Sprague-Dawley rats (age, 7–8 weeks old; gender, male; weight, 200–250 g) were purchased from Damul Science (Daejeon, Korea). All rats were provided with adequate food and water. Animal experiments were conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Chonnam National University and the protocol approved by the Committee on the Ethics of Animal Experiments of Chonnam National University (CNUHIACUC-20027). All rats were alive during the research period, and normal tympanic membranes were observed prior to LPS injection. In this study, ketamine (100 mg/kg) and xylazine (10 mg/kg) were intraperitoneally injected for the anesthesia. Anesthesia were performed before LPS injection or LED irradiation. Animals were also anesthetized before decapitation for euthanasia. The OM animal model was established by injecting LPS (2.0 mg/ml; L9143, Sigma, St. Louis, MO, United States) from Pseudomonas aeruginosa 10 into the ME of the rats through the tympanic membrane. Intra-tympanic injection was used to deliver LPS since it was the least invasive method compared to other surgical approaches opening the bulla. Rats with PBS injection served as the control. After LPS exposure for 24 h, rats with OM were randomly divided into two groups (Red/NIR LED irradiated group versus none-irradiated group).

In this study, the continuous-wave dual red and NIR LED irradiation system (HK HEALTHCARE CO., LTD., Korea) with wavelengths of 655 nm and 842 nm consisted of a control module and a battery and was connected to an LED light source with a power cable (Supplementary Figure S1). The LED light source unit was composed of an LED light source and an optical fiber. The power intensity of the LED light was 163.2 W/m². To examine the therapeutic effect of the red/NIR LED on LPS-induced AOM, rats were irradiated with the red/NIR LED through the ear canal for 30 min for 3 days after LPS injection. Optical fiber (diameter = 3 mm) was tightly placed in the external auditory canal cartilaginous portion. The direction of the optical fiber was monitored to always face the tympanic membrane during the irradiation. Since the optical fiber itself might cause otitis externa, the same optical fiber was place into the external auditory canal (under same anesthesia) without turning on the LED light for the control LPS group. The red/NIR LED system for cell experiments consisted of a power connector and a power supply unit, and the upper LED light source was composed of six LEDs. HMEECs and RAW 264.7 cells were irradiated with red and NIR wavelengths of 653 nm and 842 nm, respectively, and an intensity of 19.76 W/m² for 3 h after LPS stimulation.

HMEECs (ScienCell, Carlsbad, CA, United States) and murine macrophage RAW 264.7 cells (Korean Cell Line Bank, Seoul, Korea) were grown with EPiCM-2 and RPMI 1640, respectively. HMEECs and RAW 264.7 cells were treated with LPS at a concentration of 10 μg/mL or 1 μg/mL immediately after serum starvation.

After the rats were euthanized, tympanic bullae were harvested and fixed in 4% paraformaldehyde for 24 h at 4°C, rinsed with PBS, and decalcified in Calci-Clear Rapid (National Diagnostics, Atlanta, GA, United States) for 5 days. The softened bullae were dehydrated and embedded in paraffin. The paraffin-embedded bullae were sectioned into 7 µm longitudinal sections for staining. The sectioned bullae were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) to visualize the ME mucosa.

Immunohistochemical detection of myeloperoxidase (MPO), 7 µm-thick sections were deparaffinized and rehydrated. Citrate buffer solution was used for antigen retrieval, followed by incubation at room temperature for 10 min using 0.3% hydrogen peroxide and the removal of the endogenous peroxidase. The sections were incubated with a primary antibody at 4°C overnight and horseradish peroxidase was then applied for 1 h. Signals were developed in 3,3′-diaminobenzidine (DAB) tetrahydrochloride solution containing 0.1% H₂O₂ and observed under the microscope. The number of positive MPO neutrophils in each group was calculated by randomly selecting six fields of view at magnification ×40.

Total RNA was extracted from the ME mucosa of rats or cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. Extracted RNA was used to synthesize complementary DNA with a reverse transcription kit (Takara, Kyoto, Japan). qPCR was performed with SYBR Green (Takara) and monitored using Thermal Cycler Dice^® Real Time System III (Takara). Primers were designed to target endogenous genes, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH and Gapdh) or β-actin (ACTB and Actb) was used as the endogenous control. The crossing point of target genes with GAPDH and Gapdh was calculated using formula 2—(target gene − GAPDH and Gapdh or ACTB and Actb), and the relative amounts were quantified. The qPCR primers are listed in Supplementary Table S1.

IL-1β, IL-6, TNF-α, MPO antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, United States) or Cell Signaling Technology (Danvers, MA, United States). Total ERK, pERK, JNK, pJNK, p38, pp38 (Cell Signaling Technology), and actin (Sigma) were used with the appropriate secondary antibodies from MBL (Shirley, NY, United States). For western blotting, proteins were separated by 12% PAGE and then electrophoretically transferred onto PVDF membranes. The membranes were incubated with primary antibodies, which were diluted according to the manufacturer’s instructions at 4°C overnight. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgGs were added as secondary antibodies. The blots were reprobed with anti-actin antibody as the loading control. Finally, immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL) protocol. The protein levels of IL-1β, IL-6, and TNF-α were measured by densitometry, and the relative protein levels were compared with that of actin and depicted as bar graphs (mean ± SEM, n = 9).

The concentrations of the cytokines IL-1β, IL-6, and TNF-α in the tympanic bullae of rats were determined by ELISA using human ELISA kits from R&D Systems (Minneapolis, MN, United States), mouse ELISA kits from Elabscience (Houston, TX, United States), and rat ELISA kits from MyBioSource (San Diego, CA, United States). The results are expressed as picograms per milliliter in accordance with the manufacturer’s instructions. The results for the samples following triplicate experiments are depicted as bar graphs (mean ± SEM, n = 3).

The cell viability of HMEECs and RAW 264.7 cells was analyzed using the EZ-Cytox cell viability assay kit from DoGenBio (Seoul, Korea). In brief, the cells were plated and cultured in 96-well plates (5 × 10³ cells/well). Next, 10 µL of EZ-Cytox reagent was added to each well and incubated at 37°C for 2 h, and cell viability was examined. The absorbance was measured using an ELISA microplate reader with a wavelength of 450 nm.

We determined the statistical significance of differences by non-parametric analysis, Mann-Whitney U and Kruskal Wallis tests. When the data met normal distribution (among sample size n = 9), one-way ANOVA with post-hoc Tukey HSD or Student’s t-test was performed. Assessment of normality of data was done using Kolmogorov-Smirnov and Shapiro-Wilk test P values less than 0.05 were regarded as statistically significant. Statistical analysis was performed using SPSS 27(Chicago, IL, United States).

To verify the successful establishment of LPS-induced AOM in a rat model, we observed the tympanic membrane by otoscopy and the histologic sections by H&E staining. As shown in Figures 1A–E, the tympanic membrane of the normal control group was transparent, and there were no signs of an inflammatory reaction; however, the LPS-treated group showed an inflammatory response at 2 days after LPS treatment, and the inflammation disappeared after 7 days. The ME mucosal thickness was significantly increased on the first day after LPS treatment and recovered after 7 days (Figures 1F–O).

To investigate the therapeutic effect of the red/NIR LED on LPS-induced AOM, rats were irradiated through the ear canal using the red/NIR LED with wavelengths of 655 nm and 842 nm and an intensity of 163.2 W/m² for 3 days after LPS injection. As shown in Figures 2A–C, otoscopy revealed that red/NIR LED irradiation reduced the LPS-induced inflammation of the tympanic membrane. In comparison with the control group, the ME mucosal thickness of the LPS-treated group with OM was much thicker with increased inflammatory cell deposits (Figures 2D–I). Compared with the control group, higher degree of MPO positive cell infiltration was observed in the LPS-treated group with OM (Figures 2J–L). The ME mucosal thickness of the control group was 21.5 ± 3.53 μm, which was increased to 102.2 ± 17.81 μm in the LPS-treated OM group. On the other hand, the ME mucosal thickness was significantly reduced to 40.7 ± 8.82 μm in the red/NIR LED-irradiated OM group (Figure 2M). The number of MPO positive neutrophils was significantly decreased in the red/NIR LED-irradiated OM group (Figure 2N). The results indicated that LPS induced AOM in the rats, and the increase in the ME mucosal thickness and inflammatory cell deposits was markedly reversed by red/NIR LED irradiation.

To investigate the in vivo effects of red/NIR LED irradiation on LPS-induced AOM in rats, we designed an experiment as shown in Figure 3A. The inflammatory response is known to be crucial in ME damage under various conditions, such as bacterial exposure (Ferenbach et al., 2007). Therefore, we attempted to examine whether OM amelioration by the red/NIR LED is associated with inflammation suppression and analyzed the expression of pro-inflammatory cytokines by western blotting and ELISA in the tympanic bullae obtained from rats after LPS treatment. The results showed that the increase in inflammation of the tympanic membrane was markedly reduced by LED irradiation (Figures 3B–D), and the protein expression of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, which was increased by LPS, was significantly decreased by LED irradiation (Figure 3E). ELISA results were consistent with those of western blotting (Figures 3F–H). OM is associated with ROS levels, and ROS is associated with mitogen-activated protein kinase (MAPK) signaling. Therefore, we subsequently investigated whether MAPK signaling is inhibited by LED irradiation. The phosphorylation of ERK, p38, and JNK, which was increased in the ME tissue of rats after LPS treatment, was inhibited by LED irradiation (Figure 3I). The results demonstrated the efficacy of red/NIR LED irradiation in inhibiting pro-inflammatory cytokine expression induced by LPS, and the inhibition was highly correlated with the suppression of the MAPK signaling pathway.

We examined the in vitro effect of red/NIR LED irradiation on the expression of pro-inflammatory cytokines. The HMEEC line has been widely used in OM studies; thus, we used this cell line to investigate the effect of LED irradiation on LPS-induced cytokine elevation. The experimental design for LED irradiation with HMEECs is illustrated in Figure 4A. We first confirmed the cell viability of HMEECs after LED irradiation. The number of viable cells did not show any difference over time in the LED-irradiated group compared with the control group (Figures 4B–G). As shown in Figures 4H–J, IL-1β, IL-6, and TNF-α mRNA levels were significantly increased with LPS stimulation at 50 μg/ml for 3 h, which were greatly reduced by LED irradiation in HMEECs. Furthermore, western blot results revealed that the expression levels of IL-1β, IL-6, and TNF-α were increased by LPS exposure, and this upregulation was reversed by LED irradiation in HMEECs (Figure 4K). Additionally, the concentrations of IL-1β, IL-6, and TNF-α were determined by ELISA, which were consistent with the western blot results (Figures 4L–N). The phosphorylation of ERK, p38, and JNK, which was increased after LPS stimulation, was inhibited by LED irradiation in HMEECs (Figure 4O). Important inflammatory mediators in OM are produced by infiltrating immune cells such as macrophages (Juhn et al., 2008). LPS can induce various inflammatory mediators and stimulate local macrophages to produce soluble mediators including IL-1β, IL-6, and TNF-α. Therefore, we investigated the effect of red/NIR LED irradiation on the expression of pro-inflammatory cytokines in RAW 264.7 cells, a mouse macrophage cell line. The experimental design for RAW 264.7 cells was similar to that for HMEECs, as shown in Figure 5A. LED irradiation showed no cytotoxicity towards RAW 264.7 cells (Figures 5B–G). Likewise, the mRNA and protein expression levels of IL-1β, IL-6, and TNF-α, which were increased by LPS treatment, were markedly inhibited by LED irradiation in RAW 264.7 cells (Figures 5H–N). Moreover, the phosphorylation of ERK, JNK, and p38, which was increased by LPS, was attenuated by LED irradiation in RAW 264.7 cells (Figure 5O). These results indicated that red/NIR LED irradiation suppressed the expression of pro-inflammatory cytokines induced by LPS in the macrophage cell line RAW 264.7 through the blockade of MAPK signaling. Overall, our study demonstrated that red/NIR LED irradiation effectively suppressed inflammation caused by OM. Moreover, red/NIR LED irradiation reduced pro-inflammatory cytokine production in HMEECs and RAW 264.7 cells by blocking MAPK signaling (Figure 6).