C57BL/6J mice (SPF degree, 6-8 week) were obtained from their heterozygous littermates in the college of Public Health of Zhengzhou University, Henan, China (temperature 22℃, lights on 08:00-20:00), and were kept in stainless steel cages with water and food ad libitum. All studies were approved by the life Science Institutional Review Board of Zhengzhou University and performed strictly in accordance with the Guideline of Zhengzhou University for Animal Experiments.
Inflammation-related lung tumorigenesis mouse models and glyburide treatment
The inflammation-related lung tumorigenesis models in the mouse were induced by intratracheal instillation with B(a)p and LPS. Mice were divided into 6 groups randomly: Group 1: Vehicle control group (n=16), Group 2: B(a)p plus LPS group (n=30), Group 3: tricaprylin+Gly(0.48mg/kg) group (n=19), Group 4: B(a)P/LPS+Gly(0.48mg/kg) (n=31), Group 5: tricaprylin+Gly(0.96mg/kg) group (n=16) and Group 6: B(a)P/LPS+Gly(0.96mg/kg) (n=34). As shown in Fig.1, mice in groups B(a)p plus LPS, B(a)P/LPS+Gly(0.48mg/kg) and B(a)P/LPS+Gly(0.96mg/kg) were instilled intratracheally with B(a)p (at a dose of 1mg/mouse in 50μl tricaprylin) once a week for 4 times. 3 weeks later, these mice were instilled intratracheally with LPS (at a dose of 2.5μg/mouse in 50μl physiological saline solution) once every three weeks for 5 times, whereas mice in group Vehicle control, tricaprylin+Gly(0.48mg/kg) and tricaprylin+Gly(0.96mg/kg) were given 50μl tricaprylin in a similar manner. All instillations were administered under isoflurane (Sigma) anesthesia. To explore the potential of chronic Gly treatment as a preventive measure against tumorigenesis, mice in group tricaprylin+Gly(0.48mg/kg), B(a)P/LPS+Gly(0.48mg/kg), tricaprylin+Gly(0.96mg/kg), B(a)P/LPS+Gly(0.96mg/kg) were administered the Gly (0.48mg/kg or 0.96mg/kg, dissolved in 50μlsaline) treatment (gavage, three times a week) from one week before the mice were instilled intratracheally with B(a)p (the week of the first time of Gly treatment named Week 0) until the animal model finished. The Gly-treated mice were weighed and their fast blood glucose (FBG) were measured before the modeling process, and were also weighted and measured about once a month in subsequent studies. At week 34, the mice were anesthesia by penrobarbital sodium (1%) and then sacrificed to harvest lungs to count visible tumors on the surface of the lung tissues. The left lobes of the lungs of all groups were used for histopathological studies which were fixed by 4% paraformaldehyde, then the right lobes of the lungs were preserved in -80℃ and used for subsequent studies.
At week 34, the lung tissues were removed from sacrificed mice, washed in physiological saline solution, sucked dry by filter paper, and then weighted with electronic balance. Lung coefficient could be one of the indicators of lung injury, which was expressed as the follow equation: Lung coefficient=lung weight/body weight.
Lung pathological alterations
The left lobes of the lungs were fixed overnight by 4% paraformaldehyde, embedded in paraffin, then cut into sections having a thickness of 5μm. Eventually, the sections were observed by an electron microscope using haematoxylin and eosin(HE) staining. To evaluate the inflammation changes of lung tissues according to the literature published by Huang et al. The pathological tumor nests were identified by two experienced pathologists in a blind manner and based on the previous papers published[26, 27].
The expression of NLRP3, IL-1β, IL-18 protein in lung tissues were determined by immunohistochemistry (IHC). Lung tissue sections were blocked with goat serum for 30 mins at room temperature firstly and incubated with rabbit anti-mouse antibodies at a dilution of 1:100 including NLRP3 (Servicebio, Wuhan, China), IL-1β (Servicebio, Wuhan, China) and IL-18 (Servicebio, Wuhan, China) at 4℃ overnight, followed by incubation with biotinylated goat anti-rabbit immunoglobulin (1:100 dilution) for 50 minutes at 37℃. Positive IHC staining was reflected as brown staining and the AOD quantification was carried out using the Image-Pro Plus 6.0 software in the high-power vision fields (AOD = Integrated Optical Density [IOD] SUM/Area SUM).
Mice lung tissues were lysed using RIPA reagent (CWBio, Beijing, China) containing 100 μg/mL PMSF (CWBio, Beijing, China), protein concentration quantified using BCA Protein Assay Kit (CWbio, Beijing, China), protein samples were mixed with loading buffer and boiled for 5 minutes. Proteins were isolated by 12% or 15% SDS-PAGE and transferred onto Polyvinylidene Fluoride (PVDF) membranes. Membranes were blocked by 5% milk in TBST for 2 hours at room temperature, then incubated overnight at 4℃ with 1:1000rabbit polyclonal antibody to mouse caspase-1 (Servicebio, Wuhan, China), or 1:1000 mouse polyclonal antibody to mouse IL-1β (Servicebio, Wuhan, China), or 1:1000 mouse polyclonal antibody to mouse IL-18 (Servicebio, Wuhan, China) or 1:1000 rabbit polyclonal antibody to mouse β-actin (Servicebio, Wuhan, China), then 1:5000 goat anti-rabbit IgG (Jackson ImmunoResearch, America) or 1:10,000 goat anti-mouse IgG (Jackson ImmunoResearch, America) as secondary antibodies were used to incubate membranes at room temperature for 1 hour. Bands were detected using ECL plus western blotting detection system, and the intensity was quantified by Image J software.
Chi-square test was used to compare the tumor incidence among different groups. The results of mean tumor count, lung coefficient and pathological tumor nests were presented as mean±SEM, and the data analysis was performed by one-way ANOVAs and two-tailed Student’s t-test using SPSS21.0 (IBM, NC, USA). The P value less than 0.05 for two-tailed was considered statistically significant.