log2 FC) of the sample. D Volcano analysis showing upregulated (red) and downregulated (green) proteins between the control group and the NE group. E Heat map analysis showing upregulated (red) and downregulated (green) proteins between the control group and the NE group

GO analysis showed that the differential proteins were mainly enriched in metabolic processes, including NAD/NADP + activity, norepinephrine metabolic process, and negative regulation of inflammatory response to antigenic stimuli regulation of inflammation to an antigenic stimulus, glutathione derivative metabolic retinoid metabolic process, and retinoid metabolic process (Fig. 3).

Fig. 3.

Fig. 3

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GO analysis of the differential proteins. The activation of metabolic (blue frame), NAD activity (red frame) and immune (green frame) pathways were down-regulated during the acute acoustic trauma of the basilar membrane

In order to find the reason for the decrease of NAD level in cochlear, we detected NAD hydrolase CD38 expression in the inflammatory process.

Expression of NF-κB and CD38

The activation of NF-κB signaling pathway has been shown to mediate noise-induced cochlear inflammation [19–21]. It has been reported that NF-κB can promote the transcription of CD38 gene [22]. One day after noise exposure, the cochlear tissues are collected to conduct further experiment. We observed NF-κB expression and nuclear transfer after noise exposure by immunofluorescence staining (Fig. 4A). Basilar membrane macrophages which mainly existed in scala tympani were the main source of CD38 expression (Fig. 4B). Western blot results showed that CD38 protein expression in the NE group was higher than that in the control group (P < 0.05) (Fig. 4C).

Fig. 4.

Fig. 4

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Expression of p65 and CD38. A Nuclear transport of p65. In the control group (Ctrl), p65 was labeled around the nucleus of outer hair cell (white arrow). In the NE group (NE), p65 transferred into the nucleus (white arrow). B Immunofluorescence co-staining of CD38 and F4/80. Macrophages were labeled with Anti-F4/80 antibody (green). CD38 (red) was mainly expressed in basilar membrane macrophages (blue arrow). C CD38 protein expression in the control group (Ctrl) and the NE group (NE). Protein bands showed that CD38 protein expression in the NE group is higher than that in the control group. *Compared with control group, P < 0.05, n = 6 animals, 12 cochleae

Apigenin and Anakinra Treatment for NIHL

Then, the mice were treated with IL-1 blocker anakinra and CD38 inhibitor apigenin respectively. In the pre-experiment, three doses (5 mg/kg, 10 mg/kg, and 20 mg/kg) of anakinra were given to the animal to determine the effective dose [23]. Given by the result that low dosage (5 mg/kg, 10 mg/kg) administration showed little protective effect, we administered a dosage of 20 mg/kg to continue the following test.

After the noise exposure, the hearing threshold rose significantly. We observed that the sound intensity for the ABR threshold increased twofold, from 45.7 to 79.3 dB, while for the NE + Ana group, it increased from 37.5 to 76.7 dB (Table 1). ABR tests continued until day 14 post-exposure, and the differences in ABR thresholds between the NE + Ana group and the NE group were not significant (Fig. 5).

Table 1.

Auditory brainstem response (ABR)-click thresholds and ABR tone-burst thresholds in the NE group and NE + Ana group

Group ABR thresholds (dB SPL)
Click 4 kHz 8 kHz 16 kHz 24 kHz
Baseline 12.7 ± 4.1 13.2 ± 3.5 12.2 ± 2.1 14.5 ± 3.3 18.4 ± 4.2
Day 4 NE 46.3 ± 13.2 55.0 ± 10.6 51.3 ± 7.0 60.0 ± 9.0 75.0 ± 3.5
NE + Ana 41.3 ± 10.4 45.0 ± 11.0 54.2 ± 8.4 62.1 ± 8.3 74.6 ± 7.5
Day 7 NE 36.9 ± 3.5 43.8 ± 6.0 50..0 ± 6.1 63.8 ± 3.3 78.1 ± 3.5
NE + Ana 36.7 ± 7.2 42.1 ± 9.5 52.9 ± 8.8 63.3 ± 6.9 78.3 ± 3.1
Day 14 NE 45.7 ± 12.9 45.7 ± 7.3 60.7 ± 7.8 73.6 ± 3.5 79.3 ± 1.7
NE + Ana 37.5 ± 6.6 43.8 ± 11.9 62.5 ± 8.5 67.1 ± 5.9 76.7 ± 4.7
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Fig. 5.

Fig. 5

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The ABR results of the NE group and NE + Ana group. Baseline (A), day 4 (B), day 7 (C), and day 14 (D) ABR test results were shown. The differences between the NE + Ana group and the NE group were not significant, P > 0.05. n = 5 animals, 10 ears

After administration of apigenin, the ABR thresholds at multiple frequencies were reduced compared to the NE group. At day 7 post-exposure, the 4 kHz ABR threshold value decreased to 43.6 dB, compared to 57.5 dB in the NE group, while the 8 kHz ABR threshold value decreased to 48.0 dB, compared to 61.1 dB in the NE group (Table 2). This difference was still observed up to day 14. However, there was no significant effect of apigenin treatment on 16 k Hz and 24 k Hz hearing (Fig. 6).

Table 2.

Auditory brainstem response (ABR)-click thresholds and ABR tone-burst thresholds in the NE group and NE + Api group. *Difference compared with the NE group, P < 0.05. **Significant difference compared with the NE group P < 0.01

Group ABR thresholds (dB SPL)
Click 4 kHz 8 kHz 16 kHz 24 kHz
Baseline 12.9 ± 4.5 11.4 ± 3.5 10 ± 0 10 ± 0 13.6 ± 4.8
Day 4 NE 50.4 ± 15.4 50.0 ± 15.0 52.1 ± 15.3 73.2 ± 6.2 80.0 ± 0
NE + Api 42.5 ± 19.5 40.4 ± 17.9 45.7 ± 15.7 68.6 ± 7.4 78.9 ± 22.1
Day 7 NE 55.7 ± 17.3 57.5 ± 16.0 61.1 ± 14.4 74.6 ± 5.5 79.3 ± 1.7
NE + Api 43.6 ± 10.4* 43.6 ± 11.1* 48.0 ± 13.4* 68.6 ± 7.4 77.1 ± 4.1
Day 14 NE 55.7 ± 13.5 56.1 ± 13.0 60.4 ± 48.8 74.6 ± 5.5 79.6 ± 1.3
NE + Api 42.1 ± 9.0** 42.9 ± 10.3** 43.2 ± 13.0* 71.8 ± 8.6 79.3 ± 1.7
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Fig. 6.

Fig. 6

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The ABR results of NE group and NE + Api group. Baseline (A), day 4 (B), day 7 (C), and day 14 (D) ABR test results were shown. Significant differences were presented in click, 4 kHz and 8 kHz in 7 days after noise exposure. n = 7 animals, 14 ears. *Significant difference compared with the NE group, P < 0.05. **Significant difference compared with the NE group P < 0.01

In order to explore the effect of apigenin, we checked NF-κB (p65) and CD38 expression changes at day 7, during the peak of cochlear inflammation [24]. It was found that compared with the control group, the expression of p65 and CD38 protein were significantly increased after noise exposure, and it increased most in the NE group (P < 0.01). The protein band showed that p65 expression in the NE + Ana group was significantly lower than that in the NE group (P < 0.05), while the CD38 expression was not affected. After given apigenin, p65 (P < 0.01) and CD38 (P < 0.01) expression were decreased compared with the NE group (Fig. 7).

Fig. 7.

Fig. 7

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Western blot results of p65 (A) and CD38 (B) expression in the control group (Ctrl), the NE group (NE), the NE + Api group and the NE + Ana group. Relative protein content is shown in the histogram. #Compared with the control group, P < 0.05; ##compared with the control group, P < 0.01; *compared with NE group, P < 0.05

Scanning electron microscopy is the most direct method to detect hair cell damage. At the 7th day after noise exposure, macrophages started to consume cell fragments [25]. To better observe the effect of inflammatory response on the cochlear, samples from the NE group and the NE + Api group were collected for scanning electron microscopy observation on the 14th day, when cochlear inflammation had largely resolved. Outer hair cells in both groups showed varying degrees of morphological change. Cilia of the basal turn were severely damaged, with some nearly disappearing and few discernible preserved ciliary structures remaining. Cilia injury was less severe in the apical turn and the middle turn. There were significant differences in the morphology of outer hair cells between the NE group and the NE + Api group. The cilium of the outer hair cells in the NE group was found to be severely damaged, with most of them collapsing or even disappearing. Outer hair cell cilium of the NE + Api group was slightly scattered and rarely disappeared. These results were consistent with the ABR results. The thresholds shift was most obvious in high-frequency hearing, which consisted with the morphological changes of the basal turn cilia. The frequency at which hearing loss is greater corresponds to greater hair cell damage (Fig. 8).

Fig. 8.

Fig. 8

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Scanning electron microscopy images of hair cells in the NE group and the NE + Api group. From top to bottom, the scan pictures show the apical turn, middle turn, and basal turn of the basilar membrane. The pictures on the left were taken at a low magnification. Each row on the right side of the picture was magnified sequentially. Morphological changes were observed in the outer hair cells of both groups. Cilia of the basal turn were severely damaged, with some almost disappearing, which is consistent with the severe hearing damage observed at high frequencies. Significant differences were observed in the morphology of outer hair cells between the NE group and the NE + Api group. Hair cells in the NE + Api group showed a more complete morphology. The outer hair cell cilia in the NE group were partially disappeared, while those in the NE + Api group were slightly scattered but still distinguishable

Discussion

TMT technology is an innovative in vitro peptide tagging technology that has been applied to study various disease mechanisms [26]. In this study, we identified 3124 proteins in the mouse basilar membrane through TMT experiments, and 131 differential proteins changed after noise exposure, including 37 upregulated proteins and 94 downregulated proteins. We used GO enrichment analysis to detect changes in various metabolic reactions after noise exposure. Our findings suggest that noise exposure may induce the activation of NF-κB signaling passway, leading to inflammatory responses by activated cochlear macrophages.

As reported, the decrease in NAD levels affects the cell respiration process, resulting in damage to DNA and mitochondria [27]. NAD acts as a co-factor in the Krebs cycle and cell respiration [28]. Therefore, NAD is considered to be involved in various diseases, such as aging, neurological disorders, and cancer [29]. Hemophilus mediates various infection processes. Since it lacks the ability to synthesize NAD, it can only obtain NAD from the environment to support its physiological activities and metabolism [30]. CD38-positive immune cells can consume NAD from the surrounding environment, thereby limiting the spread of inflammation. It appears that similar signaling pathways promote the activation of macrophages during inner ear inflammation, leading to hearing impairment.

The cochlear used to be considered an “immune-free” organ due to the presence of the blood-labyrinth barrier (BLB) [31]. In recent years, it has been observed that noise exposure induces the recruitment of immune cells, immune response, and the expression of inflammatory factors in the cochlear. The NF-κB signaling pathway is involved in the inflammation of both innate and acquired immunity [21, 32]. Generally, NF-κB complex exists with the form of p50-P65 dimer binding to kappa B inhibitor protein, which activates and promotes the expression of multiple genes when received stimuli [33]. NF-κB nuclear translocation is associated with cochlear inflammatory reactions [34], and leads to apoptosis and necrosis of hair cells [35]. The NF-κB signaling pathway is closely related to the gene expression of inflammatory factors. After noise exposure, the NF-κB signaling pathway upregulates the NLRP3 inflammasome, which is activated in macrophages, leading to the transcription of various inflammatory factors [36]. Accompanying IL-6, TNF-α, and IL-1β have been shown to be important factors involved in the regulation of noise-induced cochlear inflammation [37].

The molecular mechanism by which cochlear macrophages mediate noise-induced inner ear inflammation is still unclear. CD38 is also a downstream component of the NF-κB signaling pathway and is believed to be involved in neurodegenerative diseases [38]. We labeled macrophages with the F4/80 antibody [39] and co-stained them with the CD38 antibody. CD38 was observed to be expressed on the surface of macrophages after noise exposure. Western blot quantification of cochlear protein showed that CD38 protein levels in the NE group were higher than those in the control group (P < 0.05). This suggests that activation and expression of CD38 by macrophages may be the cause of basilar membrane macrophage infiltration and NAD consumption [40].

We also investigated the effects of anakinra and apigenin on reducing cochlear inflammation. Anakinra has been found effective in NLRP3 gene mutation disorder treatment [23]. Recent clinical studies have shown that anakinra can reduce the inflammatory response in COVID-19 patients and improve survival rates without mechanical ventilation [41]. As a downstream blocker in this signaling pathway, anakinra showed limited effects on hearing protection. We suggest that the protective effect of apigenin depends on both the intensity of the noise and the duration of medication administration. It is known that high-frequency hearing is more vulnerable to noise exposure. Apigenin showed a significant protective effect on hearing at 4 kHz and 8 kHz frequencies 7 days after noise exposure, suggesting that inflammation may not be the primary cause of hair cell damage in the basal turn. Western blot showed that anakinra also inhibited NF-κB expression after noise exposure. However, we found that NF-κB (P65) expression was significantly increased in the NE + Ana group compared with the control group. The inhibiting effect of anakinra is obviously not as strong as that of apigenin. The cilium of outer cochlear hair cells can be effectively protected by apigenin, which can reduce the threshold shift by about 20 dB. Western blot analysis showed that apigenin inhibited the expression of CD38 protein in the cochlea after noise exposure and down-regulated the expression of the NF-κB signaling pathway. Blocking IL-1βalone, as one of the factors involved in NF-κB activation, cannot inhibition NF-κB expression completely. We suggest that IL-1β as well as several inflammatory factors participate in the positive feedback loop of the pathway. This may explain why anakinra showed little effect on the ABR thresholds.

Apigenin can increase the level of NAD + in vivo by inhibiting CD38, thereby treating metabolic syndrome [15], and reduce the level of mitochondrial oxidative stress by regulating the NAD/NADH ratio and Sirt3 activity [16]. As shown in the result, the ABR threshold of the NE + Api group was lower than that of the NE group, significant differences at click (P < 0.05), 4 kHz (P < 0.05), and 8 kHz (P < 0.05) were observed seven days after noise exposure (Fig. 6D). Scanning electron microscopy images proved that apigenin has an obvious protective effect on hair cell cilia (Fig. 8).

Macrophages can not only release inflammatory factors to promote inflammation, but also remove harmful substances and cell debris. Inhibiting macrophage function is a promising treatment for cochlear inflammation [42]. At present, there is no specific drug for the treatment of noise-induced hearing loss. Apigenin, as a biological drug, though may not only affect hearing by inhibiting CD38 expression, it provides evidence for the necessity of looking for similar therapeutic targets. Further studies in large animal models and clinical trials are expected. The limitation of this article is that the detailed role of CD38 in inflammatory response is not well demonstrated. Further studies such as CD38 knockout strategy would provide more meaningful results.

In summary, the TMT experiment provided a wide perspective to understand the physiological and pathological processes in cochlear. Based on proteomic analysis results, the role of NF-κB signaling pathway and CD38 played during noise-induced cochlear inflammation was discussed in this study. We considered that it was NF-κB that activated basilar membrane macrophages to express CD38, leading to cochlear inflammation and hearing loss (Fig. 9). This study provide a reference for the role of macrophages in cochlear inflammatory response and the effects of apigenin on CD38. Apigenin may be a new therapeutic strategy for NIHL.

Fig. 9.

Fig. 9

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CD38 coordinates with NF-κB to promote cochlear inflammation in NIHL. As the closest immune cell to organ of Corti, activated macrophages can express CD38 and consume NAD in organ of Corti, causing NAD + metabolism dysfunction and ultimately leads to cochlear inflammation. In this process, inflammatory factors such as IL-1, IL-6, and TNF-α promoted the activation of NF-κB signaling pathway, forming a positive feedback cycle

Author Contribution

Da Liu, Na Sai, and Ying Zhou contributed equally to designing the work, data collection, and data analysis. Weiwei Guo and Weiju Han were involving in critical reading of the article, drafting the article, and final approval of the version of the article. Ning Yu, Qing-qing Jiang, and Wei Sun were involving in critical reading, drafting the article, and critical reading.

Funding

This work was supported by National Key Research and Development Program of China (2022YFC2402705), National Natural Science Foundation of China (No.82371148), Spring City Plan: the High-level Talent Promotion and Training Project of Kunming (2022SCP001), Open Program of National Clinical Research Center for Otolaryngologic Diseases (202200007) and Beijing Nature Science Foundation (No.7242134 and No.7244412).

Data Availability

The data that support the findings of the study are not publicly available but are available from the corresponding authors.

Declarations

Ethics Approval

The study method was approved by the PLA General Hospital ethical committees.

Consent to Participate

Informed consent was obtained from all individual participants included in the study.

Consent for Publication

All authors agreed with the final version of the manuscript.

Conflict of Interest

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Da Liu, Na Sai, and Ying Zhou contributed equally to this work.

Contributor Information

Wei-ju Han, Email: hanweiju@aliyun.com.

Weiwei Guo, Email: gwent001@163.com.

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Data Availability Statement

The data that support the findings of the study are not publicly available but are available from the corresponding authors.

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