Patient and tissue samples
CRS patients were diagnosed and classified, according to EPOS2012 [27]. Nasal polyps of CRSwNP patients, sinus mucosa of CRSsNP patients, and inferior turbinate tissue of control patients without sinusitis were collected. A portion of the specimens was stored at -80 °C and reserved in biobank of clinical resources of the third affiliated hospital of Sun Yat-sen university for immunohistochemistry and immunofluorescence testing of frozen sections. The remaining fresh specimens were soaked in PBS containing 1% antibiotic-antimicrobial agent and used for fibroblast cell culture. This study was approved by the ethics committee of the Third Affiliated Hospital of Sun Yat-sen University (NO.[2016]2-16). All subjects were included in the study only after informed consent was obtained.
Human nasal mucosa primary fibroblast cell culture
Human nasal mucosal tissues were cut into 1 × 1-mm fragments and digested with dispase II (50 mg/mL, Sigma-Aldrich, St. Louis, USA) overnight at 4 °C and further digested with trypsin at 37 °C for 15 min. DMEM containing 10% fetal bovine serum was added to stop the digestion. The digested nasal tissues were then filtered through a 70-μm cell filter to obtain a cell suspension.
Real-time quantitative PCR (RT-qPCR)
RT-qPCR was used to detect the mRNA levels of the target genes coding for FN and Col Ι in the Control, CRSwNP, and CRSsNP groups, as previously described. Total RNA was extracted from cells using RNAiso Plus and was reverse transcribed to cDNA using the PrimeScript RT kit (TaKaRa Biotechnology). Real-time quantitative PCR was conducted using SYBR premix Ex Taq kit (TaKaRa Biotechnology) and the corresponding product primers (Invitrogen, Carlsbad, CA, USA). β2 microglobulin (β2M) was used as a housekeeping gene for normalization. Relevant gene expression was analyzed using the comparative CT value method.
Western Blot
Proteins were extracted from cultured fibroblasts using a loading buffer (Thermo Scientific Inc., New York, USA) and boiled for 10 min, electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Sigma-Aldrich) and finally transferred to polyvinylidene difluoride membranes (PVDF) membranes. The membranes were blocked with 5% bovine albumin V (Biotop Technologies, Beijing, China) in tris-buffered saline (Boster Biotechnology, Wuhan) with tween-20 (TBST) (Solarbio Technologies, Beijing, China) for 2 h at room temperature and were incubated with primary anti-FN (1:1000, Abcam, Cambridge, UK), Col Ι (1:1000; Abcam, Cambridge, UK), p-Smad 2/3, Smad 2/3, pIκBα, IκBα (1:1000; Cell Signaling Technology, Boston, USA) and GAPDH antibodies (1:3000; PeproTech, Rocky Hill, USA) overnight at 4 °C. After washing in TBST buffer three times, the membranes were incubated with HRP-labeled secondary antibodies (Bioworld, Nanjing, China) at room temperature for 1 h. The target proteins were visualized using the ECL reaction (Advansta, California, USA) with the FluorChem M System (protein simple, Alpha Innotech Corp). Image J software was used to analyze and quantify the density of the bands.
Immunohistochemistry
Tissues of surgical specimens from control, CRSwNP, and CRSsNP patients were fixed with 4% paraformaldehyde, paraffin-embedded, and cut into 4μm-thick sections. The sections were dewaxed, rehydrated, and subjected to heat retrieval following routine procedures. The sections were covered with 10% goat serum (Boster Biotechnology) to block non-specific binding for 30 min and then incubated with anti- wave protein (Vimentin, VIM, 1:200, Immunoway, Company of Origin), FN and Col Ι antibodies (1:200, Cell Signaling Technology, Boston, USA) overnight at 4°C. After washing three times with PBS, sections were incubated with HRP-labeled secondary antibodies (Bioworld, Nanjing, China) and detected under a 400× magnification microscope (Leica, Wetzlar, Germany). The results were expressed as staining intensity and the percentage of IHC-positive cells as percentages of the total epithelial area.
Immunofluorescence staining and confocal microscopy
For human nasal tissues, frozen slice sections of the tissues were immersed in acetone for 10min, allowed to warm up to room temperature, and then washed with TBS for 20 min on a shaker. After blocking with 5% albumin bovine V (Biotopped Science & Technology, Beijing, China) in TBST (0.1% Tween 20) for 1 h, the sections were incubated with anti-FN or Col Ι antibody (1:200; Cell Signaling Technology, Boston, USA) overnight at 4 °C after blocking with 10% goat serum blocked for 30 min. For fibroblasts, cells were fixed with 4% paraformaldehyde, closed with 1% BSA for 30 min, and then incubated with anti-FN, Col Ι, or p65 antibody (1:500; Cell Signaling Technology, Boston, USA) at 4 °C overnight. Subsequently, frozen tissue sections and fibroblasts were incubated with Alexa Fluor 594 -/- or 488-labeled secondary antibodies (1:1000; (Invitrogen, CA, USA) for 1 hour at room temperature. Finally, nuclei were labeled with 1:30,000 DAPI staining; Biolegend, CA, USA) and sealed with an anti-fade protectant (Invitrogen, CA, USA). Immunofluorescence was detected under a 400x magnification microscope (Leica, Wetzlar, Germany) or a 630x magnification confocal microscope (Carl Zeiss, Jena, Germany).
Statistical analysis
Statistical analysis was performed using IBM SPSS 20 (SPSS, Chicago, IL, USA). Three or more groups were analyzed for significance using one-way analysis of variance (ANOVA) or the Kruskal-Wallis test for comparative study. The t-test or Mann-Whitney U-test (2-tailed) was applied for comparison between groups. The paired t-test or Wilcoxon’s paired with the signed-rank test was used to compare the two groups. Significance was set at P < 0.05.