# Effect of Erdosteine on Middle Ear Effusion in Rats by Mediating TLR4 Signaling Pathway

Authors: Te Li; Wanting Zeng; Rongrong Liu
Journal: BioMed Research International
 (2021)

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## Abstract

The study aimed to investigate the effect of erdosteine on middle ear effusion in rats through mediating the Toll-like receptor 4 (TLR4) signaling pathway. Rats were injected with endotoxin to prepare the model of acute secretory otitis media (SOM). Then, they were divided into an acute SOM model group (model group,n=15) and erdosteine treatment group (18 mg/kg, gavage, treatment group, n=15). Besides, a normal group (n=15) was set up. Two weeks later, routine biochemical indicators such as aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were detected. The inflammatory effusion due to otitis media was scored. The content of myeloperoxidase (MPO), matrix metalloproteinase (MMP), and tumor necrosis factor-beta (TNF-β) in middle ear lavage fluid was detected via enzyme-linked immunosorbent assay (ELISA). Additionally, histomorphological changes were observed with the help of hematoxylin-eosin (HE) staining, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assays were carried out to measure the expression levels of TLR4 pathway genes and proteins as well as the messenger ribonucleic acid (mRNA) expression levels of key factors for otitis media (mucin 2 (MUC2) and MUC5A). In the model group, the levels of AST, ALP, and glutamic-pyruvic transaminase (GPT) were significantly increased (p<0.05). Besides, the content of MPO, MMP, and TNF-β was overtly raised in the model group (p<0.05), while it was notably lowered in the treatment group (p<0.05). In the treatment group, the cilia were slightly swollen, and inflammatory cells were fewer. The mRNA levels of MUC2, MUC5A, and pathway genes TLR4 and c-Jun N-terminal kinase (JNK) were elevated in the model group. In addition, the protein assay results revealed that the protein levels of TLR4 and JNK were evidently increased in the model group. Erdosteine can treat the middle ear effusion in rats by repressing the activation of the TLR4 signaling pathway.


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## Body

## 1. Introduction

Secretory otitis media (SOM), the most common antibiotic infection in children, is a pathological condition of the middle ear. In such a case, there is effusion in the complete tympanic membrane, accompanied by signs of acute inflammation. SOM is caused by changes in the ciliary system in the middle ear, often due to eustachian tube malfunction. It is ubiquitous, especially in children with cleft palate, and often associated with the development of URTI and other diseases [1–3]. Therefore, the diagnosis and management of SOM greatly affect the health of children, the cost of care, and the overall use of antimicrobials. Moreover, SOM brings significant social burdens and causes indirect costs. The expense of antibacterial treatment for a course of treatment is relatively high [4, 5]. Selecting a specific antimicrobial agent has become a key aspect of management for clinicians. In addition, the necessity of the use of antimicrobials is noticed by the medical field and the public because of the concerns about the rising resistance rate to antimicrobials and increasing costs of antimicrobial prescription [6, 7]. In the case of SOM, the drug resistance of many pathogens is enhanced, which has promoted the application of broad-spectrum antibiotics, and the commonly used antibacterials are relatively more expensive [8]. Furthermore, otitis media can lead to hearing loss that has not been detected for a long time, or frequent earaches, which is often found in routine screening and seriously affects the health and quality of life of patients [9, 10]. Further research is urgently needed.Secondary inflammatory damage of otitis media is induced by the activation of inflammatory cells and TLR-like inflammatory response pathways [11]. Toll-like receptor 4 (TLR4) is a cell surface sensor, which interacts with TLR4 receptors and triggers the activation of downstream pathways, leading to the phosphorylation and translocation of downstream factors [12]. TLRs are able to recognize pathogenesis- and damage-related molecular patterns, promote the activation of nonimmune leukocytes, play an important role in the innate immune system, and trigger the proinflammatory signaling pathways in microbial pathogens [13, 14]. Their downstream factors include c-Jun N-terminal kinase (JNK) that is responsible for the production of many proinflammatory cytokines and induces cell adhesion, proliferation, angiogenesis, and apoptosis [15], resulting in the late activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induction of IRF3 and thereby facilitating the production of interferon (IFN) and IFN-induced gene product [16]. It is worth noting that significant suppression on TLR4 expression inhibits interleukin-1 (IL-1) expression and IL-1β maturation and release [17]. Therefore, preventing and treating secondary inflammatory damage and inhibiting the release of inflammatory factors are more and more attractive in clinical practice and gradually become focuses of research. Erdosteine is a thiol derivative, which is recently applied in clinical practice as a well-tolerated mucolytic drug and has free radical scavenging and anti-inflammatory effects [18]. Its protective effect against oxidative tissue damage has been verified in various inflammatory models [19]. However, the specific mechanism of the effect of erdosteine ​on otitis media effusion by the TLR4 signaling pathway remains unclear. Therefore, in this study, many molecular means were employed to verify the effect of the TLR4 signaling pathway on otitis media effusion, hoping to provide experimental and theoretical bases for the prevention and treatment of otitis media through the TLR4 signaling pathway.This study is aimed at exploring the effect of erdosteine on otitis media effusion by mediating the TLR4 signaling pathway. TLR4 is an important inflammatory regulator for various diseases, but whether it participates in the pathogenesis of otitis media and regulates the middle ear effusion is rarely studied. Therefore, in this study, the potential role of TLR4 in otitis media was investigated, and its effect on otitis media was elucidated based on in vivo experiments and various molecular biological techniques. In brief, the results of this study enrich and improve the theoretical and experimental bases for the effect of the TLR4 signaling pathway on otitis media.


## 2. Materials and Methods

### 2.1. Instruments and Reagents

Tumor necrosis factor-beta (TNF-β) and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Biotechnology Co., Ltd.), TRIzol reagent, DEPC-treated water, SuperScript III reverse transcription (RT) kit, SYBR quantitative polymerase chain reaction (qPCR) Mix (ABI), RIPA lysis buffer (Beyotime), loading buffer, protease inhibitor, bicinchoninic acid (BCA) protein concentration assay kit (Biosharp), β-actin, secondary antibody (Boster Bioengineering Co., Ltd.), primary antibody (Santa), tissue homogenizer, electrophoresis system (Bio-Rad), microplate reader (Thermo Fisher Scientific Instruments Co., Ltd.), 2500 gel imager (Bio-Rad, USA), and qPCR instrument (7900 Fast, Applied Biosystems) were the instruments and reagents used.


### 2.2. Animal Modeling and Grouping

Healthy male Sprague-Dawley rats weighing about 280 g and aged about 10 weeks old were adaptively fed for 7 d to establish a rat model of acute secretory otitis media: The rats were fixed in a supine position after anesthesia, the muscles were separated along the edge of the mandible, and the auditory vesicles were exposed. Peel off the surface bone coat, and inject about 30μL of endotoxin into the middle ear cavity through the ear bubble wall. Then, lightly puncture the ear bubble 2 mm away from the perforation without puncturing to balance the pressure in the middle ear cavity and prevent the tympanic membrane from rupturing. The perforation is sealed with bone wax, and the incision is sutured. All rats were examined under an electric otoscope to exclude external ear canal and middle ear infection. Afterwards, they were divided into an acute secretory otitis media model group and erdosteine treatment group (18 mg/kg, gavage, once a day). Another normal group was set up with 15 rats in each group. The model group and the normal group were given the same dose of normal saline intragastrically. Two weeks later, the rat’s eyeballs were collected by centrifugation and the serum was collected and stored at -80 to be tested for serum biochemical indicators. After that, pentobarbital sodium was injected for anesthesia, and two appropriate amounts of middle ear tissue were carefully taken, one was used for morphological examination, and the other was stored at -80 expression levels of genes and proteins to be tested. The experimental protocol was approved by the experimental animal ethics committee of our hospital, and all animal operations were carried out in accordance with the relevant provisions of the NIH Laboratory Animal Guidance Guide.


### 2.3. Scoring of Middle Ear Inflammatory Effusion

After general anesthesia of rats in each group, the electric auriscope was used to observe bilateral tympanic membranes, and scoring [20] was conducted as per the following specific criteria: 0 point for normal tympanic membrane and no inflammation, 1 point for mild congestion and retraction of the tympanic membrane, 2 points for moderate congestion and retraction or bulging as well as mild exudation of the middle ear, 3 points for moderate to severe effusion of the middle ear, and 4 points for perforation and pus of the tympanic membrane. Score in each group was recorded in details, and the final mean indicated the severity of inflammation in each group.


### 2.4. Determination of Inflammatory Factors

On the 14th day of the experiment, the tympanic membrane was observed using a microscope after anesthesia of the rats, and then, each group of rats was decapitated. The bilateral middle ears were removed, and the middle ear cavity was lavaged with 50 mL of PBS for three times. Next, the lavage fluid (150 mL) was collected and centrifuged at 4000×g for 10 min, and the supernatant was frozen at -80°C. Thereafter, the content of myeloperoxidase (MPO), matrix metalloproteinase (MMP), and TNF-β in the ear lavage fluid in each group was detected via ELISA according to the instructions.


### 2.5. Detection of Related Gene Expression through qRT-PCR

(1) About 100 mg of sterile middle ear tissues was carefully and accurately weighed in a low-temperature environment, added with lysis buffer, and homogenized at a low temperature. Then, total ribonucleic acids (RNAs) were extracted from tissues, and the purity and concentration of RNAs were monitored qualified. (2) After that, the messenger RNAs (mRNAs) were reversely transcribed into complementary deoxyribonucleic acids (cDNAs) using the 20μL amplification system (2 μL of cDNA, 10 μL of qPCR Mix, 2 μL of primer, and 6 μL of ddH2O, for 40 cycles) and stored in a refrigerator at -80°C. (3) Later, PCR (predenaturation at 95°C for 2 min, 94°C for 20 s, 60°C for 20 s, and 72°C for 30 s) was performed for 40 cycles, and PCR amplification was conducted. The primer sequences of the target genes and the internal reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed based on those on GenBank (Table 1). The expression level of the target genes was measured by qRT-PCR. The relative expression levels of related genes in middle ear tissues in each group were calculated by 2−ΔΔCt.Table 1
Primer sequences of all indexes in qRT-PCR.
Target genePrimer sequence (5′–3′)GAPDHF: 5′-TGACTTCAACAGCGACACCCA-3′R: 5′-CACCCTGTTGCTGTAGCCAAA-3′Mucin 2 (MUC2)F: 5′-CGCTACGACCGCCAG ATTG-3′R: 5′-ACACCGTTCACCAGCAAGTC-3′MUC5AF: 5′-CTACCGCACCCGGTTACTAT-3′R: 5′-TTCCGGTTAACACGAGTGAG-3′TLR4F: 5′-CTGAACCAGGGCATACCTGT-3′R: 5′-GAGAAGTCCATGTCCGCAAT-3′JNKF: 5′-TTCCATTGTGGGTAGGTGG-3′R: 5′-CTTACAGCTTCCGCTTCAG-3′


### 2.6. Western Blotting

Tissues (200 mg) were weighed and put into a 10 mL EP tube, followed by standing on ice. Next, the tissues were added with appropriate lysis buffer prepared at a certain ratio, incubated in the refrigerator for fully lysis to release tissue proteins, and centrifuged to collect the supernatant. The protein concentration was determined according to the instructions of the BCA kit, and finally, the concentration of each protein was calculated. Western blotting was carried out as follows: the samples were loaded, subjected to electrophoresis, and transferred onto a membrane, and the membrane was then incubated with primary antibody and secondary antibody. After that, the gel imaging system was utilized for image development, and GAPDH was used to correct the level of proteins to be tested. Lastly, the gray value of the protein bands was analyzed using the Image Lab software.


### 2.7. Statistical Analysis

All raw experimental data recorded were processed using SPSS 20.0 analysis software and subjected to multiple comparisons. The obtained experimental results were expressed asmean±standarddeviation (−χ±SD), and p<0.05 suggested that the difference was statistically significant. GraphPad Prism 7.0 was used for histograms.


## 2.1. Instruments and Reagents

Tumor necrosis factor-beta (TNF-β) and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Biotechnology Co., Ltd.), TRIzol reagent, DEPC-treated water, SuperScript III reverse transcription (RT) kit, SYBR quantitative polymerase chain reaction (qPCR) Mix (ABI), RIPA lysis buffer (Beyotime), loading buffer, protease inhibitor, bicinchoninic acid (BCA) protein concentration assay kit (Biosharp), β-actin, secondary antibody (Boster Bioengineering Co., Ltd.), primary antibody (Santa), tissue homogenizer, electrophoresis system (Bio-Rad), microplate reader (Thermo Fisher Scientific Instruments Co., Ltd.), 2500 gel imager (Bio-Rad, USA), and qPCR instrument (7900 Fast, Applied Biosystems) were the instruments and reagents used.


## 2.2. Animal Modeling and Grouping

Healthy male Sprague-Dawley rats weighing about 280 g and aged about 10 weeks old were adaptively fed for 7 d to establish a rat model of acute secretory otitis media: The rats were fixed in a supine position after anesthesia, the muscles were separated along the edge of the mandible, and the auditory vesicles were exposed. Peel off the surface bone coat, and inject about 30μL of endotoxin into the middle ear cavity through the ear bubble wall. Then, lightly puncture the ear bubble 2 mm away from the perforation without puncturing to balance the pressure in the middle ear cavity and prevent the tympanic membrane from rupturing. The perforation is sealed with bone wax, and the incision is sutured. All rats were examined under an electric otoscope to exclude external ear canal and middle ear infection. Afterwards, they were divided into an acute secretory otitis media model group and erdosteine treatment group (18 mg/kg, gavage, once a day). Another normal group was set up with 15 rats in each group. The model group and the normal group were given the same dose of normal saline intragastrically. Two weeks later, the rat’s eyeballs were collected by centrifugation and the serum was collected and stored at -80 to be tested for serum biochemical indicators. After that, pentobarbital sodium was injected for anesthesia, and two appropriate amounts of middle ear tissue were carefully taken, one was used for morphological examination, and the other was stored at -80 expression levels of genes and proteins to be tested. The experimental protocol was approved by the experimental animal ethics committee of our hospital, and all animal operations were carried out in accordance with the relevant provisions of the NIH Laboratory Animal Guidance Guide.


## 2.3. Scoring of Middle Ear Inflammatory Effusion

After general anesthesia of rats in each group, the electric auriscope was used to observe bilateral tympanic membranes, and scoring [20] was conducted as per the following specific criteria: 0 point for normal tympanic membrane and no inflammation, 1 point for mild congestion and retraction of the tympanic membrane, 2 points for moderate congestion and retraction or bulging as well as mild exudation of the middle ear, 3 points for moderate to severe effusion of the middle ear, and 4 points for perforation and pus of the tympanic membrane. Score in each group was recorded in details, and the final mean indicated the severity of inflammation in each group.


## 2.4. Determination of Inflammatory Factors

On the 14th day of the experiment, the tympanic membrane was observed using a microscope after anesthesia of the rats, and then, each group of rats was decapitated. The bilateral middle ears were removed, and the middle ear cavity was lavaged with 50 mL of PBS for three times. Next, the lavage fluid (150 mL) was collected and centrifuged at 4000×g for 10 min, and the supernatant was frozen at -80°C. Thereafter, the content of myeloperoxidase (MPO), matrix metalloproteinase (MMP), and TNF-β in the ear lavage fluid in each group was detected via ELISA according to the instructions.


## 2.5. Detection of Related Gene Expression through qRT-PCR

(1) About 100 mg of sterile middle ear tissues was carefully and accurately weighed in a low-temperature environment, added with lysis buffer, and homogenized at a low temperature. Then, total ribonucleic acids (RNAs) were extracted from tissues, and the purity and concentration of RNAs were monitored qualified. (2) After that, the messenger RNAs (mRNAs) were reversely transcribed into complementary deoxyribonucleic acids (cDNAs) using the 20μL amplification system (2 μL of cDNA, 10 μL of qPCR Mix, 2 μL of primer, and 6 μL of ddH2O, for 40 cycles) and stored in a refrigerator at -80°C. (3) Later, PCR (predenaturation at 95°C for 2 min, 94°C for 20 s, 60°C for 20 s, and 72°C for 30 s) was performed for 40 cycles, and PCR amplification was conducted. The primer sequences of the target genes and the internal reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed based on those on GenBank (Table 1). The expression level of the target genes was measured by qRT-PCR. The relative expression levels of related genes in middle ear tissues in each group were calculated by 2−ΔΔCt.Table 1
Primer sequences of all indexes in qRT-PCR.
Target genePrimer sequence (5′–3′)GAPDHF: 5′-TGACTTCAACAGCGACACCCA-3′R: 5′-CACCCTGTTGCTGTAGCCAAA-3′Mucin 2 (MUC2)F: 5′-CGCTACGACCGCCAG ATTG-3′R: 5′-ACACCGTTCACCAGCAAGTC-3′MUC5AF: 5′-CTACCGCACCCGGTTACTAT-3′R: 5′-TTCCGGTTAACACGAGTGAG-3′TLR4F: 5′-CTGAACCAGGGCATACCTGT-3′R: 5′-GAGAAGTCCATGTCCGCAAT-3′JNKF: 5′-TTCCATTGTGGGTAGGTGG-3′R: 5′-CTTACAGCTTCCGCTTCAG-3′


## 2.6. Western Blotting

Tissues (200 mg) were weighed and put into a 10 mL EP tube, followed by standing on ice. Next, the tissues were added with appropriate lysis buffer prepared at a certain ratio, incubated in the refrigerator for fully lysis to release tissue proteins, and centrifuged to collect the supernatant. The protein concentration was determined according to the instructions of the BCA kit, and finally, the concentration of each protein was calculated. Western blotting was carried out as follows: the samples were loaded, subjected to electrophoresis, and transferred onto a membrane, and the membrane was then incubated with primary antibody and secondary antibody. After that, the gel imaging system was utilized for image development, and GAPDH was used to correct the level of proteins to be tested. Lastly, the gray value of the protein bands was analyzed using the Image Lab software.


## 2.7. Statistical Analysis

All raw experimental data recorded were processed using SPSS 20.0 analysis software and subjected to multiple comparisons. The obtained experimental results were expressed asmean±standarddeviation (−χ±SD), and p<0.05 suggested that the difference was statistically significant. GraphPad Prism 7.0 was used for histograms.


## 3. Experimental Results

### 3.1. Inflammatory Effusion Score of Rats in Each Group

The score was below 1 point in the normal group, without inflammation or effusion (p<0.05); close to 4 points in the model group, with severe inflammation and effusion (p<0.05); and basically close to 1 point in the treatment group, with mild inflammation and effusion (p<0.05) (Figure 1).Figure 1
Inflammatory effusion score. In the normal group, the score is below 1 point, and there is no inflammation or effusion (p<0.05). In the model group, the score is close to 4 points, and severe inflammation and effusion are observed (p<0.05). In the treatment group, the score is basically close to 1 point, and mild inflammation and effusion are detected (p<0.05). ∗p<0.05 vs. the normal group and #p<0.05 vs. the model group.


### 3.2. Results of Serum Biochemical Test

The important biochemical indicators AST, GPT, and ALP in serum play an important role in otitis media, as shown in Table2. Compared with the normal group, the levels of AST, GPT, and ALP in the model group were significantly increased (p<0.05); compared with the model group, the contents of AST, GPT, and ALP in the treatment group decreased significantly (p<0.05). The obvious change of biochemical indicators at the occurrence of otitis media is indicated, which predicts the occurrence and development of the disease.Table 2
Changes in the content of GPT, ALP, and AST (U/L).
GroupASTALPGPTNormal group74.38±3.4081.40±5.2848.78±4.37Model group161.07±5.07a208.38±2.82a124.31±5.31aTreatment group91.52±2.45b100.14±4.13b67.09±5.44bThe content of AST, GPT, and ALP is elevated markedly in the model group (p<0.05), while it clearly declines in the treatment group. ap<0.05 vs. the normal group and bp<0.05 vs. the model group.


### 3.3. Levels of TNF-β, MPO, and MMP

As shown in Table3, compared with the normal group, the levels of TNF-α, MPO, and MMP in the model group were all increased (p<0.05). Compared with the model group, the levels of TNF-α, MPO, and MMP in the treatment group were reduced and were close to those in the control group (p<0.05).Table 3
Levels of TNF-β, MPO, and MMP.
GroupTNF-β (fmol/mL)MPO (ng/mL)MMP (ng/mL)Normal group28.4±2.590.5±3.694.9±5.6Model group59.8±1.2a205.2±6.4a218.3±3.5aTreatment group38.3±3.1b109.1±2.8b116.4±5.6bThe levels of TNF-β, MPO, and MMP are elevated remarkably in the model group (p<0.05), while they are reduced in the treatment group, which are close to those in the normal group. ap<0.05 vs. the normal group and bp<0.05 vs. the model group.


### 3.4. Results of Hematoxylin-Eosin (HE) Staining

The results of HE staining (Figure2) revealed that the cilia of the middle ear had a normal structure and ordered arrangement in the normal group (Figure 2(a)). In the model group (Figure 2(b)), the ciliated epithelium was swollen, the cilia were arranged disorderly with falling off of part of them, the middle ear mucosa was thickened, and the inflammatory cells were infiltrated. The treatment group exhibited slightly swollen cilia and fewer inflammatory cells (Figure 2(c)).Figure 2
Results of hematoxylin-eosin (HE) staining. Note: the cilia of the middle ear have a normal structure and ordered arrangement in the normal group ((a) ×100). The ciliated epithelium is swollen, with disorderly arranged cilia and falling off of part of the cilia, the middle ear mucosa is thickened, and inflammatory cell infiltration is detected in model group ((b) ×100). The treatment group displays slightly swollen cilia and fewer inflammatory cells ((c) ×100).
(a)(b)(c)


### 3.5. Expression of Otitis Media-Related Genes and Pathway Molecules

The results of gene expression detection showed that the model group exhibited raised mRNA levels of MUC2, MUC5A, TLR4, and JNK (p<0.05), while the treatment group had evidently decreased such levels (p<0.05) (Figure 3), implying that in the case of otitis media, the pathway genes may be activated during the pathogenesis, which indicates the further progression of the disease.Figure 3
Results of gene expression. The mRNA levels of MUC2, MUC5A, and pathway genes TLR4 and JNK are elevated in the model group (p<0.05) but notably decline in the treatment group (p<0.05). ∗p<0.05 vs. the normal group and #p<0.05 vs. the model group.


### 3.6. Expression of Pathway Proteins

As shown in Figure4, compared with the normal group, the expression of pathway proteins TLR4 and JNK in the model group was significantly decreased and increased in the treatment group (p<0.05); compared with the model group, the expression of pathway proteins TLR4 and JNK in the treatment group decreased significantly in the model group and increased significantly in the model group (p<0.05). This shows that after treatment with erdosteine, it can promote the recovery of otitis media.Figure 4
Protein expression level. (a) The expression levels of TLR4 and JNK by Western blot. (b) Quantification analysis. The expression levels of the pathway proteins TLR4 and JNK are overtly decreased in the treatment group (p<0.05) and evidently increased in the model group (p<0.05). ∗p<0.05 vs. the normal group and #p<0.05 vs. the model group.
(a)(b)


## 3.1. Inflammatory Effusion Score of Rats in Each Group

The score was below 1 point in the normal group, without inflammation or effusion (p<0.05); close to 4 points in the model group, with severe inflammation and effusion (p<0.05); and basically close to 1 point in the treatment group, with mild inflammation and effusion (p<0.05) (Figure 1).Figure 1
Inflammatory effusion score. In the normal group, the score is below 1 point, and there is no inflammation or effusion (p<0.05). In the model group, the score is close to 4 points, and severe inflammation and effusion are observed (p<0.05). In the treatment group, the score is basically close to 1 point, and mild inflammation and effusion are detected (p<0.05). ∗p<0.05 vs. the normal group and #p<0.05 vs. the model group.


## 3.2. Results of Serum Biochemical Test

The important biochemical indicators AST, GPT, and ALP in serum play an important role in otitis media, as shown in Table2. Compared with the normal group, the levels of AST, GPT, and ALP in the model group were significantly increased (p<0.05); compared with the model group, the contents of AST, GPT, and ALP in the treatment group decreased significantly (p<0.05). The obvious change of biochemical indicators at the occurrence of otitis media is indicated, which predicts the occurrence and development of the disease.Table 2
Changes in the content of GPT, ALP, and AST (U/L).
GroupASTALPGPTNormal group74.38±3.4081.40±5.2848.78±4.37Model group161.07±5.07a208.38±2.82a124.31±5.31aTreatment group91.52±2.45b100.14±4.13b67.09±5.44bThe content of AST, GPT, and ALP is elevated markedly in the model group (p<0.05), while it clearly declines in the treatment group. ap<0.05 vs. the normal group and bp<0.05 vs. the model group.


## 3.3. Levels of TNF-β, MPO, and MMP

As shown in Table3, compared with the normal group, the levels of TNF-α, MPO, and MMP in the model group were all increased (p<0.05). Compared with the model group, the levels of TNF-α, MPO, and MMP in the treatment group were reduced and were close to those in the control group (p<0.05).Table 3
Levels of TNF-β, MPO, and MMP.
GroupTNF-β (fmol/mL)MPO (ng/mL)MMP (ng/mL)Normal group28.4±2.590.5±3.694.9±5.6Model group59.8±1.2a205.2±6.4a218.3±3.5aTreatment group38.3±3.1b109.1±2.8b116.4±5.6bThe levels of TNF-β, MPO, and MMP are elevated remarkably in the model group (p<0.05), while they are reduced in the treatment group, which are close to those in the normal group. ap<0.05 vs. the normal group and bp<0.05 vs. the model group.


## 3.4. Results of Hematoxylin-Eosin (HE) Staining

The results of HE staining (Figure2) revealed that the cilia of the middle ear had a normal structure and ordered arrangement in the normal group (Figure 2(a)). In the model group (Figure 2(b)), the ciliated epithelium was swollen, the cilia were arranged disorderly with falling off of part of them, the middle ear mucosa was thickened, and the inflammatory cells were infiltrated. The treatment group exhibited slightly swollen cilia and fewer inflammatory cells (Figure 2(c)).Figure 2
Results of hematoxylin-eosin (HE) staining. Note: the cilia of the middle ear have a normal structure and ordered arrangement in the normal group ((a) ×100). The ciliated epithelium is swollen, with disorderly arranged cilia and falling off of part of the cilia, the middle ear mucosa is thickened, and inflammatory cell infiltration is detected in model group ((b) ×100). The treatment group displays slightly swollen cilia and fewer inflammatory cells ((c) ×100).
(a)(b)(c)


## 3.5. Expression of Otitis Media-Related Genes and Pathway Molecules

The results of gene expression detection showed that the model group exhibited raised mRNA levels of MUC2, MUC5A, TLR4, and JNK (p<0.05), while the treatment group had evidently decreased such levels (p<0.05) (Figure 3), implying that in the case of otitis media, the pathway genes may be activated during the pathogenesis, which indicates the further progression of the disease.Figure 3
Results of gene expression. The mRNA levels of MUC2, MUC5A, and pathway genes TLR4 and JNK are elevated in the model group (p<0.05) but notably decline in the treatment group (p<0.05). ∗p<0.05 vs. the normal group and #p<0.05 vs. the model group.


## 3.6. Expression of Pathway Proteins

As shown in Figure4, compared with the normal group, the expression of pathway proteins TLR4 and JNK in the model group was significantly decreased and increased in the treatment group (p<0.05); compared with the model group, the expression of pathway proteins TLR4 and JNK in the treatment group decreased significantly in the model group and increased significantly in the model group (p<0.05). This shows that after treatment with erdosteine, it can promote the recovery of otitis media.Figure 4
Protein expression level. (a) The expression levels of TLR4 and JNK by Western blot. (b) Quantification analysis. The expression levels of the pathway proteins TLR4 and JNK are overtly decreased in the treatment group (p<0.05) and evidently increased in the model group (p<0.05). ∗p<0.05 vs. the normal group and #p<0.05 vs. the model group.
(a)(b)


## 4. Discussion

As a pathological condition of the middle ear caused by changes in the ciliary system in the middle ear, SOM creates significant social burdens and indirect costs. Moreover, otitis media can result in hearing loss and frequent earache, severely affecting the health and quality of life of patients [21]. However, the inflammatory mechanism of otitis media (i.e., a severer subtype) has not been studied in depth. The pathological change in the pathogenesis of otitis media is a complex process involving many factors, which needs to be further studied. In this study, acute SOM was induced by injection of endotoxin, and then, bilateral tympanic membranes were observed using the electric auriscope and scored. The results revealed that the score was 1 point in the normal group, without inflammation and effusion; close to 4 points in the model group, with severe inflammation and effusion; and basically close to 1 point in the treatment group, with mild inflammation and effusion. Major serum biochemical indicators such as AST, GPT, and ALP are important players in otitis media. It was found in this study that the levels of AST, GPT, and ALP were evidently increased in the model group, while they were distinctly reduced after treatment with erdosteine, suggesting that the obvious changes are detected in biochemical indicators in the case of otitis media, which indicates the development and progression of the disease. In addition, HE staining results showed that the normal group had neatly arranged cilia with normal structure. The model group displayed swollen ciliated epithelium, disorderly arranged cilia with partial falling off, thickened middle ear mucosa, and infiltrated inflammatory cells. The treatment group had slightly swollen cilia and fewer inflammatory cells. Ryding et al. and Dickson have obtained similar results [22, 23]. The results of this study are basically in line with the findings of previous studies.Studies have manifested that inflammation plays an indispensable role in the development and progression of otitis media, which persists with increased inflammatory cells and is characterized by a large number of neutrophils and macrophages infiltrating into the interstitial space. The number of inflammatory cells is closely correlated with the severity of the disease in biopsy [24]. MPO is mainly distributed in neutrophils whose cytoplasmic granules contain massive MPO. Therefore, elevated MPO content in lung tissues indicates the increase in neutrophil content, and its excessive accumulation will cause inflammation, so that it can act as a predictor of inflammation [25]. MMP plays an important role in the degradation of extracellular matrix and the destruction of proteolytic enzymes. Proteolytic enzymes stimulated by proinflammatory cytokines promote the production of more inflammatory factors. TNF can also induce the excessive production of other inflammatory mediators. The results of this study showed that the levels of TNF-β, MPO, and MMP were elevated in the model group, and such levels were reduced in the treatment group, which were close to those in the normal group. This suggests that the elevated levels of TNF-β, MPO, and MMP further facilitate the progression of otitis media and aggravate the inflammatory response. The condition was relieved after treatment with erdosteine. The above results are consistent with previous study findings. This indicates that erdosteine is capable of inhibiting excessive inflammatory cytokines and preventing irreversible damage to cells due to their overproduction. MUC is a class of large glycoproteins, which exerts important protective functions on the underlying epithelial cells in the middle ear, including mechanical protection and pathogen elimination achieved through elimination mechanisms and interactions with the host immune system. However, the abnormal MUC2 and MUC5A secreted by the middle ear are deemed to be important in the pathophysiological mechanism of otitis media, and their excessive secretion leads to abnormal mucociliary clearance in the middle ear, resulting in pathological phenomena including chronic otitis media and hearing loss [26, 27]. The TLR inflammatory pathway serves as an important participant in otitis media, triggering different downstream signaling cascades such as JNK and leading to the involvement of the transcription factor NF-κB in the production of downstream proinflammatory cytokines and chemokines [28, 29]. TLR4 significantly increases the level of cytokines in mouse serum, and thus, many genes or proteins regulating this pathway are expected to be potential targets for the treatment of otitis media. It was discovered in the gene detection in this study that the mRNA levels of MUC2, MUC5A and pathway genes TLR4 and JNK in the model group were raised, while those in the treatment group were evidently decreased, implying that the pathway genes may be activated during the pathogenesis of otitis media, which further indicates the further progression of the disease. The results of the pathway protein assay showed that the protein expressions of TLR4 and JNK were overtly decreased in the treatment group and markedly increased in the model group, suggesting that the treatment with erdosteine promotes the recovery of otitis media. In summary, this study confirms the effect of the TLR4 signaling pathway on the inflammatory effusion of otitis media through multiple molecular methods, thus providing new potential targets for the gene therapy of otitis media. Although such results are obtained, this study may still have some shortcomings, and in-depth subsequent studies are needed.In conclusion, it is found in this study that erdosteine is able to repress the progression of middle ear effusion of otitis media in rats by modulating the TLR4 pathway, and the therapeutic effect and prognosis can be evaluated through the TLR4 pathway. In subsequent studies, more cell lines will be introduced, and other possible mechanisms of action will be further verified and explored via gene knockout, flow cytometry, and other techniques.



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*Source: 9968907-2021-10-25.xml*