Ethics statement
Written informed consent was obtained from all patients and their guardians prior to the study. Study protocols according to the ethical principles for medical research involving human subjects of the Helsinki Declaration were approved by Ethic Committee of Linyi People’s Hospital. The animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol of animal experiments was approved by the Institutional Animal Care and Use Committee of Linyi People’s Hospital. The animal experiments were conducted based on minimized animal number and the least pains on experimental animals.
Study subjects
The AOM specimens were collected from 85 AOM patients infected with streptococcus pneumoniae who received treatment in Linyi People’s Hospital from January 2015 to December 2016. The clinical diagnostic criteria of those patients were as follow: acute occurrence (duration ≤ 48 h); fever, earache, purulent discharge in ear and other infection; tinnitus, aural fullness and hearing loss; puncture of eardrum and observed shining point detected by otoscope. All patients lived in the local area more than 3 months. Patients with symptoms for a duration over 8 weeks and immune deficiency or those who had received antibiotics therapy yet diagnosed with combined infection in other parts should be excluded. Among those patients, there were 49 male patients and 36 female patients (aged: 15 days - 10 years; average age: 4.4 ± 2.2 years). Meanwhile, the specimens of middle ear lavage fluid were collected from 40 healthy subjects as control, among which there were 21 boys and 19 girls (aged: 3 - 11 years; average age: 5.0 ± 2.0 years).
Establishment of mice model with AOM
A total of 56 specific pathogen-free (SPF) grade healthy C57BL/6 mice (half male and half female; aged: 6 - 8 weeks; weighing: 16 - 20 g) were provided by animal center. Mice were subjected to general anesthesia by intraperitoneal injection with 3% pentobarbital solution. Afterwards, bilateral external auditory canals and tissues surrounding ear were disinfected by 75% ethanol. Then a 20 μL micro pipette was used to extract 5 μL streptococcus pneumoniae suspension and inoculated into tympanum for binaural modeling after auripuncture by otoscope. Mice in the control group were injected with equal volume of phosphate buffer saline (PBS) or other prepared solutions and labeled properly. Subsequently, mice were housed in a cage in the prone position so as not to be suffocated by disturbance in respiration. Mice were further well fed, and their weight and temperature were recorded. Later, mice were collected at corresponding time points for following experiments.
Mice treatment with lentivirus and chemical
Eight mice were set as normal control, and rest 48 mice were used for establishing model. A total of 40 mice were successfully modeled. Next, 36 modeled mice were assigned into 6 groups (6 mice per group), and other 4 mice were reserved for later use. Lentivirus vector LV5-GFP was synthesized by GenePharma Company (Shanghai, China). Then lentivirus was packaged by 293T cells, which were incubated in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum (FBS) and passaged every other day. Subsequently, 2 weeks before the inoculation of streptococcus pneumoniae, mice were transduced with lentivirus vector solution (5 × 108 pfu/100 μL) or retinoic acid (RA, an inhibitor of Nrf2 signaling pathway, 40 μM) twice a week for consecutive 4 weeks [19].
Collection of middle ear lavage fluid
After mice model was successfully established, at least 3 mice were randomly collected at the 1st and 5th day. Mice underwent general anesthesia by intraperitoneal injection with 5% pentobarbital solution. Next, mice were euthanized by opening chests to collect heart blood. After the surrounding area of mouse ear was disinfected by 75% ethanol, the operating scissors sterilized under high pressure were used to cut off its auricle and expose middle ear. Subsequently, PBS containing 1% FBS was utilized to lavage middle ears of mice (10 μL per time; 6 times each ear), and then about 50 μL lavage fluid was collected and labeled. Afterwards, 5 μL lavage fluid of each ear was applied for serial dilution and bacteria counting, and rest lavage fluid was centrifuged at 4℃ and 350 × g for 5 min. The supernatant was sub-packaged, labeled and preserved at 80℃ for detection of cytokines. Cell precipitation was lysed on ice by red blood cell lysis buffer at 4℃ and 350 × g for 5 min. The supernatant was removed, and cell precipitation was resuspended by precooling PBS containing 1% FBS for cell counting, flow cytometry and the cover glasses preparation.
Isolation and culture of macrophage
Macrophages were harvested from middle ear lavage fluid, which were centrifuged at 500 × g. Next, the harvested cells were resuspended in red blood cell lysis buffer, washed with cold PBS twice and centrifuged at 500 × g for 5 min to obtain macrophages. Then cells (1 × 10 5 cells/mL) were incubated with dulbecco’s modified eagle’s medium (DMEM) (Gibco Company Grand Island, NY, USA) containing 10% FBS in a 24-well plate at 37℃ and with 5% CO2 for 4 - 6 h.
Endocytosis analysis
Fluorescein isothiocyanate (FITC; 0.5 mg/mL; Sigma, St. Louis, MO, USA) was incubated at 37℃ for 20 min to prepare FITC-labeled streptococcus pneumoniae. Next, macrophages of mice (1 × 10 5 cells) were infected by FITC-labeled sodium dodecyl sulfate (SDS) (multiplicity of infection, 10). Then cell nucleus was stained by 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen Inc., Carlsbad, CA, USA), and the images were visualized by confocal laser scanning microscope (LSM 510, Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). The ratio of endocytosis on bacteria (determined by the overlapping of green bacteria) was quantified by independent researchers based on 100 random counted cells and expressed as the percentage of cells containing bacteria.
Detection of bacterial load
The middle ear lavage fluid (0.8 mL) was rotated at 4℃ and 260 × g for 10 min, and the precipitation was resuspended in 0.5 mL sterile PBS. Next, the suspension was continuously diluted for 10 times, and 50 mL final diluent was added on a sheep blood agar plate and fixed at 37℃ and with 5% CO2. After 16 h, the colony formation unit was counted.
Colorimetric assay
The specimens were centrifuged at 1610 × g for 5 min. The supernatant was respectively added with 50 μL, 0.1 or 2 mL and 0.1 mL superoxide dismutase (SOD), nitric oxide (NO) and malondialdehyde (MDA) detection kits (20091105, NanJing JianCheng Bioengineering Institute, Nanjing, China) in strict accordance with their instructions. Initially, the specimens were fully mixed with SOD by a vortex blender and water-bathed at 37℃ for 40 min for colorimetric determination (light wavelength λ = 550 nm). Next, for NO detection, the testing, standard and blank tubes were set, completely mixed with corresponding reaction solution and allowed to stand at 37℃ for 60 min, which were then rotated for 30 s, cooled down to room temperature and centrifuged. The supernatant was visualized and allowed to stand for 10 min. The optical density (OD) value was determined at the light wavelength of 550 nm. Then the specimens were uniformly mixed with MDA by a vortex blender, water-bathed at 95℃ for 80 min and centrifuged at 2191 - 2862 × g for 10 min. The colorimetric determination was conducted for acquiring the OD value (light wavelength λ = 532 nm).
Reverse transcription quantitative polymerase chain reaction (RT-QPCR)
Total RNA was extracted using Trizol RNA kit (Invitrogen Inc., Carlsbad, CA, USA). Next, the integrity of RNA was assessed by agarose gel electrophoresis. If 28 S and 18 S bands were bright, clear and sharp, and the grey value of 28S band was twice than that of 18 S band, RNA fragment should be complete. Also, the ratio of A260/A280 between 1.8 and 2.1 indicated higher purity of RNA. PrimescriptTM RT reagent Kit (RRO37A, Takara Biotechnology Ltd., Dalian, China) was applied for reverse transcription. Fluorescence quantitative PCR instrument (ABI 7500, Applied Biosystems, Inc., CA, USA) was used to amplify target gene and internal reference. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was regarded as internal reference, and 2-ΔΔCt method was utilized to calculate mRNA expression of genes [20]. The experiment was repeated 3 times, which was also applicable for animal experiment. The primer sequences were shown in Table 1.
Western blot analysis
Cells were washed by precooling PBS and lysed by Radio-Immunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor. Next, lysis of cells was removed by a cell scraper, transferred into a centrifuge tube and ice-bathed after vibration for full lysis. The supernatant was collected after centrifugation at 4℃ and 25764 × g for 5 min and the precipitate was abandoned. Protein concentration was determined by bicinchoninic acid (BCA) method. Afterwards, 20 μg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane, which was then sealed with 5% skim milk for 2 h. Subsequently, the membrane was probed with primary antibodies against immunoresponsive gene 1 (Irg1) (ab222411, 1 : 1000), NF-E2-related factor 2 (Nrf2) (ab137550, 1 : 2000) and heme oxygenase-1 (Hmox1) (ab13243, 1 : 2000) overnight at 4℃.After washed with 0.1% tris-buffered saline with Tween-20 (TBST) 3 times, the membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (ab205718, 1 : 5000) at 37℃ for 1 h. The above antibodies were all purchased from Abcam Inc. (Cambridge, MA, USA). Afterwards, the membrane was washed 3 times and visualized by developing solution. The images were captured by enhanced chemiluminescence (ECL). Bio-Rad imaging analysis system (Bio-Rad, Inc., Hercules, CA, USA) was used to photograph, and Quantity One v4.6.2 software was utilized to analyze. The gray value ratio of target protein bands to GAPDH protein band was regarded as the relative expression of proteins. The experiment was run in triplicate.
Enzyme-linked immunoassay (ELISA)
The middle ear lavage fluid of mice was collected for determining expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β based on the manuals of TNF-α, IL-6 and IL-1β ELISA detection kits (RapidBio, Tucson, AZ, USA). The antigen was diluted by coating diluent at the ratio of 1 : 20, and each well was reacted with 100 μL standard diluent at 4℃ overnight. After the solution was discarded, the specimens were washed 3 times (1 min per time), dried and preserved at 4℃. The diluted specimens were added into reaction wells of the ELISA plate (100 μL each well), with negative and positive controls as well as duplicate wells set. Afterwards, each well was reacted with 100 μL specimen diluent diluted by certain multiple of enzyme conjugate at 37℃ for 30 min. Subsequently, the specimens were added with 100 μL HRP substrate solution and visualized in a dark place at 37℃ for 10 - 20 min. When obvious color change was observed in positive control or slight color change was observed in negative control, each well was added with 50 μL stop buffer to terminate the reaction. Within 20 min, a microplate reader (SpectraMax M5, Molecular Devices, Shanghai China) was used to determine the OD value at the light wavelength of 450 nm.
Hematoxylin-eosin (HE) staining
The middle ear tissues of mice were fixed with 10% neutral formaldehyde solution overnight, embedded in paraffin and sectioned into slices. Next, the slices were dehydrated by 100% xylene I and II respectively for 10 min, immersed in ethanol with a descending concentration (5 min each) and washed in distilled water for 3 min. Cell nucleus stained with Hansen hematoxylin for 7 - 10 min turned into bluish violet. Saline was prepared with ethanol at the ratio of 1 : 1. The slices that were quickly dipped into ammonium hydroxide turned into dark blue. After stained with eosin for 6 min, the slices were washed under running water to remove surface dyeing, dehydrated by ethanol with an ascending concentration (5 min each), cleared with 100% xylene I and II respectively for 5 min and mounted with neutral gum. At last, the cover glasses were dried, photographed and observed under an optical microscope.
Statistical analysis
All data were analyzed using a Statistic Package for the Social Science (SPSS) 21.0 statistical software (IBM Corp. Armonk, NY, USA). The enumeration data were expressed as ratio or percentage, and comparisons among groups were analyzed by chi-square test. The measurement data that conformed to normal distribution were described as mean ± standard deviation. Comparisons between two groups were analyzed by non-paired t test, while comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA) with Tukey's post hoc test. Comparisons at different time points were analyzed by ANOVA of repeated measurement with bonferroni’s post hoc test. The level of significant difference was set as p < 0.05.