Patients and samples
All patients underwent unilateral endoscopic sinus surgery between July 2017 and October 2019 at the Second Affiliated Hospital of Dalian Medical University, Liaoning, China. This study was performed on 57 cases that were equally split between three groups: normal control group (n = 19), including 7 males and 12 females with an average age of 42 y; CRS patients, without nasal polyps (n = 19), including 10 males and 9 females with an average age of 45 y; and MSFB patients (n = 19), including 8 males and 11 females with an average age of 52 y. Biopsies were obtained from the maxillary sinus ostium mucosa of all patients during endoscopic surgery. All procedures used in this study complied with the ethical standards of Dalian Medical University. The study protocol was approved by the Institutional Review Board, Dalian Medical University (2019-052). Written informed consent was obtained from all patients prior to participation.
Inclusion and exclusion criteria
Diagnosis was based on histopathological examination of surgically-collected samples from the maxillary sinus. Diagnosis was reconfirmed by CT scans and operation record. Patients who underwent functional endoscopic sinus surgery (FESS) due to simple maxillary sinus cyst but without CRS were included as control. The experimental groups were patients, diagnosed with MSFB and CRS, who underwent FESS.
Diagnostic criteria for MSFB: 1) Medical history: nasal congestion, runny nose, foul odor of nasal secretions, facial pain, and other similar symptoms associated with rhinosinusitis; 2) Onset of unilateral maxillary sinusitis; 3) Ununiform density of all or most lesions as seen in sinus CT scans. In some lesions, the CT value was higher than the soft tissue but lower than that of a calcification spot or bone. In these instances, the CT value was about 100–150 HU; 4) During surgery, sediment and caseous, brown or yellowish green, fungal masses are observed in the diseased sinuses; 5) Pathology: the mass consists of aggregates of mutually entwined fungal filaments. Diagnosis of CRS without nasal polyps was done according to the Chinese Guidelines for the Diagnosis and Treatment of Chronic Sinusitis (2018).
Patients were excluded if the lesions were located in places other than the maxillary sinus, or when lesions were concurrent with bronchial asthma, allergic rhinitis, acute rhinosinusitis within one month prior to FESS, aspirin triad, autoimmune disease, primary ciliary motor dysfunction, or cystic fibrosis.
All specimens were quickly rinsed with normal saline and then dried with filter paper. Each specimen was cut into three 4-mm parts. One part was immediately soaked in formalin for 24 h, routinely dehydrated, and embedded with paraffin. The other two parts were placed separately in cryovials and submerged quickly into liquid nitrogen.
Detection of NO
Quantity of nitrite, a stable metabolite of NO, was measured using the nitrate reductase method, using NO kit (Griess reagent; Beyotime Biotechnology Co., Ltd., Hangzhou, Zhejiang, China). Briefly, the same volume of supernatant was extracted from all samples after centrifugal. Then, 50 µL of tissue culture medium were mixed with 100 µL Griess reagent. Fresh culture medium was used as a blank control in all experiments. Subsequently, the mixture was incubated at room temperature for 10 min and absorbance at 540 nm was measured by an enzyme label microplate reader. The quantity of nitrite was determined based on a sodium nitrite standard curve.
Tissues embedded in paraffin were sliced in a cryostat into 5 µm slices. Immunostaining was carried out on these sections, using primary antibodies (mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA). For antigen retrieval, sections were rinsed with citric acid solution (pH = 6.0) and then exposed to microwave repair at 100 °C (medium-high temperature) for 25 min. Slides were then incubated with primary antibody overnight at 4 °C, according to the manufacturer’s recommendations. Color development was achieved using streptavidin–biotin amplification (Zhongshan Jinqiao kit; Solely Biotechnology, Beijing, China). Peroxidase activity was visualized using a diaminobenzidine solution. Sections were counterstained with hematoxylin. Control specimens were assayed without the primary antibody and used to verify the absence of non-specific binding. Consecutive sections were stained with hematoxylin to observe mucosal pathology and assess the degree of iNOS expression. Immunohistochemical results: iNOS immunohistochemical staining could be observed under the optical microscope, and the positive expression was yellow or brownish yellow particles under the microscope. The number of positive cells in each field of view was counted at high magnification (10 to 40 times) for each section. Perform statistical analysis.
Western blot analysis
Sinus tissues were minced and homogenized in radio immunoprecipitation assay buffer (RIPA; P0013C, Beyotime Institute of Biotechnology). The homogenate was centrifuged at 12,000 rpm and 4 °C for 15 min, and the supernatant was collected for cytosolic protein analysis. Protein concentration was determined by a BCA protein assay kit (Beyotime Institute of Biotechnology). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on equal amounts of protein samples, and proteins were transferred to polyvinylidene difluoride membrane. After blocking with 5% non-fat milk (Guangming, Shanghai, China), membranes were incubated overnight at 4 °C with anti-iNOS (ab178945; 1:1000, Abcam Biotechnology, Cambridge, United Kingdom ) or anti-β-actin (abm52614; 1:4000, Abcam Biotechnology) antibodies. On the next day, the membranes were incubated with anti-rabbit IgG (H + L) (#35568; 1:6000, Thermo Fisher Scientific, Invitrogen) antibody. The signals were subsequently imaged by Oddessy Clx. Intensity values were expressed as relative protein expression, normalized to β-actin.
Data were analyzed using GraphPad Prism7. Data were presented as means ± standard deviation. Student’s t-test was used to assess the differences between variables in any two groups. Differences between groups were considered statistically significant when p < 0.05. * p < 0.05, ** p < 0.01, and *** p < 0.001. Spearman correlation analysis was used to analyze the correlation between the expression of iNOS and the occurrence of MSFB and CRS. The “p” value should be less than 0.05, and the value range of correlation coefficient “r” is between − 1 and 1.