Human tissue procurement
Our study was approved by the Ethics Committee of the Sun Yat-sen Memorial Hospital of Sun Yat-sen University (2018-212), and written consent was obtained from each participant. Both control subjects and patients with CRSwNP which were aged from 18 to 65 were recruited from the Department of Otorhinolaryngology at the Sun Yat-sen Memorial Hospital of Sun Yat-sen University from September 2018 to July 2019. Their clinical characteristics were listed in Table 1. The diagnosis of CRSwNP was based on a European position paper on rhinosinusitis and nasal polyps published in 2012[24]. Patients with genetic defects such as primary ciliary dyskinesia or any systemic diseases were excluded from this study. Control subjects had no evidence of sinonasal mucosal inflammation but have underwent sinonasal surgery for the repair of cerebrospinal fluid rhinorrhea and optic nerve decompression. Mucosa was removed from the ethmoid sinuses (ES), which can be easily accessed during surgery. Mucosa of ethmoid sinuses from control subjects and polyp tissues from patients with CRSwNP were harvested during surgery. The samples were used for various further analyses as followed.
Air-liquid interface (ALI) cultures
The complete method for the preparation of primary sinonasal epithelial cultures can be found in our previous study[18]. Human sinonasal epithelial cells (HSECs) were transferred to Transwell inserts (Corning, 0.4µm) to initiate the ALI culture. 14 days were required to obtain the differentiated cilia via the PneumaCult™-ALI Medium (Stem cell). The ALI membranes were collected on Day-0, -7 and -14.
Quantitative RT-PCR
Quantitative real-time PCR (RT-PCR) was performed on human sinonasal epithelial cells and clinical samples. β-actin was used as a reference for normalization. Nuclear respiratory factor 1 (NRF1), nuclear respiratory factor 2 (NRF2), transcription factor A, mitochondrial (TFAM) and cytochrome c oxidase subunit 4 (COX4) PCR was performed with the Roche LightCycler 480 Real-Time PCR System using SYBR Premix Ex Taq (Takara). Relative gene expression was calculated using the 2−ΔΔCT method.
Histological staining
Immunohistochemical staining was performed on the paraffin sections. After dehydration, antigen retrieval, quenching of endogenous peroxides, and blocking, sections were incubated overnight at 4 °C with monoclonal mouse anti-NRF1 at 1:100 (Abcam, ab175932), monoclonal mouse anti-NRF2 at 1:100 (Abcam, ab62352), monoclonal mouse anti-TFAM at 1:50 (Abcam, ab131607) and polyclonal rabbit anti-COX4 at 1:200 (Cell signaling, 4850S). DAB and hematoxylin were used for staining.
Immunofluorescence staining was performed for air-liquid interface (ALI) membranes and HSECs. In brief, HSECs were incubated overnight at 4 °C with monoclonal mouse anti-beta tubulin Ⅳ at 1:100 (Abcam, ab11315), and subsequently with Alexa flour 488 secondary antibodies (Invitrogen) at 1:200 at room temperature for 1 hour, and 40,6-diamidino-2-phenylindole (DAPI) (10 mg/mL, Sigma-Aldrich) for 10 minutes.
Mitochondrial structure was detected via fluorescent MitoTracker (Invitrogen), which was cultured with HSECs at 1:500 dilution at 37 °C for 1 hour. Then the cells were fixed and stained with DAPI for 10 minutes. HSECs were observed and imaged with a Zeiss LSM 780 confocal microscope.
Western blot analysis
Total protein from nasal mucosa and HSECs were extracted in RIPA lysis buffer. Protein concentrations were determined by bicinchoninic acid assay (BCA). Samples which contained 30 µg of protein were resolved via SDS-PAGE in 10%~12% Tris-glycine gels and transferred onto a polyvinylidene fluoride membrane (Milipore) and blocked with 5% bovine serum albumin (BSA). The membranes were incubated overnight with monoclonal mouse anti-NRF1 (Abcam, ab175932), monoclonal mouse anti-NRF2 (Abcam, ab62352), monoclonal mouse anti-TFAM (Abcam, ab131607), polyclonal rabbit anti-COX4 (Cell signaling, 4850S) at 1:1000, then incubated with an appropriate secondary antibody (1: 10,000) for 1 hour. The relative protein levels were quantified by densitometric image analysis of bands using Fiji (National Institutes of Health) and were normalized against β-actin.
Scanning electron microscopy
The membranes which contained HSECs from the Transwell inserts were fixed with 2.5% glutaraldehyde for 4 hours at room temperature. Subsequently, the cells were progressively dehydrated in 30%, 50%, 70% and 90% ethanol for one time per concentration at 15-minutes intervals, 3 times in 100% ethanol for 15 minutes, and 3 times in 100% tert-butyl alcohol for 15 minutes. The filters were then glued onto scanning electron microscopic (SEM) stubs and sputter coated to a thickness of 12 nm by gold palladium. The surface of the Transwell membrane was examined at an accelerating voltage of 10 kV using a Quanta-400 scanning electron microscope (FEI).
Cell stimulation and transfection
Human sinonasal epithelial cells were transfected with siRNA targeted to WDPCP using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. HSECs were stimulated by U0126 (10 µM) or dexamethasone (0.01 mg/mL). After 24 hours of incubation, the protein was collected for further experiments.
Mitochondrial DNA copy number analysis
Total DNA was extracted from HSECs using the QIAamp DNA Mini Kit (Qiagen). The mitochondrial DNA (mtDNA) copy number was determined by amplifying genes encoding genomic DNA and mitochondrial DNA. The mtDNA levels were quantified by quantitative real-time polymerase chain reaction on a Roche Light Cycler 96 (Roche) using HV1 primers (forward: 5’-TTGCACGGTACCATAAATACTTGAC-3’, reverse: 5’- GAGTTGCAGTTGATGTGTGATAGTTG-3’). Nuclear gene β-globin primers (forward: 5’-ACACAACTGTGTTCACTAGC-3’, reverse: 5’- CAACTTCATCCACGTTCACC-3’) were used as a nuclear control. Relative quantification of mitochondrial DNA copy number was calculated after using the 2−ΔΔCT method to obtain the expression fold change.
Mitochondrial membrane potential measurement
The mitochondrial membrane potential was evaluated with JC-1 fluorescent dye (Beyotime, China). JC-1 displayed red or green fluorescence depending on the mitochondrial potential. Normal mitochondrial membrane potential exhibited red fluorescence, whereas damaged mitochondria exhibited green fluorescence. The HSECs were stained with JC-1 in culture media for 20 minutes at 37 °C and were then washed for 3 times with washing buffer. The mitochondrial membrane potential was measured by flow cytometry (BD Biosciences).
Measurement of ATP levels
An ATP Assay Kit (Beyotime, China) was used to conduct the ATP assay. Briefly, HSECs were collected in lysis buffer and centrifuged at 12,000 × g for 5 minutes at 4 °C. ATP detection reagent was added into 96-wells, then the standards and samples were added into the wells and the detection solution was mixed. Chemiluminescence was detected by a Synergy H1 Hybrid Multi-Mode Reader (BioTek, USA). The levels of ATP were calculated based on the standard curve and were normalized to the protein content.
Cilia beating frequency
Both ciliated HSECs in ALI culture and clinical samples collected within 30 minutes were used for cilia beating frequency (CBF) analysis. The room temperature was controlled at 25 ℃, and the humidity was maintained at 70-80%, which would not significantly affect the CBF[25]. A high-speed digital video camera (Basler AG) captured images at 100 frames per second and Sisson-Ammons Video Analysis (SAVA) software (National Instruments) was used for video analysis. Each measurement was obtained 3 times and recorded 15 seconds each time.
Statistical analysis
Data were presented as mean ± standard error of the mean (SEM), and analyzed using GraphPad PRISM 7 (GraphPad Software). One-way ANOVA, Student’s t-test, one sample t-tests and Fisher’s exact tests were performed for statistical analysis. A value of p <0.05 was considered statistically significant.